阅读说明：本技术 miR-325核酸类似物在制备治疗血窦内皮细胞病理性功能异常相关产品中的应用 (Application of miR-325 nucleic acid analogue in preparation of related product for treating pathological dysfunction of blood sinus endothelial cells ) 是由 李丽雅 刘文佳 于 2020-11-18 设计创作，主要内容包括：本发明提供了miR-325核酸类似物在制备治疗血窦内皮细胞病理性功能异常相关产品中的应用。通过miR-325直接和间接影响的信号通路,包括恢复细胞stabilin-2,PTPRM,YWHAQ等基因的表达,使血窦内皮细胞重新分化,促进血窦内皮细胞调节炎症,抗星状细胞激活的能力,从而使所述血管性病变得到有效缓解。(The invention provides application of miR-325 nucleic acid analogues in preparation of products related to treatment of pathological dysfunction of blood sinus endothelial cells. The signal path directly and indirectly influenced by miR-325, including the expression of genes such as cell stabilin-2, PTPRM, YWHAQ and the like, is restored, so that the endothelial cells of the blood sinuses are re-differentiated, the capacity of the endothelial cells of the blood sinuses to regulate inflammation and resist stellate cell activation is promoted, and the vascular lesion is effectively relieved.)

Application of miR-325 nucleic acid analogues in preparation of products related to treatment of pathological dysfunction of blood sinus endothelial cells.

2. The use of claim 1, wherein said pathological dysfunction of sinusoidal endothelial cells comprises hepatic sinus endothelialization, cerebral sinus endothelialization, endocrine sinus endothelialization, hematopoietic sinus endothelialization, or systemic sinus endothelialization.

3. The use according to claim 2, wherein the disease associated with endothelialization of cerebral blood sinuses comprises alzheimer's disease or cerebral arteriosclerosis.

4. The use according to claim 2, wherein the disease associated with endothelialization of the endocrine organs in the blood sinuses comprises diabetes, chronic nephritis or pancreatitis.

5. The use according to claim 2, wherein the diseases associated with endothelialization of the blood sinuses of the hematopoietic organ comprise hematopoietic disorders caused by aging, aplastic anemia, osteomyelitis or splenitis.

6. The use of claim 2, wherein the disease associated with systemic sinus endothelialization comprises hypertension, vasculitis, thrombosis, or atherosclerosis.

7. A medicament for treating pathological dysfunction of sinus endothelial cells, the medicament comprising a therapeutically effective amount of a miR-325 nucleic acid analog.

8. The medicament of claim 7, wherein the active components of the medicament comprise a miR-325 nucleic acid analog and a CXCR7 agonist.

9. The medicament of claim 8, wherein the miR-325 nucleic acid analog is at a concentration of 20nM and the CXCR7 agonist is at a concentration of 300 nM.

10. Use of a medicament as claimed in any one of claims 7 to 9 for modulating the expression level of the proteins mannase receptor, stabilin-2, alpha-SMA, F4/80, PTPRM or ywaq.

Technical Field

The invention relates to the technical field of biology, in particular to application of miR-325 nucleic acid analogues in preparation of products related to treatment of pathological dysfunction of blood sinus endothelial cells.

Background

Sinus Endothelial Cells (SEC) are specialized capillary endothelial cells with fenestrations (fenestrations) and sieve plates (sieve plates) structures without basement membrane. Liver blood sinus endothelial cells (LSEC) mainly ultrafiltrate blood through a fenestration and sieve plate structure, thereby realizing rapid nutrient and substance exchange to meet the metabolic demand of healthy Liver. Studies have demonstrated that LSEC can inhibit the activation of hepatic stellate cells, thereby inhibiting the major source of collagen deposition in the liver. In addition, LSEC controls the expansion of liver parenchymal cells by expressing, secreting, or responding to receptors and cytokines such as Hepatocyte Growth Factor (HGF), vascular endothelial growth factor receptor 2 (VEGFR 2), chemokine receptor 7 (CXCR 7). LSEC is also the first immune-privileged barrier for liver cells to contact the blood flowing through the liver, where inflammatory cells recruited by inflammatory chemokines need to adhere to and cross LSEC to reach the liver. In vivo animal experiments prove that after LSEC is induced to endothelialize and lesion under the condition of no liver injury, the LSEC loses the window hole and the sieve plate, the function is disordered, and the liver generates collagen deposition. Therefore, the liver blood sinus endothelial cell plays an important role in maintaining healthy liver homeostasis, and also plays an important role in the liver fibrosis process caused by chronic liver injury and the like. Clinically, the liver of a hepatic fibrosis patient has collagen deposition around blood vessels and blood sinuses, and the patient has hypodynamia, inappetence and lower abnormal degree of liver function than cirrhosis. The patients do not have obvious abnormal expression, so the liver function abnormality is often diagnosed to be serious, and the liver cirrhosis or liver cancer is usually diagnosed once. However, there is no specific medicine for treating liver fibrosis or cirrhosis clinically at present, and the conventional scheme adopted can only be used for carrying out antivirus and the like aiming at the liver disease cause to slow down the process of liver collagen deposition or carrying out liver protection treatment on the complications of liver fibrosis, while the liver collagen deposition can not be reversed, the normal function of the liver is difficult to recover, and the patients developing to the liver function decompensation stage only carry out liver transplantation to prolong the life as far as possible.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides the application of the miR-325 nucleic acid analogue in preparing a product related to the pathological dysfunction of the blood sinus endothelial cells.

