Expression vector for controllably up-regulating ACE2 targeting prevention and treatment of hypoxic pulmonary hypertension
阅读说明：本技术 可控性上调ace2靶向性防治低氧性肺动脉高压的表达载体 (Expression vector for controllably up-regulating ACE2 targeting prevention and treatment of hypoxic pulmonary hypertension ) 是由 刘曼玲 赵澎涛 董明清 李志超 马恒 董海莹 张博 于 2019-11-28 设计创作，主要内容包括：本发明公开了一种可控性上调ACE2靶向性防治低氧性肺动脉高压的表达载体。本发明构建了HRE增强、Tie2启动子驱动的ACE2表达载体,通过Tie2启动子特异定位于血管内皮细胞,同时,利用HIF-1α只在低氧的肺血管内皮细胞表达特异性激活HRE,从而上调ACE2的表达。本发明发现所构建的表达载体不仅具有靶向性,而且可以根据缺氧程度可控和有效地上调ACE2在低氧的肺微血管内皮细胞中的表达,进而调控肺动脉平滑肌细胞,实现低氧可控性、靶向性地抑制肺动脉的收缩和逆转肺动脉的结构重建,为防治低氧性肺动脉高压等疾病提供有效手段和策略。(The invention discloses an ACE2 expression vector driven by HRE enhanced and Tie2 promoter and specifically positioned on vascular endothelial cells through a Tie2 promoter, and meanwhile, the HRE is specifically activated only in hypoxic pulmonary capillary endothelial cells by using HIF-1 α, so that the expression of ACE2 is up-regulated.)
1. An ACE2 recombinant expression vector, comprising: comprises a hypoxia response element, a promoter, a signal peptide sequence and an ACE2 coding sequence, wherein the hypoxia response element is selected from an HRE sequence, and the promoter has specificity to vascular endothelial cells.
2. The recombinant expression vector of ACE2, according to claim 1, wherein: the repeated arrangement frequency of the HRE sequence is 1-100.
3. The recombinant expression vector of ACE2, according to claim 1, wherein: the promoter is selected from Tie2gene promoters.
4. The recombinant expression vector of ACE2, according to claim 1, wherein: the expression vector specifically comprises a 6 XHRE sequence, a Tie2gene promoter sequence, a signal peptide, a hIgG1Fc fusion marker sequence and an ACE2 coding sequence which are sequentially arranged.
5. A method of constructing the expression vector of claim 1, wherein: the method comprises the following steps:
synthesizing an HRE sequence, a promoter sequence, a signal peptide sequence and an ACE2 coding sequence; the individual sequences synthesized were cloned into an expression vector backbone.
6. Use of the expression vector of claim 1 for the preparation of a medicament for the prevention and/or treatment of hypoxic pulmonary hypertension and its complications.
7. Use according to claim 6, characterized in that: the expression vector has cell targeting property, hypoxia controllability and effectiveness of inhibiting proliferation of pulmonary artery smooth muscle cells, wherein the targeting property shows that the ACE2 is specifically and highly expressed in lung microvascular endothelial cells after the expression vector is transfected, the controllability shows that the increase rate of the expression of the ACE2 is increased along with the reduction of oxygen concentration, and the effectiveness shows that the ACE expression vector has an effect of inhibiting the proliferation of the pulmonary artery smooth muscle cells induced by hypoxia.
8. Use according to claim 6, characterized in that: the drug is selected from a lung microvascular endothelial cell culture solution containing the expression vector or adeno-associated virus packaged with the expression vector.
9. Use according to claim 6, characterized in that: the medicine adopts a nasal administration mode.
10. Use of an expression vector according to claim 1 for the preparation of a medicament for the prevention and/or treatment of hypoxia and/or ischemia related diseases, characterized in that: the disease is selected from one or more of diabetic gangrene, deep vein thrombosis and ischemic heart disease.
Technical Field
The invention relates to the field of treating hypoxic pulmonary hypertension by applying a gene recombination technology, in particular to a construction method of an HRE (high resolution factor) enhanced Tie2 promoter-driven angiotensin converting enzyme 2(ACE2) expression vector.
Background
Hypoxic Pulmonary Hypertension (HPH) is a common morbidity link in a variety of respiratory diseases, chronic plateau disease, and various diseases associated with hypoxemia. Hypoxic pulmonary vasoconstriction caused by hypoxia is one of important physiological reactions of the body, and has important significance in maintaining the ventilation/blood flow ratio around hypoxic alveoli, reducing functional shunting and improving the blood oxygen saturation. But chronic, extensive hypoxic pulmonary vasoconstriction can lead to pulmonary vascular structure remodeling. The reconstruction of the pulmonary vascular structure is a key factor for causing the continuous increase of the pulmonary artery pressure and promoting the occurrence and development of the pulmonary heart disease. Therefore, specific relaxation of pulmonary artery, inhibition or reversal of pulmonary revascularization is the main target for prevention and treatment of hypoxic pulmonary hypertension and its complications.
Studies have shown that the renin-angiotensin (RAS) system in the lung is involved in the development of hypoxic pulmonary hypertension. In hypoxic pulmonary hypertension, angiotensin ii (angii), a major effector protein of RAS, not only strongly constricts pulmonary vessels, but also significantly promotes proliferation and hypertrophy of Pulmonary Artery Smooth Muscle Cells (PASMCs), which is one of important factors causing continuous increase of pulmonary vascular resistance. Other members of the RAS, such as renin, angiotensinogen, etc., are also involved in the development of HPH in various ways. Therefore, under hypoxic pulmonary hypertension, dysfunction of the RAS in the lungs may be one of the important causes of hypoxic pulmonary vasoconstriction and structural remodeling. Recently, ACE2, a new member of RAS, is a membrane protein consisting of 805 amino acids and widely distributed in tissues of the heart, the vascular endothelial cells of the lung, the vascular smooth muscle cells, the pulmonary epithelial cells, the cardiac muscle cells and the gastrointestinal tract, the innocent bolus, the retina, the uterus, the placenta, etc. Its main action is to produce Angiotensin- (1-7) [ Angiotensin- (1-7), Ang- (1-7) ] with high efficiency. ACE2 can degrade angiotensin I (Ang I) to Ang- (1-9), and then Ang- (1-9) is subjected to neutral endopeptidase and prolyl endopeptidase to generate Ang- (1-7). ACE2 can also be used to directly enzymatically hydrolyze angiotensin II (Ang II) to generate Ang- (1-7). Ang- (1-7) has vasodilation-promoting, antiproliferative, and anti-hypertrophy effects by binding to its specific Mas receptor. By producing large amounts of Ang- (1-7), ACE2 can finely regulate the balance between vasoconstrictor and vasomotor activity in the body. In addition, ACE2 also participates in the homeostasis of the body by acting on various active substances in the body. Therefore, the expression of ACE2 in pulmonary circulation is increased, which is beneficial to improving the function disorder of RAS in lung, inhibiting the constriction of hypoxemic pulmonary vessels and reversing the reconstruction of pulmonary vessel structure, thereby playing the role of effectively preventing and treating the hypoxemic pulmonary hypertension.
