阅读说明：本技术 miR-325核酸类似物在制备改善肝胶原沉积相关产品中的应用 (Application of miR-325 nucleic acid analogue in preparation of products related to improvement of liver collagen deposition ) 是由 刘文佳 李丽雅 于 2020-11-18 设计创作，主要内容包括：本发明提供了miR-325核酸类似物在制备改善肝胶原沉积相关产品中的应用。本发明是基于肝血窦内皮细胞的去内皮化是缓解肝胶原沉积的必要条件的原理。本发明以miR-325为模板合成的miR-325核酸类似物可以纠正肝纤维化环境下肝血窦内皮细胞内皮化病变,有效恢复肝血窦内皮细胞抑制胶原沉积的功能,进而缓解肝纤维化。(The invention provides application of miR-325 nucleic acid analogues in preparation of products related to improvement of liver collagen deposition. The invention is based on the principle that de-endothelialization of liver sinusoid endothelial cells is a necessary condition for alleviating liver collagen deposition. The miR-325 nucleic acid analogue synthesized by taking miR-325 as a template can correct liver blood sinus endothelial cell endothelialization lesion under a liver fibrosis environment, effectively recover the function of inhibiting collagen deposition of liver blood sinus endothelial cells and further relieve liver fibrosis.)

Application of miR-325 nucleic acid analogues in preparation of products related to improvement of liver collagen deposition.

2. The use according to claim 1, wherein the hepatic collagen deposition is caused by at least one of fatty liver, alcoholic hepatitis, drug hepatitis, bacterial hepatitis, parasitic hepatitis, chemical viral hepatitis, cholangitis, Wilson's disease, autoimmune hepatitis, or pathological dysfunction of endothelial cells of the liver sinusoid.

3. A pharmaceutical product for improving hepatic collagen deposition, comprising a therapeutically effective amount of a miR-325 nucleic acid analog.

4. Use of the pharmaceutical product of claim 3 for the manufacture of a product for reducing serum glutamic oxaloacetic transaminase and glutamic pyruvic transaminase associated with a disorder associated with liver collagen deposition.

5. A composition comprising a miR-325 nucleic acid analog and a CXCR7 agonist.

6. The composition of claim 5, wherein the miR-325 nucleic acid analog is at a concentration of 15nM to 30nM and the CXCR7agonist is at a concentration of 200nM to 500 nM.

7. Use of the composition of claim 6 for the preparation of a product related to improvement of hepatic collagen deposition.

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 improvement of liver collagen deposition.

Background

The process of cirrhosis is the process of healing after liver damage, however, in the process, the normal metabolism of the liver is damaged, and the liver is developed towards massive secretion of collagen fibers rather than liver regeneration. Factors that cause damage to hepatocytes and cause necrosis and inflammation all contribute to liver fibrosis and progression to severe cirrhosis of the liver such that liver failure causes death of the patient. The most common factors include excessive fat accumulation, long-term intake of a large amount of alcohol, and administration of drugs which can cause liver injury (more than 1000 drugs which can cause liver injury are already known, such as chemotherapeutic drugs and partial traditional Chinese medicines). The liver is a very strong regenerative organ, which can properly secrete collagen at the early stage of damage and has certain self-protection ability, so that chronic liver diseases have no obvious symptoms at the early stage, and the phenotype of patients is more than that of patients at the later stage of cirrhosis until the liver failure occurs. At present, no specific medicine exists for clinically treating patients with liver fibrosis or liver cirrhosis, the conventional scheme adopted only can be used for resisting virus and the like aiming at the liver disease cause to slow down the liver collagen deposition process or carrying out liver protection treatment on complications of the liver fibrosis, the liver collagen deposition cannot be reversed, the normal function of the liver is difficult to recover, and the patients who develop to the liver function decompensation stage only carry out liver transplantation to prolong the life as far as possible. However, healthy liver sources are stressed and are associated with side effects of immune rejection.

Disclosure of Invention

The invention aims to provide application of miR-325 nucleic acid analogues in preparation of products related to improvement of liver collagen deposition, aiming at the problems in the prior art. The invention is based on the principle that the de-endothelialization of Liver Sinusoidal Endothelial Cells (LSEC) is a necessary condition for alleviating Liver collagen deposition. The miR-325 nucleic acid analogue synthesized by taking miR-325 as a template can correct liver blood sinus endothelial cell endothelialization lesion under a liver fibrosis environment, effectively recover the function of inhibiting collagen deposition of liver blood sinus endothelial cells and further relieve liver fibrosis.