In order to achieve the purpose, the invention provides the following technical scheme:

on the one hand, the application of the miR-325 nucleic acid analogue in preparing a product related to the pathological dysfunction of the blood sinus endothelial cells is provided.

Preferably, the pathological dysfunction of the blood sinus endothelial cells comprises brain blood sinus endothelialization lesions, endocrine organ blood sinus endothelialization lesions, hematopoietic organ blood sinus endothelialization lesions or systemic blood sinus endothelialization lesions.

Preferably, the diseases related to the endothelialization lesions of the cerebral blood sinuses comprise Alzheimer's disease and cerebral arteriosclerosis.

Preferably, the endocrine organ blood sinus endothelialization lesions comprise diabetes, chronic nephritis, pancreatitis.

Preferably, the disorder of hemopoietic organ blood sinus endothelialization comprises hematopoietic disorder caused by aging, aplastic anemia, osteomyelitis or splenitis.

Preferably, the systemic sinus endothelialization disorder comprises hypertension, vasculitis, thrombosis, or atherosclerosis.

In a second aspect, there is provided a medicament for treating pathological dysfunction of sinus endothelial cells, the medicament comprising a therapeutically effective amount of a miR-325 nucleic acid analog.

Preferably, the active components of the medicament include a miR-325 nucleic acid analog and a CXCR7 agonist.

Preferably, the miR-325 nucleic acid analog concentration is 20nM and the CXCR7 agonist concentration is 300 nM.

In a third aspect, the application of the medicine in regulating the expression level of the protein manuse receiver, stabilin-2, alpha-SMA, F4/80, PTPRM or YWHAQ is provided.

The fourth aspect of the invention provides a mesenchymal stem cell validity detection method, which comprises the following steps: before intravenous injection of the mesenchymal stem cells, the expression level of miR-325 in the mesenchymal stem cells or extracellular vesicles thereof is detected by using qPCR (expression level reference value: miR-325 Ct <30 when U6 Ct = 19), so that the mesenchymal stem cells are ensured to have the capacity of restoring the differentiation of blood sinuses.

In the fifth aspect of the invention, a mesenchymal stem cell preparation for treating the pathological dysfunction of the endothelial cells of the blood sinuses comprises mesenchymal stem cells, and the mesenchymal stem cells are transfected by miR-325.

In a sixth aspect of the invention, there is provided a medicament for treating pathological dysfunction of blood sinus endothelial cells, the medicament comprising a medicament carrier selected from exosomes, liposomes or apoptotic bodies and a miR-325 nucleic acid analog.

In a seventh aspect of the present invention, there is provided a method for improving the anti-collagen deposition function of liver sinusoid cells to inhibit vascular lesions, comprising the steps of: the medicine or the mesenchymal stem cell preparation is used for carrying out intravenous injection treatment on the patient with vascular disease.

In an eighth aspect of the present invention, there is provided a method for regulating the expression level of the proteins Mannose receiver, Stablin-2, Alpha-SMA, F4/80, PTPRM, YWHAQ, comprising the steps of: and (3) carrying out induced differentiation on the blood sinus cells with abnormal expression levels of the proteins of manose receiver, Stablin-2, PTPRM and YWHAQ by using the miR-325 mimics so as to obtain the capacity of inhibiting the expression levels of stellate cells Alpha-SMA and macrophages F4/80.

Preferably, the induced differentiation process is: carrying out in-vitro sterile culture on isolated blood sinus endothelial cells with abnormal differentiation capacity, removing normal cell culture solution when the fusion degree of the bone blood sinus endothelial cells reaches more than 70%, replacing differentiation inducing solution containing miR-325 simulant, and inducing differentiation towards the blood sinus endothelial cells with a sieve plate structure for 24-72 hours.

The characteristic of the endothelial cells of the blood sinuses with abnormal differentiation capacity is as follows: compared with normal sinus endothelial cells, the Cell membrane is thickened, the fenestration and the sieve plate structure on the Cell membrane surface are lost or reduced, the expression level of the Cell manunase, Stablin-2 is low, the expression level of PTPRM and YWHAQ is high, the proliferation capacity of the Cell is weakened, the capacity of the Cell for inhibiting macrophages is weakened, the capacity of the Cell for inhibiting the activation of stellate cells is weakened, and the intracellular Cell addition molecular signaling pathway is abnormal.