However, the conventional administration route lacks specificity, resulting in side effects of lowering systemic circulation blood pressure. Moreover, long-term single expansion of pulmonary vessels can also lead to venous blood adulteration and aggravation of hypoxemia. How to make ACE2 specifically locate in pulmonary blood vessel only and express in pulmonary blood vessel only under hypoxia condition, and controllable regulation can be realized according to degree of hypoxia, which is important and key for preventing and treating hypoxic pulmonary hypertension.
Chinese patent CN101532027 (published: 2009, 9, 16) discloses an hypoxia inducible eukaryotic gene expression vector and application thereof, wherein the hypoxia inducible gene expression vector is constructed by transforming pcDNA3.1 and replacing an enhancer of the hypoxia inducible gene expression vector with the enhancer of the hypoxia inducible gene expression vector; in order to detect the function of an hypoxia inducible gene expression vector, cDNA of hVEGF165 is inserted into the vector, a hypoxia inducible eukaryotic gene expression vector p6HRE-hVEGF165 containing hVEGF165 is constructed, the vector is transferred into BHK cells, the BHK cells expressing hVEGF are cultured in a hypoxia environment, the content of hVEGF in culture supernatant is detected by an ELISA method, and the result proves that the expression quantity of hVEGF after hypoxia induction is increased; the p6HRE-hVEGF165 is used for gene therapy of rabbit limb ischemic diseases, can effectively promote the formation of new blood vessels and collateral circulation of the affected limb, and enables the tibial arterial pressure of the affected limb to recover.
But the pulmonary circulation vessels have significantly different properties from the systemic circulation vessels of the above patents. The pulmonary vessels contract hypoxia during short-term hypoxia so as to maintain the ventilation/blood flow ratio around hypoxic alveoli, reduce functional shunting and improve the blood oxygen saturation; long-term hypoxia causes excessive secretion of vasoactive substances such as endothelin, 5-hydroxytryptamine blood vessels, endothelial growth factor (VEGF) and the like, and further participates in pulmonary vessel reconstruction, aggravates the degree of ischemia and hypoxia of lungs, and is the pathophysiological characteristic of causing hypoxic pulmonary hypertension. The systemic circulation blood vessel is obviously dilated under the condition of hypoxia, and the blood flow is increased to relieve the symptoms of ischemia and hypoxia. Hypoxic pulmonary arterial hypertension (HPH) therefore requires more precise expression regulation to be given depending on the degree of hypoxia.
Disclosure of Invention
The invention aims to provide an expression vector for controllably up-regulating ACE2 targeting prevention and treatment of hypoxic pulmonary hypertension.
In order to achieve the purpose, the invention adopts the following technical scheme:
an ACE2 recombinant expression vector (HTSFCACE2 for short) comprises a hypoxia response element, a promoter, a Signal Peptide (Signal Peptide) sequence and an ACE2 coding sequence, wherein the hypoxia response element is selected from an HRE sequence, and the ACE2 expression has vascular endothelial cell specificity by utilizing the promoter while playing the role of HRE.
Preferably, the repeated arrangement frequency of the HRE sequence is 1-100.
Preferably, the promoter is selected from Tie2gene promoters.
Preferably, the expression vector specifically comprises a 6 × HRE sequence, a Tie2gene promoter sequence, a signal peptide, an hIgG1Fc fusion marker sequence and an ACE2 coding sequence which are sequentially arranged.
Preferably, the expression vector is constructed on an adeno-associated viral vector backbone or a eukaryotic expression vector backbone.
The construction method of the expression vector comprises the following steps: synthesizing a 6 XHRE sequence, a Tie2gene promoter sequence, a signal peptide, a hIgG1Fc fusion marker sequence and an ACE2 coding sequence; each of the synthesized sequences was cloned into an expression vector backbone (e.g., pAAV-MCS), and the resulting recombinant plasmid was designated HRE-Tie2-FC-ACE2 (HTSFCACE2 for short). The recombinant plasmid without the ACE2 coding sequence was used as a control plasmid and was designated HRE-Tie2-FC (HTSFC for short).
Use of the above expression vector (HTSFCACE2) for the preparation of a medicament for the prevention and/or treatment of hypoxic pulmonary hypertension and/or complications thereof.
Preferably, the expression vector has cell targeting, normoxia (21% O)2Concentration) was only allowed to specifically express ACE2 in transfected lung microvascular endothelial cells (PMVECs).
Preferably, the expression vector has the controllability of hypoxia (oxygen concentration below 21%, i.e., hypoxia), at various concentrations of hypoxia (10%, 5% and 1% O)2) Under stimulation, the expression level of ACE2 in transfected PMVECs and cell supernatant is increased obviously, and the increase rate is increased along with the decrease of oxygen concentration.
Preferably, the expression vector has the effect of inhibiting the proliferation of the PASMCs, for example, after PMVECs are transfected into HTSFCACE2, hypoxia stimulation with 10% oxygen concentration is given, and the supernatant of PMVECs cells collected after the PMVECs are cultured for 24 hours continuously is PMVECs hypoxia condition culture solution containing the expression vector HTSFCACE2, and the PMVECs hypoxia condition culture solution has obvious inhibition effect on the proliferation of hypoxia-induced Pulmonary Artery Smooth Muscle Cells (PASMCs).