The purpose of the invention is realized by the following technical scheme:

on one hand, the invention provides application of the miR-325 nucleic acid analogue in preparation of a product related to improvement of liver collagen deposition.

Preferably, the hepatic collagen deposition is caused by at least one disease selected from fatty liver, alcoholic hepatitis, drug hepatitis, bacterial hepatitis, parasitic hepatitis, chemical toxic hepatitis, cholangitis, Wilson's disease, autoimmune hepatitis, and pathological dysfunction of endothelial cells of liver blood sinuses.

Preferably, the invention provides application of the miR-325 nucleic acid analogue in preparation of a product for improving liver blood sinus endothelial cell endothelialization.

Preferably, the sinus endothelial cell differentiation refers to the occurrence of lamina cribosa on the cell surface, and the sinus function refers to the ability of sinus ultrafiltration phagocytosis, the ability to supply nutrients and exchange substances, the ability to inhibit macrophages, the ability to inhibit stellate cell activation, and the ability to respond to and send signals that promote cell regeneration, which refers to the response to CXCR7 agonists, and the restoration of VEGFR2, VEGFR3 expression.

Preferably, the factor causing said hepatic collagen deposition does not include viral hepatitis.

In a second aspect, there is provided a pharmaceutical product for improving hepatic collagen deposition, the pharmaceutical product comprising a therapeutically effective amount of a miR-325 nucleic acid analog.

In a third aspect, there is provided the use of said medicament for the manufacture of a product for reducing serum glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase associated with a disease associated with hepatic collagen deposition in an individual suffering therefrom.

In a fourth aspect, a composition is provided that includes a miR-325 nucleic acid analog and a CXCR7 agonist.

Preferably, the concentration of the miR-325 nucleic acid analogue is 20nM, the concentration of the CXCR7agonist is 300nM, and the purity standard requirement of the CXCR7agonist is more than or equal to 95%.

In a fifth aspect, the application of the composition in preparing products related to improving liver collagen deposition is provided.

In a sixth aspect, there is provided a method for improving hepatic collagen deposition, comprising the steps of dissolving the above product or the above composition in sterile-treated physiological saline, and intravenously injecting the product or the composition into a subject suffering from the above disease to reduce hepatic collagen deposition in the subject.

In a seventh aspect, there is provided a method of modulating the expression level of a gene manuse receiver, stabilin-2, α -SMA, F4/80, PTPRM or yhaq comprising the steps of: dissolving the product or the composition in sterile physiological saline, and performing intravenous injection on individuals suffering from the diseases to induce the differentiation and recovery of blood sinuses of the individuals.

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

the invention proves that the pathological dedifferentiation state of liver sinusoidal endothelial cells in liver tissues of an individual with hepatic fibrosis can be reversed, the dedifferentiation state can obviously reduce the hepatic fibrosis after being reversed, the invention proves that the miR-325 nucleic acid analogue can be used for reversing the pathological dedifferentiation state of the liver sinusoidal endothelial cells, the miR-325 nucleic acid analogue is used for intravenous injection to act on the individual with the dedifferentiation state of the liver sinusoidal endothelial cells or the individual with hepatic fibrosis, and the signal path directly and indirectly influenced by miR-325 is used for restoring the expression of genes such as cells stabilin-2, PTPRM, YWHAQ and the like, so that the liver sinusoidal endothelial cells are redifferentiated, the regulation inflammation of the liver sinusoidal endothelial cells is promoted, and the capability of resisting the activation of hepatic stellate cells is realized, thereby the hepatic fibrosis is; 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 hepatic fibrosis individual function-deficient hepatic sinus endothelial cells, and provides potential ideas and schemes for treating hepatic fibrosis and other diseases related to sinus dedifferentiation clinically.

Drawings

FIG. 1A shows the result of staining Tianlang's red liver leaves in the patients with liver diseases in the examples, showing significant red collagen deposition;

fig. 1B shows the results of sirius red staining of the liver disease group administered by miR-325 intravenous injection in the example, and the red collagen deposition was significantly reduced.

FIG. 2 is a signal path of significant changes in hepatic sinus endothelial iTRAQ protein expression profiles before and after treatment with miR-325 in example.

