Use of mesenchymal stem cells in preventing or treating liver disease

Abstract

The present invention relates to the technical field of liver disease treatment, and in particular to the use of mesenchymal stem cells in preventing or treating liver disease. The present invention uses miR-3059-5p as a therapeutic target, constructs umbilical cord mesenchymal stem cells transfected with miR-3059-5p, and verifies that the umbilical cord mesenchymal stem cells prepared by the present invention have good liver repair ability and can be used for the treatment of patients with acute liver injury.

Description

Technical Field

[0001] The present invention relates to the technical field of liver disease treatment, and in particular to use of mesenchymal stem cells in preventing or treating liver disease. Background Art

[0002] The liver is the largest solid organ in the abdominal cavity and is responsible for important physiological functions of the human body. Liver cells have poor tolerance to hypoxia, so the hepatic artery and portal vein provide abundant blood supply, and bile is transported by large and small bile ducts and blood vessels. It is located deep in the right upper abdomen and is protected by the lower chest wall and diaphragm. However, due to its large size and brittle texture, the liver is easily damaged once it is subjected to violence, resulting in intra-abdominal bleeding or bile leakage, causing hemorrhagic shock or bile peritonitis, with serious consequences, and must be diagnosed and treated in time.

[0003] Umbilical cord-mesenchymal stem cells (UC-MSCs) are adult stem cells isolated from umbilical cord tissue with multidirectional differentiation potential and multiple biological functions. Compared with human bone marrow and fat tissues, obtaining MSCs from the umbilical cord has many advantages, including abundant sources, high proportion of MSCs, strong proliferation ability of MSCs, lower immunogenicity, stronger immunosuppression, and no trauma to the donor. MSCs can secrete more nutritional growth factors, such as hepatocyte growth factor, which is very beneficial for MSCs to play the role of tissue and organ repair.

[0004] Relevant researchers are using stem cells to treat a variety of liver diseases. Studies have shown that mesenchymal stem cell transplantation can improve liver function, reduce liver fibrosis, and repair liver damage. However, its therapeutic effect is not ideal, and genetic improvements are still needed to overcome its technical defects. Summary of the invention

[0005] In view of the technical problems existing in the prior art, the present invention provides a use of mesenchymal stem cells in preventing or treating liver disease. The present invention uses miR-3059-5p as a therapeutic target, constructs umbilical cord mesenchymal stem cells transfected with miR-3059-5p, and verifies that the umbilical cord mesenchymal stem cells prepared by the present invention have good liver repair ability and can be used to treat patients with acute liver injury.

[0006] One of the purposes of the present invention is to use miR-3059-5p in preparing drugs for preventing or treating liver diseases. The sequence of miR-3059-5p is shown in SEQ ID NO.1.

[0007] Preferably, the liver disease is acute liver injury.

[0008] Preferably, the drug is a substance that upregulates the expression of miR-3059-5p.

[0009] Furthermore, the present invention provides a mesenchymal stem cell, wherein the mesenchymal stem cell specifically overexpresses miR-3059-5p.

[0010] Preferably, the mesenchymal stem cells are selected from umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells or adipose umbilical cord mesenchymal stem cells.

[0011] Preferably, the mesenchymal stem cells are transfected with miR-3059-5p.

[0012] Furthermore, the present invention also provides the use of the above-mentioned mesenchymal stem cells in the preparation of drugs for preventing or treating liver diseases.

[0013] Preferably, the mesenchymal stem cells are administered by intravenous injection.

[0014] Preferably, the liver disease is acute liver injury.

[0015] Furthermore, the present invention also provides a use of mesenchymal stem cells in preventing or treating liver disease, which comprises administering the miR-3059-5p or mesenchymal stem cells of the present invention.

[0016] The advantages of the present invention are as follows: The present invention first discovered that the expression of miR-3059-5p was significantly downregulated in patients with acute liver injury, and thus can be used as a therapeutic target for the development of liver disease drugs. To this end, the present invention constructed umbilical cord mesenchymal stem cells transfected with miR-3059-5p based on isolated umbilical cord mesenchymal stem cells, and further verified through an acute liver injury mouse model that the umbilical cord mesenchymal stem cells can effectively restore the liver index, increase the survival rate of liver cells, and repair damaged liver tissue, confirming that the umbilical cord mesenchymal stem cells prepared by the present invention have good liver repair ability and can be used for the treatment of patients with acute liver injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 . Expression of miR-3059-5p in patients with acute liver injury;

[0018] Figure 2 .Analysis of miR-3059-5p expression before and after transfection of umbilical cord mesenchymal stem cells. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0020] Example 1 RT-PCR detection of miR-3059-5p gene expression in patients with acute liver injury.