Preferably, the HTSFCACE2 expression vector packaged as the adeno-associated virus can reduce the right ventricular pressure of rats, relieve the hypertrophy degree of the right ventricle, inhibit the structural change of pulmonary arterioles and improve the reconstruction of pulmonary artery structures after being applied to hypoxic pulmonary hypertension rats in a nasal drip manner at one time, and the virus can increase the expression of ACE2 only in lung tissues after entering the bodies of the rats, reduce the content of angiotensin II (AngII) in the lung tissues and increase the content of angiotensin (1-7) [ Ang (1-7) ], so that the virus has tissue specificity and effectiveness.
The expression vector (HTSFCACE2) can be used for preparing medicine for preventing and/or treating diabetic gangrene, deep vein thrombosis, ischemic heart disease, etc. and a series of diseases related to hypoxia and/or ischemia.
The invention has the beneficial effects that:
the specificity of the promoter driving expression positioned in vascular endothelial cells, the hypoxia conditional expression of HRE and the characteristic that ACE2 has the fine regulation of a renin-angiotensin (RAS) system in lung are utilized to construct an ACE2 expression vector driven by the HRE enhancement and the promoter (such as a Tie2gene promoter), so that ACE2 can be expressed in hypoxic PMVECs in a targeted, controllable and effective manner, PASMCs are regulated and controlled, contraction and structural reconstruction of pulmonary arteries are inhibited, the effect of preventing and treating hypoxic pulmonary hypertension is achieved, a new strategy and an important experimental basis are provided for researching gene therapy of hypoxic pulmonary hypertension and complications of hypoxic pulmonary hypertension, and the application prospect is good.
Furthermore, the invention improves the tissue specificity of expression by using the Tie2gene promoter (Tie2 promoter), solves the side effect of reducing the systemic circulation blood pressure caused by the existing eukaryotic expression vector promoter, and avoids the problems of long-term single reduction of venous blood doping caused by pulmonary vascular pressure and aggravation of hypoxemia of patients. Meanwhile, expression controllability and accuracy are improved.
Furthermore, the invention ensures the full combination with the subsequent sequence in the case of hypoxia by optimizing the determined HRE sequence repetition times, and improves the controllability and the accuracy of expression.
Furthermore, the experiment of the invention verifies that the recombinant plasmid HTSFCACE2 can be highly expressed in the hypoxic pulmonary artery endothelial cells, so as to regulate and control the pulmonary artery smooth muscle cells, realize the hypoxic controllability and the targeted inhibition of the contraction of the pulmonary artery and the reversal of the structural reconstruction of the pulmonary artery. The recombinant plasmid is also suitable for a series of diseases related to hypoxia or/and ischemia, such as diabetic gangrene, deep venous thrombosis, ischemic heart disease and the like.
Drawings
FIG. 1 is a schematic diagram of the structure of the recombinant plasmid HTSFCACE2 and a control plasmid.
FIG. 2 shows the restriction enzyme identification result of
FIG. 3 shows the restriction enzyme identification result of the control plasmid HTSFC; lane M is marker,
FIG. 4 shows normal oxygen (21% O)2) Changes in expression of ACE2 following transfection of HTSFCACE2 with different types of cells under conditions; wherein: (A) and (B) expression and quantification profiles of ACE2 in different cell types, P<0.05vs. untraduced pmvecs cells, n ═ 5; (C) for the change in the amount of ACE2 in the supernatants of different cell types<0.05vs.untransduced PMVECs cells,n＝5。
FIG. 5 shows the recombinant plasmid HTSFCACE2 vs. normoxia (21% O)2) And varying degrees of hypoxia (10%, 5% and 1% O)2) Effect of ACE2 expression levels in stimulated PMVECs cells; wherein: (A) and (B) is a representation and quantification of ACE2 expression in PMVECs cells<0.05vs. untransformed PMVECs cells, n ═ 5; (C) change in the rate of increase of ACE2 expression in PMVECs cells<0.05vs.Normoxia+transduced HTSFCACE2,n＝5。
FIG. 6 shows the recombinant plasmid HTSFCACE2 vs. normoxia (21% O)2) And varying degrees of hypoxia (10%, 5% and 1% O)2) Effect of ACE2 content in stimulated PMVECs cell supernatants; wherein: (A) the change of ACE2 content in PMVECs cell supernatant<0.05vs. untransformed PMVECs cells, n ═ 5; (B) the change of the increase rate of the ACE2 content in the supernatant of PMVECs cells<0.05vs.Normoxia+transduced HTSFCACE2,n＝5。
FIG. 7 is a graph of PMVECs hypoxic conditioned medium versus normoxic (21% O) medium containing HTSFCACE22) And varying degrees of hypoxia (10%, 5% and 1% O)2) Effects of changes in proliferation of stimulated PASMCs; wherein: (A) is normoxia (21% O)2) And varying degrees of hypoxia (10%, 5% and 1% O)2) (iii) the effect of stimulation on proliferation changes of PASMCs,. P<Normoxia group, n ═ 5; (B) is normoxia (21% O)2) Proliferation change of PMVECs (human serum leukocytes) hypoxia culture solution containing HTSFCACE2 on PASMCs under the conditionThe influence of (a); (C) is a mixture of varying degrees of hypoxia (10%, 5% and 1% O)2) Effect of hypoxic conditioned cultures of PMVECs containing HTSFCACE2 on proliferation of PASMCs<(iii) 0.05vs. untraduced, n-5; (D) effect of different concentrations of hypoxic conditioned culture of PMVECs containing HTSFCACE2 on proliferation changes of PASMCs under 10% hypoxic conditions<Untransformed group, n is 5.
FIG. 8 shows that hypoxia promotes progression of pulmonary hypertension in rats; wherein: (A) effect of hypoxia on mean left common carotid pressure (mCAP) in rats for 4 weeks; (B) effect of hypoxia on rat Right Ventricular Pressure (RVSP) for 4 weeks; (C) is the effect of hypoxia on the right heart hypertrophy index [ RV/(LV + S) ] of rats for 4 weeks; (D) effect of hypoxia on pulmonary arteriole area ratio (WA%) in rats for 4 weeks; (E) effect of hypoxia on the ratio of vessel diameter (WT%) of pulmonary arterioles in rats for 4 weeks; (F) the effect of hypoxia on rat lung tissue for 4 weeks; p <0.05vs. con group (normoxia), n-8.