FIG. 3 shows the normal group (WT) and the liver disease group (PBS + CCl) in the examples₄) miR-325 group for liver diseases by intravenous injection (miR-325+ CCl)₄) The level of liver function.

FIG. 4A is a diagram of the liver disease group (PBS + CCl) in example₄) Liver Paraffin section H&E staining results, a large number of liver cell necrotic vacuoles and inflammatory infiltrates were visible.

FIG. 4B shows the liver disease group with intravenous miR-325 (miR-325+ CCl) in example₄) Liver slice H&And E, a staining result shows that a small amount of hepatocyte necrotic vacuoles can be seen, and the inflammatory infiltration condition is obviously reduced.

Fig. 4C shows H & E staining of liver sections from the CXCR7agonist injected liver disease group (CXCR 7 aginst + CCl 4) with infiltration of inflammatory cells but morphologically normal liver cells and decreased necrotic cells.

FIG. 4D shows the result of H & E staining of liver sections of a liver disease group (miR-325+ CXCR7 aginst + CCl 4) injected with the miR-325 and CXCR7 composition in the example, inflammation is substantially eliminated, necrotic vacuoles of hepatocytes disappear, cell arrangement is regular, and the liver is substantially restored to normal.

FIG. 5A shows the liver disease group (PBS + CCl) in example₄) The liver frozen section was stained for the sinus differentiation marker, stabilin-2, showing severe sinus loss and pathological dedifferentiation.

FIG. 5B shows the example of the treatment of liver disease by intravenous injection of umbilical cord mesenchymal stem cell-derived Extracellular vesicles (extracelluar vesicles + CCl)₄) The blood sinus differentiation marker stabilin-2 of the frozen section is stained, and the blood sinus density and differentiation are recovered to a certain degree.

FIG. 5C shows that after the umbilical cord mesenchymal stem cells are transfected with miR-325 nucleic acid analogues in vitro and the extracellular vesicles of the umbilical cord mesenchymal stem cells are used for treating liver diseases, the blood sinus differentiation marker stabilin-2 of a liver frozen section is stained, and the blood sinus density and differentiation are greatly improved.

FIG. 5D shows that after the umbilical cord mesenchymal stem cells are transfected with miR-325 nucleic acid inhibitor in vitro and the extracellular vesicles of the umbilical cord mesenchymal stem cells are used for treating liver diseases, the blood sinus differentiation marker stabilin-2 of the frozen liver slices is stained, the density of the visible blood sinuses is reduced, and differentiation is inhibited.

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.

Cell culture media (DMEM medium and alpha-MEM medium) in the following examples were purchased from Gibco, USA.

Liberase and OptiPrep in the examples described below were purchased from sigma.

MACS sorting CD11b + magnetic beads and sorting columns in the examples described below were purchased from Miltenyibitec, Inc.

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

miR-325 nucleic acid analogs in the examples described below were purchased from Ruibo organisms.

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 liver blood sinuses:

1. staining biopsy tissue with sirius red to detect collagen deposition around hepatic blood sinus

The method can use frozen biopsy tissue stored for a long time, or fresh tissue. After the tissue is fixed, the tissue is subjected to conventional sirius red staining, and the result is shown in figure 1, and after the liver leaf sirius red staining of a liver disease group, the red collagen deposition is obvious; in the liver disease group of miR-325 intravenous injection Tianlang red staining, red collagen deposition is obviously reduced.

Detection of regeneration key signal path after separation culture of primary hepatic sinus cells

Cutting fresh individual biopsy tissue, and using Liberase^TMDigesting for 1 hour in DMEM culture medium dissolved in 2% fetal bovine serum, gently blowing and beating to form single cells, centrifuging at low speed of 280rpm for 5 minutes to remove liver parenchymal cells, centrifuging the supernatant at 1800rpm for 10 minutes to obtain liver parenchymal cells including liver blood sinus endothelial cells, performing gradient centrifugation by using OptiPrep to obtain a liver blood sinus endothelial cell layer, finally sorting by using MACS magnetic beads, negatively selecting CD11b + cells to remove hybrid cells, and obtaining primary blood sinus endothelial cells with expanded adherent walls. Protein mass spectrometry detection is carried out by using the iTRAQ technology, and key proteins in a Hippo pathway in primary liver blood sinus cells after miR-325 treatment are shown in figure 2Significant changes suggest: during development, the signal pathway Hippo signaling that regulates organ size is activated.

