A preparation of umbilical cord mesenchymal stem cells and its use in the treatment of liver diseases

Abstract

The present invention belongs to the field of biomedical technology, and particularly relates to a preparation of umbilical cord mesenchymal stem cells and its use in the treatment of liver diseases. The preparation of umbilical cord mesenchymal stem cells includes umbilical cord mesenchymal stem cells of passages 3 to 6, a GPR55 peptide conjugate, and a pharmaceutically acceptable excipient. The amino acid sequence of the GPR55 peptide conjugate is selected from CKKNSPTLC, CKKNTPTQC or CKKNSPTGC. The preparation of umbilical cord mesenchymal stem cells of the present invention can significantly reduce the secretion of α-SMA and COL-I by hepatic stellate cells, inhibit the activation of hepatic stellate cells, and further verify its anti-hepatic fibrosis activity in a CCl4-induced liver fibrosis model.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, relates to a stem cell preparation, and particularly relates to a preparation of umbilical cord mesenchymal stem cells and its use in the treatment of liver diseases. Background Art

[0002] Hepatic fibrosis refers to the excessive deposition of extracellular matrix (especially collagen) diffusely in the liver. Hepatic fibrosis is a major problem in the field of liver disease treatment. It is not an independent disease, but many chronic liver diseases can cause hepatic fibrosis. Its causes can be roughly divided into infectious (chronic hepatitis B, C, and D virus infections, schistosomiasis, etc.), congenital metabolic defects (Wilson's disease, hemochromatosis, α1-antitrypsin deficiency, etc.), and chemical metabolic defects (chronic alcoholic liver disease, chronic drug-induced liver disease), as well as autoimmune hepatitis, primary biliary cirrhosis, and primary sclerosing cholangitis, etc.

[0003] The pathological changes of hepatic fibrosis are manifested as an increase in the synthesis of extracellular matrix in the liver and a decrease in degradation. If the treatment is improper, it can mainly form cirrhosis through two major hazards: one is that due to the destruction of the hepatic tissue structure, the blood vessels in the liver are compressed, distorted, blocked, or there is a "short circuit" anastomosis between arteries and veins, resulting in an increase in the vascular resistance of the portal vein system, forming portal hypertension, leading to splenomegaly, ascites formation, and gastric fundus and esophageal varices, with the potential risk of rupture and bleeding of varicose veins in the upper digestive tract; the other is that the blood microcirculation channels between normal hepatocytes are blocked due to the deposition of fibrous tissue components, affecting the blood supply of hepatocytes, making the damaged hepatocytes due to inflammation difficult to repair or even aggravating the damage, until there are fewer and fewer normal-functioning hepatocytes, and finally leading to liver failure.

[0004] Mesenchymal stem cells have the characteristics of self-renewal, multi-directional differentiation and immunomodulation, and have shown good curative effects in the treatment of liver diseases, attracting the attention of researchers from various countries. Human Umbilical Cord Mesenchymal Stem Cells (hUC-MSCs) are a type of pluripotent stem cells existing in neonatal umbilical cord tissue. hUC-MSCs have multi-directional differentiation potential, can differentiate into various cell types such as osteoblasts, chondrocytes, muscle cells, and nerve cells, and can also regulate immune responses by secreting a variety of bioactive factors, with anti-inflammatory and immunosuppressive properties. Compared with other mesenchymal stem cells, hUC-MSCs have lower immunogenicity, which means they are not easily induced to cause immune rejection in the host during transplantation. Compared with stem cells derived from bone marrow or adipose tissue, the acquisition process of hUC-MSCs is simpler, usually extracted from the umbilical cord after the birth of a neonate, without harm to the donor. Due to their multi-directional differentiation potential, low immunogenicity, rich sources and few ethical controversies, umbilical cord mesenchymal stem cells are regarded as the "treasure house of mesenchymal stem cells", and have achieved clinical research results in the treatment of various diseases, showing great application potential in regenerative medicine and tissue engineering.

[0005] The mainstream drugs for anti-hepatic fibrosis include drugs that reduce the proliferation and activation of hepatic stellate cells (HSCs), drugs acting on matrix metalloproteinase (MMP) / tissue inhibitor of metalloproteinase (TIMP), drugs that inhibit inflammatory responses and regulate immune responses, drugs for oxidative stress, targeted anti-fibrosis drugs, gene therapy, and traditional Chinese medicine preparations, etc. Although there are a variety of drugs for anti-hepatic fibrosis, there is still a lack of specific and effective targeted drugs, and the road to the research and development of anti-hepatic fibrosis drugs is still long.