Compared with the prior art, the invention has the beneficial effects that:

the pathological dedifferentiation of the endothelial cells of the blood sinuses in the tissues of individuals with aging, chronic inflammation or cardiovascular diseases, which can be reversed with a significant reduction in the corresponding pathologies. The invention has been proved through the experiment, use miR-325 nucleic acid analog can reverse the pathologic dedifferentiation state of the endothelial cell of blood sinus, the invention uses miR-325 nucleic acid analog intravenous injection to act on the individual or said vascular lesion individual of the dedifferentiation state of the endothelial cell of blood sinus, through miR-325 direct and indirect signal path of influence, including restoring expression of cell stablin-2, PTPRM, YWHAQ, etc., make the endothelial cell of blood sinus differentiate again, promote the endothelial cell of blood sinus to regulate the inflammation, the ability to resist stellate cell activation, thus make said vascular lesion get and relieved effectively; in addition, the invention adopts the nucleic acid analogue of miR-325, has the characteristics of simple operation, low cost, obvious effect and the like, realizes the re-differentiation of the endothelial cells of the blood sinuses with functional defects, and provides a potential thought and scheme for treating patients with diseases related to the dedifferentiation of the blood sinuses clinically.

Drawings

FIG. 1A is the surface of the endothelial cells of the liver sinusoids, which are scanned by a scanning electron microscope of the liver disease group in the example, and which lose the differentiation characteristic structure due to liver damage;

in the embodiment of FIG. 1B, the liver disease group injected with miR-325 intravenously reappears a plurality of differentiated characteristic sieve plate structures on the surface of liver blood sinus endothelial cells shot by a scanning electron microscope;

FIG. 2A shows that the tissue of the example has an immunofluorescence detecting sinus differentiation marker, stabilin-2 indicating healthy liver sinus differentiation;

FIG. 2B is an immunofluorescence assay of tissues in examples showing the differentiation of antrum differentiation marker stablin-2 indicating differentiation of hepatic sinusoids in a liver fibrosis model;

FIG. 2C is an immunofluorescence assay of tissues in examples showing sinus differentiation marker stablin-2 indicating sinus differentiation of liver after miR-325 analog system injection treatment;

FIG. 3A is a graph showing the distribution of F4/80 positive macrophages in healthy liver in the examples;

FIG. 3B shows infiltration of F4/80 positive macrophages (inflammation) before sinus recovery in the examples;

FIG. 3C is a graph showing the modulation of F4/80 positive macrophages (inflammation) following sinus recovery in the examples;

FIG. 4A is the distribution of α -SMA positive hepatic stellate cells of healthy liver in the examples;

FIG. 4B is the case of α -SMA positive hepatic stellate cells (anti-collagen secretion) before sinus recovery in the examples;

FIG. 4C is the anti- α -SMA positive hepatic stellate cells (anti-collagen secretion) after sinus recovery in the examples;

FIG. 5 shows examples of normal group (WT) and liver disease group (PBS + CCl)₄) miR-325 group for liver diseases by intravenous injection (miR-325+ CCl)₄) The level of liver function of;

FIG. 6 shows the expression of active YAP in pathological group, miR-325 mimetic treatment group, and miR-325 inhibition group at the liver regeneration-related signal activity during the Western blot detection period in the example.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The technical solutions in the embodiments of the present invention will be described in detail below with reference to the accompanying drawings in the embodiments of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the following examples cell culture dishes were purchased from Corning, usa.

The PBS buffer in the following examples was purchased from Beijing kang, century Biotechnology Ltd.

The cell culture medium (DMEM medium) in the following examples was purchased from Gibco, USA.

The activity detection kit for aspartate Aminotransferase (AST) in the following examples was purchased from Nanjing institute of bioengineering (96T, C010-2).

Active YAP antibodies in the examples described below were obtained from Abcam, USA (ab 205270).

The stabilin-2 antibody in the examples described below was purchased from MBL International (D317-3).

The α -SMA antibodies in the following examples were purchased from Abcam, Inc. (ab 5694) of USA.

The F4/80 antibody in the examples described below was purchased from Abcam, Inc. (ab 111101) in USA.

In the following examples, the nucleic acid analogues of miR-325 were purchased from Ruibo, under product number (miR 40004640-4-5, miR 10000771-1-5), and 80% pure.

Cell culture conditions were used in the following examples: CO 2₂Concentration of 5%, O₂A humidified cell culture chamber at 37 ℃ and a concentration of 20%.