Table 1 shows the change of the expression levels of ywaq and PTPRM proteins directly regulated by miR-325 detected by protein mass spectrometry, where 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 of the liver disease group treated by miR-325 to Mean of the liver disease group), and less than 0.8 indicates that the listed proteins are significantly down-regulated 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

Application of miR-325 nucleic acid analogue or composition in treatment of liver collagen deposition related abnormality

1. Intravenous injection for restoring liver function

The severity of the dysfunction can be determined by performing the test in exemplary step one, and deciding to use the miR-325 nucleic acid analog alone, or the miR-325 nucleic acid analog and CXCR7agonist combination. The specific implementation is that the intravenous injection is carried out on the individual patient with abnormal liver collagen deposition. The concentration of the miR-325 nucleic acid analogue is 20 nM. The purity standard requirement of the CXCR7agonist is more than or equal to 95 percent, and the grade of the CXCR7agonist is required to be used for cell culture; the CXCR7agonist was aseptically processed prior to use and the CXCR7agonist was at a concentration of 300 nM. The product is not mixed with statin drugs, melatonin, and other drugs for interfering with vascular function. The composition is not useful for individuals with cancer. 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 3, 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. The results of the combination are shown in fig. 4A-4D, wherein in H & E staining of livers of each group, fig. 4A is a liver fibrosis lesion group, fig. 4B is a miR-325 nucleic acid analog-treated group, fig. 4C is a CXCR7 agonist-treated group, and fig. 4D is a miR-325 nucleic acid analog and CXCR7agonist combination-treated group. The staining result indicates that the hepatic parenchymal cell necrosis vacuoles are obviously reduced, the inflammatory infiltration is relieved and the arrangement of the hepatic units is recovered compared with a fibrosis pathological group after single treatment, and further, the hepatic parenchymal cell necrosis vacuoles and the inflammation basically disappear in a combined treatment group, the arrangement of the hepatic units is clear and ordered, and the pathological microenvironment of hepatic fibrosis is obviously reversed.

The method can also be used as an effectiveness index of the mesenchymal stem cells to detect the treatment capacity of the mesenchymal stem cells before intravenous injection of the mesenchymal stem cells. The fact that expression of miR-325 in the mesenchymal stem cells or extracellular vesicles thereof is detected by using qPCR (expression level is referred to as U6 Ct =19, miR-325 Ct < 30) can ensure that the mesenchymal stem cells restore liver function by restoring sinus differentiation.

The method can also be used in combination with mesenchymal stem cell treatment, after the umbilical cord mesenchymal stem cells cultured in vitro are transfected with the miR-325 nucleic acid analogue, the transfected mesenchymal stem cells or mesenchymal stem cell outer vesicles are used for intravenous injection, and the final concentration of the miR-325 nucleic acid analogue in the culture solution after transfection is up to 20 nM. The mesenchymal stem cell culture method comprises the steps of taking a fresh umbilical cord, cutting off small segments, carefully peeling off vascular nerves, cutting into small pieces after obtaining Fahrenheit jelly, performing primary culture by using a serum-free alpha-MEM culture medium, carrying out passage and amplification. The passage should be less than 5 passages, i.e. less than 5 times of cryopreservation or pancreatin to make a single cell suspension. Mesenchymal stem cell outer vesicles were obtained by ultracentrifugation using less than 5 passages of cell culture supernatant. The ultracentrifugation step comprises centrifuging at normal temperature at 800rpm to remove cell debris, and ultracentrifuging at 130,000rpm to obtain extracellular vesicles. As shown in fig. 5A to 5D, the differentiation marker stablin-2 of liver sinusoid endothelial cells was stained with immunofluorescence, fig. 5A is a liver fibrosis lesion group, fig. 5B is an extracellular vesicle treatment group alone, fig. 5C is an extracellular vesicle treatment group extracted using a mesenchymal stem cell supernatant transfected with a miR-325 nucleic acid analog, and fig. 5D is an extracellular vesicle treatment group extracted using a mesenchymal stem cell supernatant transfected with a miR-325 nucleic acid inhibitor. The staining results indicate that up-regulation of expression of the miR-325 nucleic acid analog in the extracellular vesicles induces differentiation of liver sinusoids to promote regression of liver fibrosis, and inhibition of miR-325 in the extracellular vesicles inhibits differentiation of liver sinusoids to block the therapeutic effect of liver fibrosis.

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.