[0006] G protein-coupled receptor 55 (GPR55) is a regulatory factor that has gradually attracted attention in the research of liver diseases in recent years. Studies have shown that GPR55 is related to the pathophysiological mechanism of non-alcoholic fatty liver disease (NAFLD) (Jiang Zhongchun, Xie Zhiqin, Tang Caixi, FONDEVILA MF, FERNANDEZ U, GONZALEZ-RELLAN MJ. The L-α-lysophosphatidylinositol / GPR55 system induces the development of non-alcoholic steatosis and steatohepatitis [J]. Journal of Clinical Hepatology, 2020). GPR55 is also closely related to the processes of liver lipid metabolism, inflammatory response, and fibrosis. In the treatment of liver inflammation and fibrosis, scientists have discovered a cyclic peptide P1-1, which can antagonize GPR55 and effectively inhibit the secretion of collagen in hepatic stellate cells. P1-1 improves acute liver inflammation and fibrosis by reducing the production of reactive oxygen species (ROS), alleviating endoplasmic reticulum (ER) stress, and inhibiting mitochondria-related hepatocyte apoptosis (Zihan Shi et al. Discovery of Novel Peptide Antagonists Targeting GPR55 for Liver Inflammation and Fibrosis. Journal of Medicinal Chemistry 2024). This study verifies that GPR55 can be an effective target for the treatment of liver fibrosis.

[0007] Based on this, the present invention aims to provide an effective preparation, using umbilical cord mesenchymal stem cells and GPR55 peptide conjugate as the active ingredients, to provide effective guarantee for the clinical treatment of liver fibrosis. Summary of the Invention

[0008] Based on the deficiencies in the prior art, the present invention provides an umbilical cord mesenchymal stem cell preparation and its use in the treatment of liver diseases, specifically, its use in the treatment of liver fibrosis diseases.

[0009] To achieve the above object, the technical solution of the present invention is realized as follows:

[0010] In the first aspect, the present invention provides an umbilical cord mesenchymal stem cell composition, comprising the following active components: umbilical cord mesenchymal stem cells of the 3rd to 6th generation, and GPR55 peptide conjugate.

[0011] Further, the mass ratio of the umbilical cord mesenchymal stem cells to the GPR55 peptide conjugate is 1:(1 - 3).

[0012] Further, the amino acid sequence of the GPR55 peptide conjugate is selected from CKKNSPTLC (SEQ ID NO.1), CKKNTPTQC (SEQ ID NO.2), or CKKNSPTGC (SEQ ID NO.3).

[0013] Further, the method for isolating the umbilical cord mesenchymal stem cells comprises the following steps:

[0014] S1. Take 8 - 12 cm of fresh human umbilical cord, rinse it with PBS to remove residual blood and impurities;

[0015] S2. Cut the umbilical cord into small segments of 2 - 3 cm, rinse it again with PBS, longitudinally cut open the umbilical cord, and strip Wharton's jelly;

[0016] S3. Cut the stripped Wharton's jelly into tissue fragments of 1 mm 3 , place them in a culture flask containing a culture medium, and statically culture them in an incubator at 37 °C and 5% CO2; the culture flask is pre - coated with polylysine; the culture medium is DMEM medium supplemented with 10 - 12% FBS, 100 U / mL penicillin, and 100 mg / L streptomycin;

[0017] S4. Observe the cell growth condition every day under an inverted microscope, perform a full medium change for the first time after 5 - 7 days, and then perform a half medium change every 2 - 3 days; when the cell growth reaches a confluence of 80 - 90%, digest and passage the cells, and inoculate them into a culture flask containing a culture medium. When the cells grow to a confluence of 80% again, the first - generation umbilical cord mesenchymal stem cells are obtained.

[0018] Digest and passage the first - generation umbilical cord mesenchymal stem cells with 0.25% trypsin, and inoculate them into a culture flask containing a culture medium. When the cells grow to a confluence of 80% again, the second - generation umbilical cord mesenchymal stem cells are obtained; by using the same culture steps as described above, the third - to sixth - generation umbilical cord mesenchymal stem cells can be obtained in sequence.

