Umbilical cord mesenchymal stem cell preparation and application thereof in treatment of hepatic diseases

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an umbilical cord mesenchymal stem cell preparation and application thereof to treatment of hepatic diseases. The umbilical cord mesenchymal stem cell preparation comprises umbilical cord mesenchymal stem cells from the third generation to the sixth generation, a GPR55 peptide conjugate and pharmaceutically acceptable auxiliary materials. And the amino acid sequence of the GPR55 peptide conjugate is selected from CKKNSPTLC, CKKNTPTQC or CKKNSPTGC. The umbilical cord mesenchymal stem cell preparation can obviously reduce alpha-SMA and COL-I secreted by hepatic stellate cells and inhibit activation of the hepatic stellate cells, and the fact that the umbilical cord mesenchymal stem cell preparation has anti-hepatic fibrosis activity is further verified in a CCl4 induced hepatic fibrosis model.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to stem cell preparations, and in particular to an umbilical cord mesenchymal stem cell preparation and its use in treating liver diseases. Background Art

[0002] Hepatic fibrosis refers to the excessive deposition of extracellular matrix (especially collagen) in the liver. Hepatic fibrosis is a major problem in the treatment of liver diseases. 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, 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) and autoimmune hepatitis, primary hepatic cirrhosis and primary sclerosing cholangitis.

[0003] The pathological changes of liver fibrosis are characterized by increased synthesis of liver extracellular matrix and decreased degradation. If improperly treated, liver cirrhosis can occur through two major hazards: first, due to the destruction of liver tissue structure, the intrahepatic blood vessels are compressed and twisted, closed, or a "short-circuit" anastomosis occurs between arteries and veins, causing increased vascular resistance in the portal vein system and portal hypertension, leading to splenomegaly, ascites formation, and esophageal varices at the fundus of the stomach, with the potential risk of varicose vein rupture and bleeding in the upper gastrointestinal tract; second, the blood microcirculation channels between normal liver cells are blocked by the deposition of fibrous tissue components, affecting the blood supply to liver cells, making it difficult to repair liver cells damaged by inflammation or even aggravating the damage, until there are fewer and fewer liver cells with normal function, and finally leading to liver failure.

[0004] Mesenchymal stem cells have the characteristics of self-renewal, multidirectional differentiation and immunoregulation. They have shown good therapeutic effects in the treatment of liver diseases and have attracted the attention of researchers from various countries. Human Umbilical Cord Mesenchymal Stem Cells (hUC-MSCs) are a type of multipotent stem cells found in the umbilical cord tissue of newborns. hUC-MSCs have multidirectional differentiation potential and can differentiate into various cell types such as osteocytes, chondrocytes, muscle cells, and nerve cells. They can also regulate immune responses by secreting a variety of bioactive factors and have anti-inflammatory and immunosuppressive properties. Compared with other mesenchymal stem cells, hUC-MSCs have lower immunogenicity, which means that they are not likely to cause immune rejection reactions in the host when transplanted. Compared with stem cells derived from bone marrow or adipose tissue, the acquisition process of hUC-MSCs is simpler. They are usually extracted from the umbilical cord of newborns after birth and are harmless to the donor. Umbilical cord mesenchymal stem cells are regarded as a "treasure trove of mesenchymal stem cells" due to their multidirectional differentiation potential, low immunogenicity, abundant sources and few ethical controversies. They have achieved clinical research results in the treatment of various diseases and have shown great application potential in regenerative medicine and tissue engineering.

[0005] Mainstream drugs for anti-liver fibrosis include drugs that reduce the proliferation and activation of hepatic stellate cells (HSC), drugs that act 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 many types of anti-liver fibrosis drugs, there is still a lack of targeted drugs with strong specificity and good efficacy, and the road to anti-liver fibrosis drug research and development is still long.

[0006] G protein-coupled receptor 55 (GPR55) is a regulatory factor that has gradually attracted attention in liver disease research 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. L-α-lysophosphatidylinositol / GPR55 system induces the occurrence of non-alcoholic fatty degeneration and fatty hepatitis [J]. Journal of Clinical Hepatobiliary Diseases, 2020). GPR55 is also closely related to liver lipid metabolism, inflammatory response and fibrosis. In the treatment of liver inflammation and fibrosis, scientists have discovered a cyclic peptide P1-1 that can antagonize GPR55 and effectively inhibit collagen secretion in hepatic stellate cells. P1-1 improved 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 validated that GPR55 can be used as an effective target for the treatment of liver fibrosis.