FIG. 9 is a graph showing that hypoxia reduces the expression of ACE2 and promotes progression of pulmonary hypertension in rats; wherein: (A) and (B) is a graph of the influence and quantification of the hypoxia on the ACE2 protein expression level in rat lung tissues; (C) the effect of hypoxia on the content of AngII in the homogenate of rat lung tissue; (D) and (E) reducing the amount of ACE2 and Ang (1-7) in rat lung homogenate for hypoxia; p <0.05vs. con group (normoxia), n-8.
FIG. 10 shows the result of agarose gel electrophoresis of the PCR product; lane M is marker,
FIG. 11 is a graph of the progression of AAV-HTSFCACE2 in ameliorating hypoxic pulmonary hypertension in rats; wherein: (A) is the effect of AAV-HTSFCACE2 on mean left common carotid artery pressure (mCAP) in rats; (B) is the effect of AAV-HTSFCACE2 on rat right ventricular pressure (RVSP); (C) is the influence of AAV-HTSFCACE2 on the right heart hypertrophy index [ RV/(LV + S) ] of rats; (D) the effect of AAV-HTSFCACE2 on the ratio of vessels to arterioles in rat lung (WT%); (E) the effect of AAV-HTSFCACE2 on the area ratio (WA%) of pulmonary arterioles in rats; (F) the effect of AAV-HTSFCACE2 on rat lung tissue; p <0.05vs. normoxia, P <0.05vs. hypoxia, n-8.
FIG. 12 is a graph showing that AAV-HTSFCACE2 upregulates ACE2 expression in lung tissue of hypoxic pulmonary hypertension rats; wherein: (A) and (B) the effect and quantification result of AAV-HTSFCACE2 on the expression of ACE2 in rat lung tissue; (C) is the effect of AAV-HTSFCACE2 on the amount of AngII in rat lung homogenate; (D) is the effect of AAV-HTSFCACE2 on the content of Ang (1-7) in rat lung homogenate; (E) the influence of AAV-HTSFCACE2 on the content of ACE2 in tissue homogenates of rat heart, liver, lung, kidney and the like; (F) testing the effect of AAV-HTSFCACE2 on rat lung tissue for immunohistochemical staining; (G) quantification of immunohistochemical staining; p <0.05vs. normoxia, P <0.05vs. hypoxia, n-8.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Firstly, construction, identification and sequencing verification of recombinant plasmid HRE-Tie2-FC-ACE2
The 6 XHRE sequence (rat HRE: 5' GACTCCACAGTGCATACGTGGGCTTCCACAGGTCGTCTC3', see SEQ. ID. NO.1) and the promoter of Tie gene (Tyrosine kinase with Id EGF homology domains) (Mus mululus receptor type kinase Tie2gene,5' -cloning generation, access number AF022456.1, location: AF022456.1: 1-223, Tie2 promoter for short) were synthesized from the whole gene. Meanwhile, synthesizing a signal peptide and a hIgG1Fc fusion tag (SPFC:5'ATGAAACATCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGATGGGTCCTGTCC3', see SEQ. ID. NO.2, the signal peptide and the fusion tag are fused in a section of sequence, and the sequence has two corresponding functions) and a rat ACE2 coding sequence (the specific sequence is see SEQ. ID. NO.5), and cloning the sequences to pUC57 (Shenzhen Baien vitamin science and technology Co., Ltd.) to obtain a template plasmid pUC57-HRE-Tie2-FC-
The recombinant plasmid HTSFCACE2 and the control plasmid HTSFC are subjected to double enzyme digestion identification by MluI and BamHI, and are subjected to agarose gel electrophoresis to obtain target gene bands of 2953bp and 541bp respectively (figure 2 and figure 3). And respectively recovering the plasmid pAAV-MCS enzyme digestion large fragment and the recombinant target gene, and carrying out sequencing verification, wherein the result is completely the same as the original sequence, and the recombinant plasmid HTSFCACE2 and the reference plasmid HTSFC are successfully synthesized.
Second, cell experiment
1. Primary culture and characterization of rat pulmonary microvascular endothelial cells (PMVECs) and rat Pulmonary Artery Smooth Muscle Cells (PASMCs)
Primary PMVECs were cultured according to the modified tissue block method. The method comprises the following specific steps: anesthetizing and killing rat (purchased from fourth department of military medical university animal center), rapidly taking out lung tissue, taking out lung tissue block at outer edge of lung in superclean bench, rinsing lung tissue block for several times with precooled serum-free DMEM culture solution, and shearing into pieces with volume of about 1mm3The tissue blocks are uniformly planted in culture flasks. Add about 2ml DMEM medium containing 20% FBS, 90U/ml heparin sodium, 100U/ml penicillin and 100U/ml streptomycin, place the flask upright for 1h and turn over the flask, let the medium slowly submerge, cover the tissue mass. The culture was continued for 24h, and then the medium was changed to aspirate the blood cells. And (4) observing the forms of the ECs, and removing the mixed cells by adopting differential digestion and differential adherence. And (4) carrying out passage after the bottom cell is basically confluent in a monolayer. PMVECs were identified by immunohistochemical staining with factor VIII.
And (3) culturing the primary PASMCs by adopting a tissue block adherence method. The method comprises the following specific steps: anesthetizing and killing rat, quickly taking out lung tissue, quickly separating main pulmonary artery and second and third pulmonary arteries in a superclean bench, rinsing with precooled serum-free DMEM culture solution, stripping adventitia of blood vessel, and destroying bloodCutting the inner tube into 1mm3Uniformly planting the tissue blocks with the sizes in a culture bottle, adding 2ml of DMEM culture solution containing 15% fetal calf serum, vertically placing the culture bottle for 2-4h, turning over the culture bottle, slowly immersing the culture solution to cover the tissue blocks, continuously culturing for 24h, changing the culture solution, observing the growth condition of the PASMCs under a microscope, allowing the PASMCs with elongated fusiform shapes to climb out from the periphery of the tissue blocks after about 7-10d, carrying out passage when the PASMCs grow to 80% fusion, detecting the expression of α -SM-actin in the PASMCs by an immunohistochemical method, and identifying the PASMCs.
2. Normal oxygen (21% O)2) Effect of transient transfection of HTSFCACE2 and control plasmid on ACE2 expression levels in different cell types and cell supernatants under conditions
50. mu.l of each of the bacterial solutions containing the recombinant plasmid HTSFCACE2 and the control plasmid HTSFC were added to the culture broth containing Amp+The OD600 was determined to be about 0.4 to 0.6 in a sterile Erlenmeyer flask with LB medium, shaking overnight at 37 ℃ at 250 rpm. Plasmids were extracted according to kit instructions, OD values were determined, quantitated, and stored at-20 ℃ until use.