[0019] In a second aspect, the present invention provides the use of an umbilical cord mesenchymal stem cell composition in the preparation of a drug for treating liver diseases.

[0020] Further, the umbilical cord mesenchymal stem cell composition comprises the following active components: the third - to sixth - generation umbilical cord mesenchymal stem cells, and a GPR55 peptide conjugate.

[0021] Further, the mass ratio of the umbilical cord mesenchymal stem cells to the GPR55 peptide conjugate is 1:(1 - 3).

[0022] Further, the amino acid sequence of the GPR55 peptide conjugate is selected from CKKNSPTLC (SEQ ID NO.1), CKKNTPTQC (SEQ ID NO.2) or CKKNSPTGC (SEQ ID NO.3).

[0023] Further, the liver disease is liver fibrosis.

[0024] In a third aspect, the present invention provides a preparation of umbilical cord mesenchymal stem cells, comprising: umbilical cord mesenchymal stem cells of passages 3-6, a GPR55 peptide conjugate, and a pharmaceutically acceptable excipient.

[0025] Further, the mass ratio of the umbilical cord mesenchymal stem cells to the GPR55 peptide conjugate is 1:(1-3).

[0026] Further, the amino acid sequence of the GPR55 peptide conjugate is selected from CKKNSPTLC (SEQ ID NO.1), CKKNTPTQC (SEQ ID NO.2) or CKKNSPTGC (SEQ ID NO.3).

[0027] In a fourth aspect, the present invention provides the use of a preparation of umbilical cord mesenchymal stem cells in the manufacture of a medicament for treating liver diseases.

[0028] Further, the preparation of umbilical cord mesenchymal stem cells comprises: umbilical cord mesenchymal stem cells of passages 3-6, a GPR55 peptide conjugate, and a pharmaceutically acceptable excipient.

[0029] Further, the mass ratio of the umbilical cord mesenchymal stem cells to the GPR55 peptide conjugate is 1:(1-3).

[0030] Further, the amino acid sequence of the GPR55 peptide conjugate is selected from CKKNSPTLC (SEQ ID NO.1), CKKNTPTQC (SEQ ID NO.2) or CKKNSPTGC (SEQ ID NO.3).

[0031] Further, the liver disease is liver fibrosis.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows: The activation of hepatic stellate cells is a hallmark of liver fibrosis. The composition and preparation of umbilical cord mesenchymal stem cells combined with a GPR55 peptide conjugate can significantly reduce the secretion of α-SMA and COL-I by hepatic stellate cells, inhibit the activation of hepatic stellate cells, and further verify the anti-liver fibrosis activity of the combined preparation of umbilical cord mesenchymal stem cells and the GPR55 peptide conjugate in a CCl4-induced liver fibrosis model. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Relative expression levels of α-SMA and COL-I in LX-2 human hepatic stellate cells.

[0034] Figure 2 Relative expression level of hepatic α-SMA in a CCl4-induced mouse liver fibrosis model.

[0035] Figure 3 Content of serum SOD in a CCl4-induced mouse liver fibrosis model. Detailed implementation manners

[0036] The present invention will be further described below in conjunction with specific implementation cases, but the present invention is not limited to these embodiments.

[0037] The GPR55 peptide conjugate in the present invention is based on prior research (Mangini M et al. Peptide-guided targeting of GPR55 for anti-cancer therapy. Oncotarget. 2017), and is obtained through structure optimization and repeated improvement. The synthesis of the GPR55 peptide conjugate involved in the present invention adopts the Fmoc-SPPS synthesis method.

[0038] Experimental example 1 Isolation and identification of human umbilical cord mesenchymal stem cells

[0039] Take the umbilical cord of a healthy full-term pregnant woman in the obstetrics and gynecology department of the hospital, and obtain the informed consent of the parturient and her family members.

[0040] The isolation method of human umbilical cord mesenchymal stem cells includes the following steps:

[0041] S1. Take 10 cm of fresh human umbilical cord, rinse it with PBS to remove residual blood and impurities;

[0042] S2. Cut the umbilical cord into 2-cm segments, rinse it again with PBS, longitudinally cut open the umbilical cord, and peel off Wharton's jelly;

[0043] S3. Cut the peeled Wharton's jelly into 1-mm 3 tissue fragments, place them in a culture flask containing a culture medium, and statically culture them in an incubator at 37°C and 5% CO2; the culture flask is pre-coated with polylysine; the culture medium is a DMEM medium supplemented with 12% FBS, 100 U / mL penicillin, and 100 mg / L streptomycin;

[0044] S4. Observe the cell growth condition with an inverted microscope every day. Perform a full medium change for the first time after 6 days, and then perform a half medium change every 2 days thereafter. When the cell confluence reaches 80%, digest and passage the cells with 0.25% trypsin, and inoculate them into a T25 culture flask containing culture medium. When the cells grow to a confluence of 80% again, the umbilical cord mesenchymal stem cells of the first passage are obtained.