[0021] 1. Preparation of experimental materials

[0022] Blood samples were collected from 30 patients diagnosed with acute liver injury by clinicians from January to December 2022 for future use; blood samples from 30 healthy people were collected during the same period as controls.

[0023] 2. Test methods

[0024] 1) Peripheral blood mRNA extraction: The total mRNA from blood samples of patients and healthy subjects was extracted using a peripheral blood mRNA extraction kit (Dynabeads™ mRNA purification kit, Thermo Fisher Scientific) and stored at -20°C for future use;

[0025] 2) Reverse transcription cDNA synthesis: The total RNA in step 1) was reverse transcribed using a reverse transcription kit PrimeScript RT reagent Kit (brand: Takara; number: RR037Q) to obtain cDNA, which was stored at -20°C for future use; wherein the reverse transcription primer sequence is shown in SEQ ID NO.2;

[0026] 3) Real-time quantitative PCR: Specific primers were designed based on the nucleic acid sequence of miR-3059-5p (SEQ ID NO.1), wherein the upstream primer and downstream primer of miR-3059-5p were SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0027] The 25 μL PCR reaction system included: 10 μL 10× PCR Buffer, 2 μL SYBR Green I fluorescent dye, 4 μL dNTP, 1 μL each of the upstream and downstream primers of miR-376a-5p, 2 μL Taq DNA polymerase and 5 μL cDNA; the PCR reaction program was 95°C pre-denaturation for 5 min; 95°C for 40 s, 58°C for 45 s, and 72°C extension for 40 s, for a total of 38 cycles; 72°C extension for 10 min;

[0028] According to formula 2 -ΔΔCt The relative expression of miR-3059-5p was calculated, and the results are shown in Figure 1 ;Depend on Figure 1 It can be seen that compared with healthy people, the expression level of miR-3059-5p detected in the blood samples of patients with acute liver injury showed a significant downward trend.

[0029] Example 2 Analysis of the improvement of acute liver injury by upregulating miR-3059-5p

[0030] 1. Preparation of Mouse Model of Acute Liver Injury

[0031] Thirty BALB / C mice were subcutaneously injected with a mixture of equal volumes of CCl4 and olive oil (1:1) at a dose of 5 ml / kg. Transplantation experiments were performed 24 hours after modeling.

[0032] 2. Test methods

[0033] 2.1 Isolation and preparation of umbilical cord mesenchymal stem cells

[0034] 1) Under sterile conditions, take a fresh umbilical cord, wash it thoroughly with sterile saline, remove the umbilical artery and umbilical vein, peel off the outer membrane, and cut it into small pieces with sharp scissors for later use;

[0035] 2) adding a digestion solution containing 0.5-1.5% hyaluronidase and 0.1-1.0% trypsin to the umbilical cord cut into small pieces in step 1) for enzymatic digestion, and incubating at 37° C. for 1-2 hours for later use;

[0036] 3) Add an equal volume of low-glucose DMEM medium to the digestion solution to terminate the enzymatic digestion, filter with a 100-200 mesh filter, collect the filtrate, centrifuge the collected filtrate at 1000g for 5-10 minutes, discard the supernatant, and wash with low-glucose DMEM medium 2-3 times to obtain umbilical cord mesenchymal stem cells;

[0037] 4) The umbilical cord mesenchymal stem cells separated in step 3) are continuously passaged for 3 times, and the morphology and immunophenotype are identified. After the identification is qualified, the cells are collected by centrifugation for later use;

[0038] 5) Artificially synthesize 100nM miR-3059-5p, and take 10μL miR-3059-5p and 10μL Lipofectamine™ 2000 transfection reagent (brand: ThermoFisher Invitrogen, manufacturer: Thermo, catalog number 11668019) and mix them in 200μL low-glucose DMEM medium for later use;

[0039] 6) Add the mixed solution from step 5) to 2 mL of 1×10 7 cell / mL umbilical cord mesenchymal stem cell suspension for transfection, and incubate at 37°C, 100 rpm for 2-4 h for later use;

[0040] 7) After transfection, replace the medium with fresh low-glucose DMEM and inoculate the cell suspension into a culture flask for continued culture.