[0007] Based on this, the present invention aims to provide an effective preparation, which uses umbilical cord mesenchymal stem cells and a GPR55 peptide conjugate as active ingredients to provide effective protection 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 treating liver diseases, specifically, provides its use in treating liver fibrosis.

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

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

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

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

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

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

[0015] S2, cut the umbilical cord into small pieces of 2-3 cm, rinse with PBS again, cut the umbilical cord longitudinally, and remove Wharton's jelly;

[0016] S3. Cut the peeled Wharton's glue into 1mm pieces. 3 The tissue fragments are placed in a culture bottle containing a culture medium, and statically cultured in an incubator at 37°C and 5% CO2; the culture bottle is pre-coated with poly-lysine; the culture medium is a DMEM medium supplemented with 10-12% FBS, 100U / mL penicillin, and 100mg / L streptomycin;

[0017] S4. Observe the cell growth every day under an inverted microscope. Perform the first full medium change after 5 to 7 days, and perform half medium change every 2 to 3 days thereafter. When the cells grow to a confluence of 80 to 90%, digest and subculture them, and inoculate them into culture bottles containing culture medium. When the cells grow to a confluence of 80% again, the first generation of umbilical cord mesenchymal stem cells are obtained.

[0018] The first generation of umbilical cord mesenchymal stem cells are digested and passaged with 0.25% trypsin and inoculated into a culture bottle containing culture medium. When the cells grow again to a confluence of 80%, the second generation of umbilical cord mesenchymal stem cells are obtained. The same culture steps as above can be used to obtain the third to sixth generations of umbilical cord mesenchymal stem cells in sequence.

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

[0020] Furthermore, the umbilical cord mesenchymal stem cell composition comprises the following active components: umbilical cord mesenchymal stem cells of passages 3 to 6, and a GPR55 peptide conjugate.

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

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

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

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

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

[0026] Furthermore, the amino acid sequence of the GPR55 peptide binder 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 a use of an umbilical cord mesenchymal stem cell preparation in the preparation of a medicament for treating liver diseases.

[0028] Furthermore, the umbilical cord mesenchymal stem cell preparation includes: umbilical cord mesenchymal stem cells of passages 3 to 6, a GPR55 peptide conjugate, and pharmaceutically acceptable excipients.

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

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

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

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows: hepatic stellate cell activation is a sign of liver fibrosis, and the composition of umbilical cord mesenchymal stem cells combined with GPR55 peptide conjugates and their preparations 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 GPR55 peptide conjugates in a CCl4-induced liver fibrosis model. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Relative expression levels of α-SMA and COL-I in LX-2 human hepatic stellate cells. Figure 2 Relative expression of α-SMA in the liver in the CCl4-induced mouse liver fibrosis model. Figure 3 Serum SOD content in CCl4-induced mouse liver fibrosis model. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with specific implementation cases, but the present invention is not limited to these embodiments.

[0034] The GPR55 peptide conjugate of the present invention is developed based on the previous study (Mangini M et al. Peptide-guided targeting of GPR55 for anti-cancer therapy. Oncotarget. 2017), and is obtained through structural optimization and repeated improvements. The synthesis of the GPR55 peptide conjugate of the present invention adopts the Fmoc-SPPS synthesis method.

[0035] Experimental Example 1 Isolation and Identification of Human Umbilical Cord Mesenchymal Stem Cells

[0036] The umbilical cords of healthy full-term pregnant women in the obstetrics and gynecology department of the hospital were obtained, and informed consent was obtained from the pregnant women and their families.