[0045] Digest and passage the umbilical cord mesenchymal stem cells of the first passage with 0.25% trypsin, and inoculate them into a T25 culture flask containing culture medium. When the cells grow to a confluence of 80% again, the umbilical cord mesenchymal stem cells of the second passage are obtained. The umbilical cord mesenchymal stem cells of the 3rd - 6th passages can be obtained successively by using the same culture procedure.

[0046] Through morphological observation, the isolated umbilical cord mesenchymal stem cells are adherent cells, and the cell morphology is fibroblast - like. The flow cytometry results show that the umbilical cord mesenchymal stem cells of the 1st - 2nd passages are positive for CD29, CD90, and CD105, and express a certain degree of CD19, CD34, and CD45. The umbilical cord mesenchymal stem cells of the 3rd - 6th passages are positive for CD29, CD90, and CD105, and negative for CD19, CD34, and CD45, which conforms to the identification of the surface markers of umbilical cord mesenchymal stem cells. The in vitro culture period of primary umbilical cord mesenchymal stem cells is limited. For umbilical cord mesenchymal stem cells beyond the 6th passage, most of the cell morphologies will change from fibroblast - like to irregular or polygonal, and cell differentiation leads to a significant reduction in differentiation potential.

[0047] Therefore, the present invention uses the umbilical cord mesenchymal stem cells of the 3rd - 6th passages as the active component to ensure the curative effect.

[0048] Experimental Example 2 Preparation of Umbilical Cord Mesenchymal Stem Cell Composition

[0049] Example 1: Take the umbilical cord mesenchymal stem cells of the 4th passage prepared in Experimental Example 1 and the GPR55 peptide conjugate, and mix them evenly according to a mass ratio of 1:2 to obtain the umbilical cord mesenchymal stem cell composition; the amino acid sequence of the GPR55 peptide conjugate is CKKNSPTLC (SEQ ID NO.1).

[0050] Example 2: Take the umbilical cord mesenchymal stem cells of the 4th passage prepared in Experimental Example 1 and the GPR55 peptide conjugate, and mix them evenly according to a mass ratio of 1:2 to obtain the umbilical cord mesenchymal stem cell composition; the amino acid sequence of the GPR55 peptide conjugate is CKKNTPTQC (SEQ ID NO.2).

[0051] Example 3: Take the umbilical cord mesenchymal stem cells of the 4th generation prepared in Experimental Example 1 and the GPR55 peptide conjugate, and mix them evenly according to a mass ratio of 1:2 to obtain an umbilical cord mesenchymal stem cell composition; the amino acid sequence of the GPR55 peptide conjugate is CKKNSPTGC (SEQ ID NO.3).

[0052] Comparative Example 1: Only the GPR55 peptide conjugate, with the amino acid sequence CKKNSPTLC (SEQ ID NO.1).

[0053] Comparative Example 2: Only the GPR55 peptide conjugate, with the amino acid sequence CKKNTPTQC (SEQ ID NO.2).

[0054] Comparative Example 3: Only the GPR55 peptide conjugate, with the amino acid sequence CKKNSPTGC (SEQ ID NO.3).

[0055] Effect verification of the LX-2 human hepatic stellate cell model in Experimental Example 3

[0056] The LX-2 human hepatic stellate cells were purchased from the Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection, catalog number: SCSP-527. The complete culture medium formula for LX-2 cells: 98% DMEM medium (Gibco, product number 11995065), 2% fetal bovine serum FBS (Gibco), 100 U / ml penicillin and 100 U / ml streptomycin. The LX-2 cells were cultured in an incubator at 37°C and 5% CO2. After the cell state was stable, the LX-2 cells with a confluence of about 70% were incubated with 8 ng / mL TGF-β1 for 24 h to activate them.