The A549, 293 and NIH-3T3 cells (purchased from ATCC in USA) were recovered and cultured in DMEM containing 10% FBS. 2-3 passages of PASMCs, PMVECs, A549, 293 and NIH-3T3 cells were used.
The results of the above experiments were: under the condition of normal oxygen (21% O)2Concentration) A549 cells, 293 cells, Lung motilityThe cells and cell supernatants of pulse microvascular endothelial cells (PMVECs) and Pulmonary Artery Smooth Muscle Cells (PASMCs) both contained a small amount of ACE2 expressed, while the ACE2 expression was lower in the cells and cell supernatants of fibroblasts (NIH-3T3) (FIG. 4). The four cells were cultured under normoxic conditions (21% O)2Concentration) of ACE2 in cells and cell supernatant after transfection of recombinant plasmid HTSFCACE2, the expression level of ACE2 was not significantly increased in a549, 293, NIH-3T3 and PASMCs cells, but significantly increased in PMVECs (fig. 4). After the control plasmid is transfected, the expression level of ACE2 in the cell and the cell supernatant of each type of cells is not obviously influenced. The experimental result shows that the recombinant plasmid HTSFCACE2 is specifically and highly expressed only in the transfected PMVECs and has cell targeting property.
3. PMVECs were transfected with the recombinant plasmid HTSFCACE2 and the control plasmid HTSFC, respectively, and then given different hypoxia (10%, 5%, and 1% O)2) Detecting the content of ACE2 in cell supernatant after stimulation
Selecting PMVECs cultured in 3 rd-4 th generation according to the ratio of 105The cells were inoculated into 6-well plates at a concentration of one ml and transfected until the cells reached 80% (specific procedure:
The results of the above experiments were: the expression level of ACE2 in PMVECs without plasmid transfection decreased gradually with decreasing oxygen concentration. After transfection of the recombinant plasmid HTSFCACE2, the expression level of ACE2 in PMVECs cells was significantly increased (fig. 5A and B), and the rate of increase of the expression level of ACE2 increased with decreasing oxygen concentration (fig. 5C).
The content of ACE2 in the supernatant of PMVECs cells in the case of untransfected plasmids gradually decreased with decreasing oxygen concentration. After transfection of the recombinant plasmid HTSFCACE2, the content of ACE2 in the supernatant of PMVECs cells was significantly increased (FIG. 6A), and the increase rate of the ACE2 expression amount was increased with the decrease of the oxygen concentration (FIG. 6B).
After transfection of the control plasmid, the expression level of ACE2 in PMVECs cells and cell supernatant is not obviously influenced. The recombinant plasmid HTSFCACE2 therefore has hypoxia controllability at the cellular level.
4. Preparation of hypoxic conditioned PMVECs culture Medium, addition to PASMCs for Normal oxygen (21% O)2) And different concentrations of hypoxia (10%, 5% and 1% O)2) Proliferative changes in PASMCs following stimulation
PMVECs in accordance with 105The plasmid was inoculated into a 6-well plate at a concentration of one ml and cultured, and transfection was carried out after 80% growth (specific procedure:
Collecting growth-promoting PASMCs at 5 × 103Each well was inoculated into a 96-well plate, and DMEM medium containing 10% FBS was added. And when the cells grow to be full of 60%, replacing serum-free DMEM culture solution to culture for 24 hours to synchronize. Mixing a newly prepared PMVECs hypoxia condition culture solution or control condition culture solution containing recombinant plasmid HTSFCACE2 and a DMEM culture solution containing 10% FBS according to different proportions, and adding the mixture into the holes, wherein the mixing proportions of the PMVECs hypoxia condition culture solution or control condition culture solution are respectively 0%, 25%, 50% and 75%:
① 0% group, adding 200 μ L DMEM culture solution containing 10% FBS and 0 μ L of the PMVECs hypoxic condition culture solution or control condition culture solution into each well;
② 25% PMVECs hypoxic condition culture medium group/control condition culture medium group, adding 150 μ L DMEM culture solution containing 10% FBS and 50 μ L PMVECs hypoxic condition culture solution or control condition culture solution into each well;
③ 50% PMVECs hypoxic condition culture medium group/control condition culture medium group, adding 100 μ L10% FBS DMEM culture medium and 100 μ L PMVECs hypoxic condition culture medium or control condition culture medium into each well;
④ 75% PMVECs hypoxic condition culture medium group/control condition culture medium group, adding 50 μ L DMEM culture solution containing 10% FBS +150 μ L PMVECs hypoxic condition culture solution or control condition culture solution per well;
the above groups of PASMCs with or without the hypoxic conditioned medium of PMVECs or the control conditioned medium were then subjected to different oxygen concentrations (21%, 10%, 5% and 1% O)2) The culture was continued with 5 replicates per oxygen concentration. Adding MTT (5mg/mL) for further incubation for 4h, then adding 150 μ l of DMSO solution into each well, shaking for 5min, measuring the absorbance OD value of each well at 490nm wavelength on a full-automatic enzyme standard instrument, taking 3 measurement results from each well, and calculating the average value.
The results of the above experiments were: pulmonary Artery Smooth Muscle Cells (PASMCs) in normoxia (21% O)2) And different concentrations of hypoxic stimuli (10%, 5% and 1% O)2) After 48 hours of culture, there was a variable degree of proliferation of the PASMCs with decreasing oxygen concentration, suggesting that hypoxia induced proliferation of the PASMCs (fig. 7A). The recombinant plasmid HTSFCACE2 transfects PMVECs, and after the PMVECs are subjected to hypoxia stimulation with the oxygen concentration of 10% for 24 hours, the collected PMVECs cell supernatant is the PMVECs hypoxia condition culture solution (namely the PMVECs hypoxia condition culture solution containing HTSFCACE 2). Fresh PMVECs hypoxic conditioned Medium was immediately added to the PASMCs, followed by normoxic (21%, O)2) And different concentrations of hypoxia (10%, 5% and 1% O)2) Proliferation changes of the PASMCs were detected after 48 hours of continued culture under the conditions. PMVECs hypoxic conditioned Medium containing HTSFCACE2 vs. normoxic (21% O)2) Proliferation of the PASMCs cultured under the conditions was not significantly affected (fig. 7B). But under different concentrations of hypoxic conditions (10%, 5% and 1% O)2) The PMVECs hypoxic condition culture fluid containing HTSFCACE2 has obvious inhibition effect on hypoxia-induced proliferation of the PASMCs (figure 7C), and the inhibition effect is dose-dependent, and the PMVECs are added according to the proportion of 50 percentHypoxia-induced proliferation of PASMCs was significantly inhibited by hypoxic conditioned medium (fig. 7D). PMVECs are transfected by the control plasmid HTSFC, and after the PMVECs are subjected to hypoxia stimulation with 10% oxygen concentration for 24 hours, collected PMVECs cell supernatant is the control conditioned culture solution. The control conditioned medium had no effect on inhibiting proliferation of the PASMCs as described above.
