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Method for inducing human umbilical cord mesenchymal stem cells in vitro into liver cells and application thereof

A technology of mesenchymal stem cells and human umbilical cords, applied in the direction of artificial cell constructs, animal cells, vertebrate cells, etc., can solve the problems of complicated induction methods, and achieve good induction and differentiation effects, good differentiation effects, and short duration effects

Inactive Publication Date: 2011-08-31
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that mesenchymal stem cells have the potential to differentiate into liver cells. At present, the main induction methods include: in vitro induction (adding a variety of cytokines to the culture medium, induced by chemical drugs), co-cultivation with hepatocytes or liver non-parenchymal cells , conditional culture, or in vivo transplantation, and transgenic methods. The method of growth factor induction is more commonly used, and the method of step-by-step induction is more common. The two-step method is more common, and these induction methods are relatively complicated.

Method used

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  • Method for inducing human umbilical cord mesenchymal stem cells in vitro into liver cells and application thereof
  • Method for inducing human umbilical cord mesenchymal stem cells in vitro into liver cells and application thereof
  • Method for inducing human umbilical cord mesenchymal stem cells in vitro into liver cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Isolation, culture and identification of human umbilical cord mesenchymal stem cells

[0036] (1) Isolation and culture of human umbilical cord mesenchymal stem cells. Obtain human umbilical cord under aseptic conditions (aseptically obtained from the First Affiliated Hospital of Shantou University, the umbilical cords in this experiment were all from healthy pregnant women, with the consent of volunteers and family members), and the cells were separated within 6 hours. After immersing the human umbilical cord with 0.25% iodophor by volume for 3 minutes, wash away the blood clots on the surface and in the blood vessels with normal saline (try to completely remove the blood clots to reduce blood cell contamination), use surgical forceps to tear the umbilical cord, find two arteries and one Root vein, use tweezers to pick the Walton gel (also called umbilical cord matrix) around the blood vessel, and cut into 1~2mm 3 The size of the tissue pieces, and evenly added...

Embodiment 2

[0053] Example 2: Inducing human umbilical cord mesenchymal stem cells to differentiate into hepatocytes in vitro

[0054] The purpose of this example is to promote the differentiation of human umbilical cord mesenchymal stem cells into liver-like cells by culturing the induction medium added with inducing factors. Take the human umbilical cord mesenchymal stem cells from the 3rd to 8th generation, and use 1×10 5 The cell density of cells / well is seeded in 6 wells or 1×10 4 The cell density of the cells / well is seeded in a 24-well culture plate. When the cell density reaches 70%, the normal growth medium is discarded and replaced with a hepatocyte differentiation medium to induce differentiation to obtain hepatocytes. Hepatocyte differentiation medium is IMDM culture medium containing: 1% fetal bovine serum, 100U / ml penicillin, 100ug / ml streptomycin, 1μg / ml amphotericin, HGF (PeproTech) 40ng / ml, FGF-4 ( PeproTech) 10ng / ml, OSM (Sigma-Aldrich) 20ng / ml, bFGF (PeproTech) 10ng / ml, EG...

Embodiment 3

[0055] Example 3: Identification of differentiated cells

[0056] (1) Immunofluorescence to detect the expression of hepatocyte specific protein

[0057] According to the method in Example 2, human umbilical cord mesenchymal stem cells were cultured in the induction differentiation medium for 21 days, digested with 0.25% trypsin, and resuspended in the induction differentiation medium, and then the cells were resuspended in 1×10 4 Cells / cm 2 Inoculate in a 24-well culture plate at 37°C, 5% CO 2 Cultivate overnight in an incubator. Undifferentiated human umbilical cord mesenchymal stem cells cultured in normal growth medium were used as a negative control. Immunocytochemistry was used to detect hepatocyte-specific proteins: α-fetoprotein (AFP), albumin (ALB) and cytokeratin 18 (CK-18). After cell fixation, mouse anti-human monoclonal antibody α-fetoprotein (AFP), albumin (ALB) and cytokeratin 18 (CK-18) were added dropwise and incubated at 37°C for 1 hour, and then FITC or PE-label...

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Abstract

The invention discloses a method for inducing human umbilical cord mesenchymal stem cells in vitro into liver cells and application thereof. The method comprises the following steps of: at the culture logarithmic growth phase of human umbilical cord mesenchymal stem cells, adding a liver cell induced differentiation culture medium and performing differentiated induction to obtain the liver cells formed by the differentiation of the human umbilical cord mesenchymal stem cells. The liver cell induced differentiation culture medium consists of the basic culture medium is an IMDM culture medium containing 10 to 70 ng / ml of liver cell growth factor, 3 to 30 ng / ml of fibroblast growth factor, 4.5 to 50 ng / ml of oncostatin, 2 to 40 ng / ml of alkali fibroblast growth factor, 20 ng / ml of epidermal growth factor, 1 mu M of dexamethasone and 50 mg / ml of ITS+premix. The method has the characteristics of one step method, simplicity, high repeatability, short duration time of differentiation process and good induced differentiation effect. The method can be applied to the research on turning mesenchymal stem cells into liver cells by induced differentiation.

Description

Technical field [0001] The invention relates to the field of biotechnology for inducing differentiation of mesenchymal stem cells, in particular to a method and application for inducing human umbilical cord mesenchymal stem cells to become hepatocytes in vitro. Background technique [0002] The liver is one of the most important vital organs of the body, with functions such as metabolism, detoxification, and circulation regulation. At the same time, liver disease is also an important disease that affects human life and health. Liver cirrhosis, hepatitis, liver failure, liver cancer, etc. are all common clinical diseases, especially my country is a high-risk area of ​​viral hepatitis. The mortality rate of severe hepatitis caused by viral hepatitis can reach 50-70% or higher. For the treatment of these serious diseases, orthotopic liver transplantation is the most ideal and currently the only feasible treatment method. However, due to the lack of donor liver and xenoimmune rejecti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/071
Inventor 魏星吴征征
Owner JINAN UNIVERSITY
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