[0037] The method for isolating human umbilical cord mesenchymal stem cells comprises the following steps:

[0038] S1. Take 10 cm of fresh human umbilical cord and rinse it with PBS to remove residual blood and impurities;

[0039] S2, cut the umbilical cord into 2 cm segments, rinse with PBS again, cut the umbilical cord longitudinally, and remove Wharton's jelly;

[0040] S3. Cut the peeled Wharton's glue into 1mm pieces. 3 The tissue fragments are placed in a culture bottle containing culture medium, and cultured in a 37°C, 5% CO2 incubator; the culture bottle is pre-coated with poly-lysine; the culture medium is DMEM medium supplemented with 12% FBS, 100U / mL penicillin, and 100mg / L streptomycin;

[0041] S4. Observe the cell growth every day under an inverted microscope. Perform the first full medium change after 6 days, and perform half medium change every 2 days thereafter. When the cells grow to a confluence of 80%, digest them with 0.25% trypsin and subculture them. Inoculate them into a T25 culture flask containing culture medium. When the cells grow to a confluence of 80% again, the first generation of umbilical cord mesenchymal stem cells are obtained.

[0042] The first generation of umbilical cord mesenchymal stem cells are digested and passaged with 0.25% trypsin, and inoculated into a T25 culture flask containing culture medium. When the cells grow again to a confluence of 80%, the second generation of umbilical cord mesenchymal stem cells are obtained; the same culture steps can be used to obtain the third to sixth generations of umbilical cord mesenchymal stem cells in sequence.

[0043] Morphological observation showed that the isolated umbilical cord mesenchymal stem cells were adherent cells with a fibroblast-like morphology. Flow cytometry results showed that the 1st to 2nd generation umbilical cord mesenchymal stem cells were CD29, CD90, and CD105 positive, and also expressed a certain degree of CD19, CD34, and CD45. The 3rd to 6th generation umbilical cord mesenchymal stem cells were CD29, CD90, and CD105 positive, and CD19, CD34, and CD45 negative, which was consistent with the identification of umbilical cord mesenchymal stem cell surface markers. The in vitro culture cycle of primary umbilical cord mesenchymal stem cells is limited. Most umbilical cord mesenchymal stem cells that exceed the 6th generation will change from fibroblast-like to irregular or polygonal in morphology, and the differentiation potential of the cells will be significantly reduced due to cell differentiation.

[0044] Therefore, the present invention uses umbilical cord mesenchymal stem cells of the 3rd to 6th generations as active components to ensure the therapeutic effect.

[0045] Experimental Example 2 Preparation of Umbilical Cord Mesenchymal Stem Cell Composition

[0046] Example 1: The 4th generation umbilical cord mesenchymal stem cells and the GPR55 peptide conjugate prepared in Experimental Example 1 were mixed evenly at a mass ratio of 1:2 to obtain an umbilical cord mesenchymal stem cell composition; the amino acid sequence of the GPR55 peptide conjugate was CKKNSPTLC (SEQ ID NO.1).

[0047] Example 2: The 4th generation umbilical cord mesenchymal stem cells and the GPR55 peptide conjugate prepared in Experimental Example 1 were mixed evenly at a mass ratio of 1:2 to obtain an umbilical cord mesenchymal stem cell composition; the amino acid sequence of the GPR55 peptide conjugate was CKKNTPTQC (SEQ ID NO.2).

[0048] Example 3: The 4th generation umbilical cord mesenchymal stem cells and the GPR55 peptide conjugate prepared in Experimental Example 1 were mixed evenly at a mass ratio of 1:2 to obtain an umbilical cord mesenchymal stem cell composition; the amino acid sequence of the GPR55 peptide conjugate was CKKNSPTGC (SEQ ID NO.3).

[0049] Comparative Example 1: GPR55 peptide binder only, the amino acid sequence is CKKNSPTLC (SEQ ID NO. 1).

[0050] Comparative Example 2: GPR55 peptide binder only, the amino acid sequence is CKKNTPTQC (SEQ ID NO. 2).

[0051] Comparative Example 3: GPR55 peptide binder only, the amino acid sequence is CKKNSPTGC (SEQ ID NO. 3).

[0052] Experimental Example 3: Verification of the effect of LX-2 human hepatic stellate cell model

[0053] LX-2 human hepatic stellate cells were purchased from the Cell Bank of the Chinese Academy of Sciences, Catalog Number: SCSP-527. The complete culture medium of LX-2 cells is composed of 98% DMEM medium (Gibco, Catalog Number 11995065), 2% fetal bovine serum FBS (Gibco), 100 U / ml penicillin and 100 U / ml streptomycin. LX-2 cells were cultured in an incubator at 37°C and 5% CO2. After the cells were stabilized, LX-2 cells grown to a confluence of about 70% were incubated with 8 ng / mL TGF-β1 for 24 h to activate them.