Examples

Firstly, detecting abnormal function of blood sinuses:

1. differentiation characterization detection is carried out after primary cells of blood sinuses are isolated and cultured

Cutting fresh individual biopsy tissue, and using Liberase^TMDissolving in DMEM medium containing 2% fetal calf serum, digesting for 1 hour, gently blowing and beating to obtain single cells, culturing in DMEM medium containing 10% fetal calf serum, and changing the culture solution about 4-7 days according to the adherent condition to obtain primary sinus endothelial cells with extended adherent walls. After culturing to a suitable density for single cell shooting (about 20%), cells in the culture dish were fixed, dehydrated, and sprayed with gold, and then examined under a scanning electron microscope whether the cell surface had lost differentiation features-sieve plate and fenestration, the results are shown in FIGS. 1A-1B.

Biopsy tissue for sinus differentiation and sinus function marker staining

A small 1 cm square piece of relatively fresh biopsy was taken and blood was washed clean with PBS and fixed with 4% paraformaldehyde overnight at four degrees. After 30% sucrose solution settled at 4 degrees for 1 to 2 days, cryosections were taken and stablin-2, F4/80 and α -SMA were immunofluorescent stained. Results as shown in FIGS. 2A-2C, the sinus endothelial cell differentiation marker, stablin-2 (FIG. 2B), was reduced in liver tissue with abnormal differentiation compared to healthy liver (FIG. 2A), while there was a significant increase in stablin-2 after miR-325 treatment (FIG. 2C); as shown in fig. 3A-3C, the increase of F4/80 positive macrophages in fibrotic liver tissue (fig. 3B) over healthy liver (fig. 3A) showed increased inflammatory infiltration, while F4/80 (fig. 3C) significantly decreased to near healthy liver levels after miR-325 treatment; as shown in fig. 4A-4C, the main sources of fibrotic liver collagen: active stellate cell marker α -SMA (fig. 4B) was increased compared to healthy liver (fig. 4A), while α -SMA (fig. 4C) was significantly reduced to near healthy liver levels after treatment with miR-325.

Liver function detection after sinus function improvement

Taking venous blood, standing at normal temperature for half an hour, transferring the separated upper serum to a sterile Eppendorf tube, centrifuging at 4 ℃, 2000rpm for 20 minutes, carefully not touching erythrocyte sediment at the bottom of the tube, and transferring the finally obtained serum to the sterile Eppendorf tube. Taking 5 microliters of serum, carrying out conventional AST activity detection by using an AST activity detection kit, wherein the result is shown in figure 5, and the AST activity in the serum of the miR-325 intravenous liver disease group is remarkably reduced compared with that in the serum of a control liver disease group, so that the liver function is prompted to be relieved.

Application of miR-325 nucleic acid analogue or composition in treatment of abnormal blood sinus function

1. Detection of regeneration key signal pathway of dedifferentiated pathological blood sinuses

Cutting fresh individual biopsy tissue, and using Liberase^TMDissolving in DMEM medium containing 2% fetal calf serum, digesting for 1 hour, gently blowing and beating to obtain single cells, culturing in DMEM medium containing 10% fetal calf serum, and changing the culture solution about 4-7 days according to the adherent condition to obtain primary sinus endothelial cells with extended adherent walls. After culturing to a suitable density (about 80%) of protein single cells, conventional Western blot detection is performed by using protein lysate, and as shown in FIG. 6, the activated YAP protein level in primary blood sinus cells is up-regulated after miR-325 treatment, which indicates that the developmental stage expression and the signal pathway Hippo signaling for regulating the size of organs are activated.

Accounting analogs or compositions for sinus abnormalities in liver fibrosis

The severity of the dysfunction can be judged according to the detection result in the first implementation example step, and the miR-325 nucleic acid analogue is determined to be used. The specific implementation is to carry out intravenous injection on hepatic fibrosis patients with abnormal blood sinus function. The miR-325 nucleic acid mimic concentration is 20 nM. In practice, the product is not used in combination with statins, melatonin, and other drugs that interfere with vascular function. Table 1 shows the changes of the expression levels of ywaq and PTPRM proteins directly and indirectly regulated by miR-325 detected by protein mass spectrometry, wherein protein ID is the unique number of the listed proteins in the Uniprot database, protein name is the common name of the listed proteins, Mean _ Ratio is the Ratio of the expression amounts of the listed proteins (Mean value of the liver disease group of miR-325 intravenous injection to Mean value of the liver disease group), and less than 0.8 indicates that the listed proteins are significantly reduced after miR-325 injection.

TABLE 1

Protein ID	Name of protein	Mean_Ratio	Pvalue	SD
					P28828	PTPRM	0.80	0.02323	0.073
P68254	YWHAQ	0.74	0.03696	0.122

The above description is only an embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions within the technical scope of the present disclosure should be covered by the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.