[0041] The results showed that the external morphology of the umbilical cord mesenchymal stem cells isolated by the method of the present invention showed a typical spindle-like fibroblast morphology, and the cell surface specifically expressed CD44, CD90, and CD105, while low expression of CD34, CD45, and CD80. It was confirmed that the cells isolated by the method of the present invention were umbilical cord mesenchymal stem cells, which could meet the requirements of subsequent experimental analysis. At the same time, after transfecting umbilical cord mesenchymal stem cells with miR-3059-5p, RT-PCR analysis found that compared with untransfected cells, the expression of miR-3059-5p in umbilical cord mesenchymal stem cells after transfection was significantly increased, and the difference was significant ( Figure 2 ).

[0042] 2.2 Analysis of umbilical cord mesenchymal stem cell transplantation

[0043] The mice were randomly divided into three groups. The treated group was intravenously injected with 1×10 4 The umbilical cord mesenchymal stem cells were transfected with miR-3059-5p, and the control group mice were intravenously injected with 1×10 4 cell umbilical cord mesenchymal stem cells, and the blank group mice were intravenously injected with an equal amount of normal saline. The liver function indexes, hepatocyte apoptosis rate, and serum ALT, AST, AKP, TC, TG, LDL-C, and HDL-C levels of the mice were observed and recorded 7 days after transplantation.

[0044] The results are as follows: As can be seen from Table 1, the liver index in the treatment group is significantly higher than that in the control group, and the apoptosis rate of stem cells is the lowest, which confirms that the umbilical cord mesenchymal stem cells prepared by the present invention can effectively restore the liver index, improve the survival rate of liver cells, and repair damaged liver tissue. In addition, by further analyzing the changes in the levels of AST, AKP, TC, TG, LDL-C and HDL-C in mouse serum, it can be concluded that the levels of AST, AKP, TC, TG, and LDL-C in the mice in the treatment group of the present invention are significantly lower than those in the control group, while the HDL-C level is significantly increased, further confirming that the umbilical cord mesenchymal stem cells prepared by the present invention have good liver repair ability and can be used to treat patients with acute liver injury.

[0045] Table 1 Liver index and hepatocyte apoptosis rate

[0046] Liver index Hepatocyte apoptosis (%) Blank group 6.23±0.20 89.65±2.68 Control group 4.98±0.56 56.89±3.58 Treatment Group 3.56±0.12 5.89±0.12

[0047] Table 2 Serum AST, AKP, TC, TG, LDL-C and HDL-C levels

[0048] ALT (U / L) AST (U / L) AKP (King Unit / 100mL) TG (mmol / L) TC (mmol / L) LDL-C (mmol / L) HDL-C (mmol / L) Blank group 1.12±0.23 1.59±0.36 2.89±0.69 0.98±0.06 3.56±0.25 2.23±0.18 0.60±0.32 Control group 0.62±0.55 1.02±0.13 1.35±0.25 0.69±0.08 2.15±0.24 1.56±0.16 0.88±0.21 Treatment Group 0.32±0.06 0.68±0.24 0.80±0.15 0.30±0.02 1.62±0.05 0.45±0.05 1.56±0.28

[0049] The above embodiments are intended to illustrate the essential content of the present invention, but are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solution of the present invention.

Claims

1. Use of miR-3059-5p in preparing a drug for preventing or treating liver disease, characterized in that: The sequence of miR-3059-5p is shown in SEQ ID NO.1, and the liver disease is acute liver injury.

2. Use of mesenchymal stem cells in the preparation of a drug for preventing or treating liver disease, wherein: The liver disease is acute liver injury, wherein the mesenchymal stem cells are transfected with miR-3059-5p and specifically overexpress miR-3059-5p; the sequence of the miR-3059-5p is shown in SEQ ID NO.

1.

3. The use according to claim 2, characterized in that The mesenchymal stem cells are selected from umbilical cord mesenchymal stem cells.

4. The use according to claim 2, characterized in that The mesenchymal stem cells are administered by intravenous injection.

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