The cell experiment results show that the recombinant plasmid HTSFCACE2 can only increase the expression level of ACE2 in PMVECs according to the degree of hypoxia, and the PMVECs hypoxia condition culture solution containing HTSFCACE2 can reduce the proliferation of the PASMCs in a dose-dependent mode, and has cell-level targeting property, hypoxia controllability and effectiveness.
Second, in vivo animal experiment
1. Duplicating rat hypoxic pulmonary hypertension model, experimental grouping and detecting various indexes
SD rats were randomly divided into 5 groups of 8 animals each:
① normoxic group (Normoxia). rats were placed in normoxic environment for 28 days.
② hypoxic 7-day group (
③ hypoxic group for 14 days (
④ hypoxic 21-day group (
⑤ hypoxic 28-day group (
Rats in normoxic group were placed in animal chamber and raised under natural conditions (atmospheric pressure of about 718mm Hg, pO in the Western region)2150.6mmHg, oxygen concentration about 21%); the hypoxic rats were placed in a hypoxic chamber (cabin pressure 380mm Hg, pO)2Reduced to 79.6mmHg, equivalent to an oxygen content of 5540 meters above sea level, with an oxygen concentration reduction of about 10%) for 8h per day for 28 consecutive days; deodorizing in a low-pressure low-oxygen chamber with soda lime and desiccant to absorb CO2. After 28 days, the relevant indexes of pulmonary hypertension of rats in each group are detected.
Detection of hemodynamic index: anaesthetizing and fixing the rat, making an incision in the middle of the neck, separating and ligating the distal ends of the left common carotid artery and the right external jugular vein, clamping the proximal end of the blood vessel, cutting a small opening between the two by using an ophthalmic scissors, respectively inserting a polyethylene catheter filled with 0.5% heparin solution into the left common carotid artery and the right external jugular vein, leaving one end of the catheter in the blood vessel, knotting and fixing the catheter, connecting the other end of the catheter with a pressure transducer, and recording the average left common carotid artery pressure (mCAP) and the right ventricular systolic pressure peak value (RVSP) of the rat.
Measurement of the right ventricular hypertrophy index RV/(LV + S). times.100%: the rat sternum is cut open to expose the heart; the tissue and blood vessels surrounding the heart, as well as the left and right atria, atrial appendage, etc., are removed, the pulmonary artery cone is found, and the Right Ventricle (RV) is cut down the pulmonary artery cone and weighed. The remaining tissue was also weighed, i.e., the weight of the left ventricle and the ventricular septum (LV + S). The right ventricular hypertrophy index [ RV/(LV + S). times.100% ] was calculated from the measurement values to reflect the degree of right ventricular hypertrophy.
Preparation of lung tissue paraffin section and HE staining: the materials are taken along the transverse section of the pulmonary portal, tissue blocks of about 1cm multiplied by 2cm of the upper right lung lobes of the rats are cut and placed in an embedding frame, the embedding frame and the embedding frame are placed in 10% neutral formaldehyde buffer solution for fixation for 24 hours, and then the embedding frame is taken out and placed in 70% ethanol solution. Then dehydrated, embedded, and made into paraffin blocks. And slicing the paraffin blocks, performing HE staining after dewaxing to water, and detecting the change of the pulmonary arterioles.
Lung tissue section image analysis: observing the HE stained section under a microscope, selecting small pulmonary arteries with the outer diameter of less than 50-100 μm, collecting and analyzing blood vessel images by using image analysis software, respectively measuring the inner diameter, the outer diameter, the wall thickness and the blood vessel area of the blood vessel, and respectively calculating two indexes reflecting the thickening of the blood vessel wall according to the measured values, namely WT% (the wall thickness/the outer diameter multiplied by 100%) and WA% (the wall area/the total area multiplied by 100%).
ELISA method for detecting ACE2, AngII and Ang (1-7) content in lung homogenate: a small piece of lung tissue was cut at a uniform position, 100mg of the tissue was accurately weighed and placed in an EP tube, and 1ml of physiological saline was added, and ground with a hand-held homogenizer to prepare a tissue homogenate. The EP tube was then centrifuged at 4500rpm at 4 ℃ for 10min and the supernatant was aspirated. The levels of ACE2, AngII and Ang (1-7) were measured in lung homogenates according to the ELISA kit protocol.
The Western blot method for detecting the expression level of the lung tissue ACE2 includes shearing a small piece of lung tissue at a uniform position, accurately weighing 100mg of the tissue, adding lysis solution RIPA at a ratio of tissue/lysis solution of 10mg/100ul, shearing the tissue as soon as possible with small ophthalmic scissors, homogenizing the tissue thoroughly, placing the tissue on ice for lysis for 30 minutes, placing the specimen in a 4-temperature environment at 12000rpm, centrifuging the 5-minute centrifuge, collecting the supernatant, which is a cellular protein, quantifying the protein content with the Coomassie Brilliant blue method, adding the prepared protein standard and protein sample into a 24-well plate with a pipette, measuring the OD value of the sample at a wavelength of 595nm, combining the protein standard concentration, compiling a protein concentration standard curve, calculating the corresponding protein concentration according to the ACE curve and the absorbance of the protein sample, determining the upper sample volume of the protein sample, adding deionized water and 5 × SDS upper sample buffer which are not marked with the same as the protein sample concentration, adding deionized water and 5 × SDS buffer, placing the buffer into a 95 ℃ water bath pan, facilitating denaturation of the protein denaturation in accordance with ACE 32, preparing the ACE, preparing the power supply, preparing the protein, preparing the corresponding protein concentration of the protein, adding the protein, rinsing min, placing the protein, placing the gel buffer into a gel buffer, placing the gel buffer into a gel buffer, placing the gel buffer into a gel buffer, placing into a gel buffer with a power supply, placing the gel buffer with a power supply, placing the gel buffer at a power supply of 10-transferring the gel buffer at a power supply of 10-transferring the gel buffer of a 200-10 rpm, transferring the gel buffer of a 200-10 rpm, placing the gel buffer of a 200-10 rpm, placing the electrophoresis buffer of a 200-10 rpm, placing the gel buffer of a 200-10 rpm, placing the No. 10-10 rpm, placing the electrophoresis buffer, placing the gel buffer of a 200-10 rpm, placing the electrophoresis buffer, placing the gel buffer of a 200-10 rpm, placing the electrophoresis buffer of a 200-10 rpm, placing the gel-10 rpm, placing the electrophoresis.