[0057] The test was set up with a control group and a dosing group. In the control group, only the culture medium was changed. In the dosing group, the compositions of Examples 1-3 and Comparative Examples 1-3 were added respectively while changing the culture medium. The compositions of Examples 1-3 were added with a final concentration of 20 μM of the GPR55 peptide conjugate, and the final concentration of the GPR55 peptide conjugate in Comparative Examples 1-3 was 25 μM, and incubated for 24 hours.

[0058] Hepatic stellate cell activation is a hallmark of liver fibrosis. Hepatic stellate cells are activated between hepatocytes and hepatic sinusoidal endothelial cells and have the ability to store vitamins and regulate hepatic sinusoidal blood flow under static conditions. When the liver is damaged, on the one hand, neighboring cells release inflammatory factors in a paracrine manner to activate hepatic stellate cells, and on the other hand, the inflammatory environment can also stimulate hepatic macrophages to secrete IL-1β and IL-6 to further activate hepatic stellate cells. Activated hepatic stellate cells show characteristics such as cell proliferation, increased contractility, and a large amount of expression of α-smooth muscle actin (α-SMA), and begin to synthesize a large amount of extracellular matrix, such as type I collagen (COL-I) and type III collagen (COL-III), resulting in changes in liver tissue structure and impaired function. According to Figure 1 the test results, the compositions of Examples 1-3 can significantly reduce the relative expression levels of α-SMA and COL-I in activated LX-2 human hepatic stellate cells, and have a better inhibitory activity compared to those of Comparative Examples 1-3, reflecting the synergistic effect of the combination of umbilical cord mesenchymal stem cells and GPR55 peptide.

[0059] Experimental Example 4 Verification of the effect of the CCl4-induced mouse liver fibrosis model

[0060] Male C57BL / 6J mice at 8 weeks of age were selected as experimental animals and randomly divided into a control group, a model group, groups of Examples 1-3, and groups of Comparative Examples 1-3, with 6-8 mice in each group. The model group, the groups of Examples, and the groups of Comparative Examples were intraperitoneally injected with a 25% CCl4 solution diluted with corn oil at a dose of 3 ml / kg every other day for liver fibrosis modeling, and the control group was injected with an equal amount of corn oil in the same manner. The modeling lasted for 6 weeks. The groups of Examples 1-3 and the groups of Comparative Examples 1-3 were intraperitoneally injected with the corresponding compositions or GPR55 peptide conjugates prepared in Experimental Example 2 at a dose of 250 μg / kg every day for 4 weeks. The mice were anesthetized and blood samples were collected from the retro-orbital venous plexus. The SOD content in the serum was detected using a kit; the liver tissues were separated and the relative expression level of α-SMA was detected by Western blotting.

[0061] The anti-liver fibrosis activity of the combination of umbilical cord mesenchymal stem cells and GPR55 peptide conjugate was verified in a CCl4-induced liver fibrosis model, and the results are shown in Figure 2 and Figure 3 as follows. In addition to reducing the relative expression level of α-SMA in the CCl4-induced liver fibrosis model, the compositions of Examples 1-3 also alleviated further liver injury caused by oxidative stress by increasing the production of SOD. In contrast, although the GPR55 peptide conjugates of Comparative Examples 1-3 could also produce significant anti-liver fibrosis activity, there were still obvious differences in activity compared to the compositions of Examples 1-3.

[0062] The basic principles, main features and advantages of the present invention have been described above. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification is only the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection required by the present invention is defined by the appended claims and their equivalents.

Claims

1. An umbilical cord mesenchymal stem cell composition, comprising the following active ingredients: umbilical cord mesenchymal stem cells of passages 3 to 6, and a GPR55 peptide conjugate; The mass ratio of the umbilical cord mesenchymal stem cells to the GPR55 peptide conjugate is 1:2; The amino acid sequence of the GPR55 peptide binder is shown in SEQ ID NO.2 or SEQ ID NO.

3.

2. An umbilical cord mesenchymal stem cell preparation, comprising: The umbilical cord mesenchymal stem cell composition according to claim 1, and a pharmaceutically acceptable excipient.

3. Use of the umbilical cord mesenchymal stem cell composition according to claim 1 in the preparation of a medicament for treating liver disease; the liver disease is liver fibrosis.

4. Use of the umbilical cord mesenchymal stem cell preparation according to claim 2 in the preparation of a medicament for treating liver disease; the liver disease is liver fibrosis.

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