Mesenchymal stem cells combined with traditional Chinese medicines for activating amplification and preparation method and application of mesenchymal stem cells

A mesenchymal stem cell and traditional Chinese medicine technology, applied in mesenchymal stem cells and its application fields, can solve the problems of mesenchymal stem cell activity and anti-HBV infection ability, and cannot repair damaged liver cells well, so as to achieve anti-HBV Improvement of infection ability, reduction of liver damage, and strong cell activity

Active Publication Date: 2016-10-05
SIMPSON INT LIFE TECH (TIANJIN) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Aiming at the above-mentioned problems existing in the prior art, the present invention provides a combination of traditional Chinese medicine activated and expanded mesenchymal stem cells and its preparation method and its application in the preparation of drugs for liver fibrosis cell transplantation, which can effectively solve the problem of mesenchymal stem cell activity. And the ability to resist HBV infection is not strong, and the problem of damaged liver cells cannot be well repaired

Method used

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  • Mesenchymal stem cells combined with traditional Chinese medicines for activating amplification and preparation method and application of mesenchymal stem cells
  • Mesenchymal stem cells combined with traditional Chinese medicines for activating amplification and preparation method and application of mesenchymal stem cells
  • Mesenchymal stem cells combined with traditional Chinese medicines for activating amplification and preparation method and application of mesenchymal stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1 Preparation of activated and expanded mesenchymal stem cells combined with traditional Chinese medicine

[0026] A mesenchymal stem cell activated and expanded in combination with traditional Chinese medicine, the preparation method of which comprises the following steps:

[0027] (1) Pretreatment is the separation of mesenchymal stem cells: under sterile conditions, wash the human umbilical cord with PBS buffer 2-3 times, cut it into pieces, and obtain 1-3cm 3 Tissue fragments were then mixed with low-sugar DMEM (Gibco) mixture containing trypsin (w / v 0.05%) and type IV collagenase (1.5mg / mL) at a volume ratio of 1:3, Digest at 37°C for 1-2h, then add hyaluronidase at a final concentration of 25ug / mL for 5-10min, centrifuge at 100-200r / min for 5-10min, discard the supernatant, and culture in low-sugar DMEM containing 10% FBS The base was washed and precipitated once, centrifuged at 100-200r / min for 5-10min, the supernatant was removed, and the precipitate wa...

Embodiment 2

[0033] Embodiment 2Q-PCR detects the expression of microRNAs

[0034] The relative expression of anti-virus-related microRNAs (miR-122, miR-141, miR-199a, miR-210) in the mesenchymal stem cells activated and amplified by combined traditional Chinese medicine obtained in Example 1 was detected by Taqman probe method.

[0035] The specific method is as follows: respectively extract the RNA of the Chinese medicine treatment group (the mesenchymal stem cells activated and amplified by the combined traditional Chinese medicine prepared by the present invention) and the control group mesenchymal stem cells in Example 1, and reverse transcribe to obtain cDNA; use cDNA as a template , perform Q-PCR reaction under the guidance of specific primers and probes; use MicroRNA Assay kit, the reaction system is: 0.67 μL cDNA, 1 μL 10×Taqman Universal PCR master mix, 1 μL primers and probe mix, 7.33 μL ddH 2 O, the total volume was 10 μL; the reaction conditions were: pre-denaturation at 95°...

Embodiment 3

[0056] In vitro experimental analysis of mesenchymal stem cells treated with traditional Chinese medicine to enhance liver cell resistance to HBV, the specific steps are as follows:

[0057] (1) In vitro model of HBV infection of liver cells

[0058] Primary human hepatocytes (primary human hepatocytes, PHH) and HepG2-hNTCP-C4 cell line were used as experimental objects, and 10 g.e / cell and 100 g.e / cell (genomeequivalents per cell) of HBV were added to the cells, respectively. Add 4% PEG 8000 to the culture medium, and 1 / 2 volume of mesenchymal stem cell culture supernatant (MSC) without traditional Chinese medicine treatment or 1 / 2 volume of mesenchymal stem cell supernatant (MSC- CM), cultured at 37°C for 24 hours;

[0059] (2) Q-PCR detection of HBV-related DNA

[0060] The specific method is: collect hepatocytes in step (1), use cell lysate (pH7.4, Tris-HCl with a concentration of 50mM, 1mM EDTA, 1% NP-40) to lyse, and centrifuge at 15000r / min for 5min (4°C ), the super...

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Abstract

The invention provides mesenchymal stem cells combined with traditional Chinese medicines for activating amplification, and a preparation method of the mesenchymal stem cells comprises the following steps: (1) pretreatment: digesting tissue pieces with pancreatin and IV collagenase, separating primary mesenchymal stem cells; (2) culture: adding the cells obtained in the step (1) to a low sugar DMEM culture medium containing components of 50-80mg/mL hemianthus micranthemoides extract, 50-80mg/mL Chinese gall extract, 1.5-3mmol/mL L-glutamine and 1.5-3ng/mL bFGF, culturing in a wet incubator of 37 DEG C with 5% CO2, changing the culture medium once every 3-4 days; and (3) collection of cells and subculture. Shown by in vitro tests, the traditional Chinese medicines activated mesenchymal stem cells can effectively inhibit reproduction of HBV and promote repair of damaged hepatic cells.

Description

technical field [0001] The invention belongs to the technical field of mesenchymal stem cells and their application, and in particular relates to a mesenchymal stem cell activated and expanded by combining traditional Chinese medicine, a preparation method and an application thereof. Background technique [0002] Liver cirrhosis is an important stage in the progress of many chronic liver diseases. In my country, viral hepatitis is the main cause of liver cirrhosis. The HBsAg positive rate of stem cells in patients with liver cirrhosis is as high as 76.7%. The common virus that causes liver cirrhosis is hepatitis B HBV or hepatitis C virus HCV, and the HBV infection rate in our country has been as high as 10%. Develop liver cirrhosis, of which 50,000-200,000 may develop liver cancer. The main pathogenesis of liver cirrhosis is based on the long-term chronic inflammation and death of liver cells caused by viruses invading the liver, and the development of liver tissue fibrosi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K35/28A61P1/16
CPCA61K35/28C12N5/0668C12N2500/34C12N2500/76C12N2501/115
Inventor 王仲
Owner SIMPSON INT LIFE TECH (TIANJIN) CO LTD
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