The results of the above experiments were: in the process of duplicating the rat model of hypoxic pulmonary hypertension for 4 weeks, the peak value of Right Ventricular Systolic Pressure (RVSP) of the rat is gradually increased along with the prolongation of the time of hypoxia (fig. 8B), which reflects the index [ RV/(LV + S) ]% of right heart hypertrophy is gradually increased (fig. 8C), and hypoxia causes the smooth muscle layer of pulmonary arteriole of the rat to be gradually thickened and the lumen to be narrowed (fig. 8F), which represents the indexes of pulmonary arteriole thickening that WA% and WT% are both obviously increased (fig. 8D, E). And hypoxia induced a significant decrease in ACE2 expression in lung tissue over time (fig. 9A and B), a significant increase in AngII levels in lung homogenate (fig. 9C), and a significant decrease in ACE2 and Ang (1-7) (fig. 9D and E).
The results of the above in vivo animal experiments indicate that hypoxia causes the RAS system in the lung to be disturbed, ACE2 expression is significantly reduced, and AngII is relatively hyperactive, thereby inducing the development of hypoxic pulmonary hypertension.
2. AAV-HTSFCACE2 for adeno-associated virus and AAV-HTSFC construction, collection and virus titer determination
Recovering the frozen 293AAV cells (Shenzhen Baien vitamin science and technology Co., Ltd.), culturing with DMEM culture solution containing 10% FBS, inoculating into 10cm culture dish, adjusting the number of 293AAV cells to about 1-5 × 106On the other hand, the cells were cultured continuously to reach a cell length of 80% or more. 2h of cell fluid change before transfection, and then HET transfection reagent of Shenzhen Bainwei companyAnd (3) carrying out plasmid DNA transfection on the 293AAV cells by the cassette instruction, continuously culturing the transfected 293AAV cells for 4-6h, and then changing the liquid. After 12-18h of transfection, the culture medium in the petri dish was aspirated off by a pipette, and 10ml of fresh complete culture medium containing 1% of double antibody was added to continue the culture. Culturing for 48 hr, collecting cell mixture, placing into a centrifuge tube, repeatedly freezing and thawing in water bath at-80 deg.C and 37 deg.C for 3 times, centrifuging at 3000rpm for 10min, collecting supernatant, concentrating, purifying, and storing at-80 deg.C.
Designing Real time PCR primers:
hGHpolyA Forward: 5'-CAAGCGATTCTCCTGCCTCA-3' (see SEQ. ID. NO.4)
hGHpolyA Reverse: 5'-ACGCCTGTAATCCCAGCAAT-3' (see SEQ. ID. NO.3)
According to the experimental requirements, 20ul of AAV-HTSFCACE2 and AAV-HTSFC concentrated virus liquid to be detected are respectively taken and respectively made into 103、104、105、106、107Diluting, constructing a Realtime PCR reaction system, carrying out PCR reaction, and calculating the copy number in the AAV sample according to the measured Ct value.
3. Characterization of adeno-associated virus AAV-HTSFCACE2/AAV-HTSFC
Extraction of viral genomic DNA: 293 cells are cultured until more than 80 percent of cells are fused, and 200 mu l of AAV-HTSFCACE2 or AAV-HTSFC virus frozen stock solution is added respectively to continue culturing for 36 h. When the cells were rounded but not yet rinsed, the medium was pipetted out and the cells were rinsed once with PBS. Mu.l of cell lysate (containing 0.6% SDS, 10mM EDTA and 100. mu.g/ml proteinase K) was added to each flask, incubated at 56 ℃ for 1h, followed by addition of 200. mu.l of 5M NaCl to each flask, mixed well, placed on ice for 1h, and the cells were collected and centrifuged at 12000rpm for 10min at 4 ℃. The supernatant was aspirated, an equal volume of a mixture of phenol, chloroform and isoamyl alcohol (mixed at a ratio of 25:24: 1) was added, centrifugation was carried out at 12000rpm for 15min, the supernatant was collected, and 1/9 volumes of 3M NaAc and 2 volumes of absolute ethanol were added, and the mixture was left at-20 ℃ for 30 min. Then, the mixture was centrifuged at 12000rpm for 15min, the supernatant was discarded, and water was removed by vacuum suction. Then 1ml of 70% ethanol is added for resuspension, centrifugation is carried out at 10000rpm for 10min, and the supernatant is discarded. The EP tube was placed at room temperature for 10min to dry the water, 40ul of deionized water was then added to dissolve the extracted DNA, and the DNA was stored at-20 ℃.
And (3) PCR identification: and (3) constructing a PCR reaction system, carrying out PCR reaction, respectively taking 5mL of PCR products to carry out 1% agarose gel electrophoresis, and checking the PCR result.
According to the above experiment, 293AAV cells were transfected with the recombinant plasmid HTSFCACE2 and the control plasmid HTSFC, respectively, and the cell mixture was collected, centrifuged, packaged into adeno-associated virus, and the virus titer was determined. Extracting virus genome DNA to perform Real-timePCR, respectively taking PCR products to perform 1% agarose gel electrophoresis, and performing virus identification. The results of PCR product identification are shown in FIG. 10, and the adeno-associated virus titers are shown in Table 1. The results show that the virus packaging is successful, the virus titer is better, and the virus packaging can be used in animals.