[0054] The experiment set up a control group and a drug administration group. The control group only replaced the culture medium, and the drug administration group added the compositions of Examples 1-3 and Comparative Examples 1-3 while replacing the culture medium. The compositions of Examples 1-3 were added at a final concentration of 20 μM of the GPR55 peptide binder, and the final concentration of the GPR55 peptide binder of Comparative Examples 1-3 was 25 μM, and incubated for 24 hours.

[0055] Activation of hepatic stellate cells is a hallmark of liver fibrosis. Hepatic stellate cell activation is active between hepatocytes and sinusoidal endothelial cells, and has the ability to store vitamins and regulate 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. On the other hand, the inflammatory environment can also stimulate liver 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 large expression of α-smooth 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), leading to changes in liver tissue structure and impaired function. According to Figure 1 The test results show that 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 better reducing activity than the effects of Comparative Examples 1-3, reflecting the synergistic effect of the umbilical cord mesenchymal stem cells and the GPR55 peptide conjugate.

[0056] Experimental Example 4: Verification of the effect of CCl4-induced mouse liver fibrosis model

[0057] Experimental animals 8-week-old male C57BL / 6J mice were selected and randomly divided into a control group, a modeling group, an embodiment 1-3 group, and a comparative example 1-3 group, with 6-8 mice in each group. The modeling group, the embodiment and the comparative example group 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 way, and the modeling lasted for 6 weeks. The corresponding composition or GPR55 peptide conjugate prepared in Experimental Example 2 was intraperitoneally injected with 250 μg / kg in the embodiment 1-3 group and the comparative example 1-3 group every day for 4 weeks. The mice were anesthetized and blood samples were collected from the posterior venous plexus of the eyes, and the SOD content in the serum was detected using a kit; the liver tissue was separated, and the relative expression of α-SMA was detected by Western blotting.

[0058] The anti-fibrotic activity of umbilical cord mesenchymal stem cells combined with GPR55 peptide conjugates was verified in a CCl4-induced liver fibrosis model. Figure 2 and Figure 3 As shown. The compositions of Examples 1-3 not only reduce the relative expression of α-SMA in the CCl4-induced liver fibrosis model, but also alleviate further liver damage caused by oxidative stress by increasing the production of SOD. In contrast, the GPR55 peptide conjugates of Comparative Examples 1-3, although they can also produce significant anti-liver fibrosis activity, still have obvious activity differences compared to the compositions of Examples 1-3.

[0059] The above describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and the description only describe the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached 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.

2. The composition according to claim 1, It is characterized in that The mass ratio of the umbilical cord mesenchymal stem cells to the GPR55 peptide conjugate is 1:(1-3).

3. The composition according to claim 2, It is characterized in that The mass ratio of the umbilical cord mesenchymal stem cells to the GPR55 peptide conjugate is 1:

2.

4. The composition according to claim 1, It is characterized in that The amino acid sequence of the GPR55 peptide binder is selected from CKKNSPTLC (SEQ ID NO. 1), CKKNTPTQC (SEQ ID NO. 2) or CKKNSPTGC (SEQ ID NO. 3).

5. An umbilical cord mesenchymal stem cell preparation, include: Umbilical cord mesenchymal stem cells of passages 3 to 6, GPR55 peptide conjugates, and pharmaceutically acceptable excipients.

6. The preparation according to claim 5, It is characterized in that The mass ratio of the umbilical cord mesenchymal stem cells to the GPR55 peptide conjugate is 1:(1-3).

7. The preparation according to claim 6, It is characterized in that The amino acid sequence of the GPR55 peptide binder is selected from CKKNSPTLC (SEQ ID NO. 1), CKKNTPTQC (SEQ ID NO. 2) or CKKNSPTGC (SEQ ID NO. 3).

8. Use of the umbilical cord mesenchymal stem cell composition according to claim 1 in preparing a medicament for treating liver diseases.

9. Use of the umbilical cord mesenchymal stem cell preparation according to claim 5 in the preparation of a medicament for treating liver diseases.

10. The use according to claim 8 or 9, It is characterized in that The liver disease is liver fibrosis.

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