TABLE 1 titer of AAV viruses
4. Adeno-associated virus AAV-HTSFCACE2 improves hypoxic pulmonary hypertension progression in rats
SD rats were randomly divided into 4 groups of 8 animals each:
① normoxic group (Normoxia). rats were placed in normoxic environment for 28 days.
② hypoxic group (Hypoxia) rats were placed in a hypoxic chamber with 10% oxygen concentration for 8h per day for 28 days in succession, replicating the hypoxic pulmonary hypertension model of rats.
③ hypoxia + AAV-HTSFCACE2 group, rats were placed in a low-pressure hypoxia chamber with 10% oxygen concentration for 8h each day, and AAV-HTSFCACE2 was added dropwise to the rat at 15d, and hypoxia was continued to 28 d.
④ hypoxia + AAV-HTSFC group, rats were placed in a hypoxic chamber with 10% oxygen concentration for 8h each day, and AAV-HTSFC was administered by nasal drip once at 15d, and hypoxia was continued to 28 d.
When a rat is dripped into the nose, the rat is fixed in a supine position after anesthesia, the head is lifted, a pipettor is used for sucking a specific amount of recombinant adeno-associated virus, the recombinant adeno-associated virus is slowly dripped into the nostril of the rat, and the mouth is ensured to be closed when the rat is dripped into the nose, so that liquid is ensuredAnd (4) sucking. The virus amount per rat is about
The hypoxic treatment is as described above. After 28 days, the relevant indexes of pulmonary hypertension of rats in each group are detected, which is the same as the above. And detecting the expression quantity of ACE2 in lung tissue by using a Western blot method, detecting the contents of ACE2, AngII and Ang (1-7) in lung tissue homogenate by using an ELISA method, and detecting the expression of ACE2 in pulmonary arterioles by using an immunohistochemical method.
Immunohistochemistry method for detecting the expression of ACE2 in small pulmonary artery of rat: dewaxing the prepared rat lung tissue paraffin section to water, and then operating according to the following steps:
(1) washing with xylene for 10min × 4;
(2) washing with 100% ethanol for 10min × 2;
(3) washing with 95% ethanol for 5 min;
(4) washing with 90% ethanol for 5 min;
(5) washing with tap water for 5 min;
(6)3％H2O2incubating with deionized water at room temperature for 30 min;
(7) washing with distilled water for 5min × 3;
(8) soaking the slices in 0.01M citrate buffer solution, adding into microwave oven for antigen retrieval, heating to boil with high fire power, maintaining the medium fire power for 10min, taking out, and naturally cooling at room temperature;
(9) adding normal rabbit serum working solution for sealing dropwise, standing at room temperature for 30min, and pouring off excessive liquid without washing;
(10) dropwise adding 1: rabbit anti-rat ACE2 monoclonal antibody (1: 200) diluted at 50, wet-box overnight at 4 deg.C, washed with PBS for 5min × 3;
(11) taking out the wet box, and rewarming at 37 ℃ for 1 h;
(12) dropwise adding 1: 50 diluted peroxidase-labeled goat anti-rabbit IgG is washed with PBS for 5min × 3 at 37 ℃ for 30 min;
(13) DAB color development, and tap water full flushing;
(14) counterstaining with hematoxylin, dehydrating, clearing, and sealing
(15) When observed under a light mirror, the cell membrane is seen to be yellow brown and is ACE2 positive cell. Under 400 times visual field, 3 pulmonary arterioles with the diameter of 200-500 μm are selected and analyzed by measuring and analyzing the average Integrated Optical Density (IOD) of the positive particles of the pulmonary arteriole smooth muscle layer by using an IMAGE-PRO plus6 microscopic IMAGE analysis system.
The results of the above experiments were: the hypoxic pulmonary hypertension rat model was replicated for 4 weeks and adeno-associated virus AAV-HTSFCACE2 or control virus AAV-HTSFC was applied to rats at one time, nasally, at
In addition, AAV-HTSFCACE2 was able to up-regulate ACE2 expression in lung tissue (FIGS. 12A and B), reduce AngII levels in lung homogenates (FIG. 12C), and increase Ang (1-7) levels (FIG. 12D). Immunohistochemical staining results showed increased ACE2 expression in the endothelial tissue of pulmonary arterioles following administration of adeno-associated virus AAV-HTSFCACE2 (fig. 12F and G). And the content of ACE2 in the heart, liver, lung, kidney and other tissues of the rat is detected by an ELISA method, and the ACE2 content is only obviously increased in the lung tissue and is not obviously changed in other tissues after the AAV-HTSFCACE2 is administered to the rat (figure 12E), which suggests that the ACE2 is only increased in the pulmonary arteriolar endothelial tissue after the AAV-HTSFCACE2 enters the rat body, and has tissue specificity and effectiveness. Rats given control virus AAV-HTSFC showed no such expression.
The above results in vivo animal experiments indicate that adeno-associated virus AAV-HTSFCACE2 improves the progression of hypoxic pulmonary hypertension at the animal level.
In conclusion, the gene recombination technology is utilized, the characteristics that ACE2 has the function of finely regulating a renin-angiotensin (RAS) system in a lung are combined, the expression of the Tie2gene has the specificity of vascular endothelial cells, and a Hypoxia Response Element (HRE) is regulated by HIF-1 α (the expression of Hypoxia induction factor-1 α (Hypoxia-induibile factor-1 α -1 α) is increased and is related to the reduction of oxygen partial pressure in Hypoxia, and the expression of HIF-1 α is only increased in pulmonary vessels and is less increased in systemic circulation in Hypoxia), and the like, so that the ACE2 expression vector driven by the HRE enhanced Tie2 promoter is constructed, the ACE2 is highly expressed in hypoxic pulmonary artery endothelial cells, and the pulmonary artery smooth muscle cells are regulated and controlled through paracrine action, the Hypoxia controllability is realized, the pulmonary artery contraction is targetedly inhibited, the pulmonary artery structural reconstruction is reversed, a new means and a new pulmonary artery hypertension strategy are provided for preventing and treating low-oxygen-associated diseases such as diabetes, deep gangrene, deep vein, thrombosis, heart disease, ischemia and ischemic heart disease.
<110> the fourth military medical university of the Chinese people liberation army
<120> expression vector for controllably up-regulating ACE2 targeting prevention and treatment of hypoxic pulmonary hypertension
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