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Method for autocrine secretion of extracellular matrix by stem cells and induction of stem cells to become hepatocytes

A technology of stem cells and extracellular matrix, applied in the field of stem cell differentiation, can solve the problems of inability to meet basic research and clinical treatment, affect the physiological function of cultured cells, and poor differentiation effect, and achieve good urea synthesis ability, good differentiation effect, and glycogen. The effect of strong synthesis ability

Inactive Publication Date: 2013-11-20
THE THIRD AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, there are many ways to induce BMSCs to differentiate into hepatocytes, but most of them are to inoculate BMSCs on ordinary culture substrates. Under this two-dimensional condition, the cells can only grow in a single layer, the cell density is low, and the differentiation effect is not good. This greatly affects the physiological functions of cultured cells, making differentiated cells only have some functions of hepatocytes, which cannot meet the needs of basic research and clinical treatment.

Method used

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  • Method for autocrine secretion of extracellular matrix by stem cells and induction of stem cells to become hepatocytes
  • Method for autocrine secretion of extracellular matrix by stem cells and induction of stem cells to become hepatocytes
  • Method for autocrine secretion of extracellular matrix by stem cells and induction of stem cells to become hepatocytes

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Induced production of hepatocytes

[0036] S1. Cross-linking modification of gelatin coating:

[0037] First, add 0.2% gelatin solution to the 12-well plate and place it at 37°C, 5% CO 2 Let stand in the incubator for 1 hour, then add 1% glutaraldehyde solution and 1M ethanolamine solution, and let stand at room temperature for 30 minutes.

[0038] S2. Induce bone marrow mesenchymal stem cells to secrete extracellular matrix, the process is as follows figure 1 Shown:

[0039] Bone marrow mesenchymal stem cells were treated at 3,000 cells / cm 2 The density was inoculated into gelatin cross-linked cell culture substrate containing complete medium, complete medium was (α-MEM basal medium, 10% fetal bovine serum, 100U / ml penicillin, 100U / ml streptomycin and 0.25 μg / ml amphotericin B). When the cell density reached 90% confluence, 50 μg / mL ascorbic acid was added to the culture medium to continue the culture. To obtain decellularized ECM, the ECM secreted by b...

Embodiment 2

[0043] Example 2 Microscopic observation of extracellular matrix

[0044] The ECM was first observed and photographed under a light microscope, and then after being fixed with 4% paraformaldehyde and dehydrated with gradient alcohol (50%, 75%, 80%, 95%, 100%), the morphology and structure were observed under an electron microscope. The results are as follows image 3 shown.

[0045] Example 2 Fluorescence staining of extracellular matrix

[0046] Before and after the decellularization treatment, the ECM was first fixed with ice methanol, then blocked with 1% bovine serum albumin, and added primary antibodies of type I collagen, type III collagen, fibronectin, decorin, and laminin antibodies. After incubation and washing with PBS for three times, the secondary antibody was added and observed with an immunofluorescence microscope. The results were as follows: image 3 shown.

Embodiment 4

[0047] Example 4 Identification of Differentiated Cells

[0048] 1. Identification of Differentiated Hepatocytes Using Glycogen Synthesis Capacity

[0049] Cells were collected on the 14th and 28th day of induction of differentiation, first fixed with 4% paraformaldehyde, then incubated with 1% periodic acid solution at room temperature for 5 minutes, then washed with PBS, stained with magenta reagent for 15 minutes, and observed under the microscope , the result is as Figure 4 shown

[0050] 2. Identification of Differentiated Hepatocytes Using Urea Synthesis Capacity

[0051] The culture supernatants were collected on the 7th, 14th, 21st, and 28th days of induction of differentiation, and placed in a -80°C low-temperature refrigerator. After the experiment was over, the concentration of urea in the supernatant was detected by spectrophotometry. The results were as follows: Figure 5 shown.

[0052] 3. Using Real-time RT-PCR to identify the expression...

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Abstract

The invention provides an application of a method for the autocrine secretion of an extracellular matrix by stem cells and the induction of the stem cells to become hepatocytes. The extracellular matrix is secreted by inducing bone marrow mesenchymal stem cells and removes the matrix of the bone marrow mesenchymal stem cells. The invention concretely provides the method for inducing the bone marrow mesenchymal stem cells into hepatocytes by the extracellular matrix according to actual demands. The method for generating the hepatocytes through induction has the advantages of high efficiency, practicality, high induction efficiency and good differentiation effect, and the glycogen synthesis capability and the urea synthesis capability of the hepatocytes obtained after the induction are strong.

Description

technical field [0001] The invention relates to the field of stem cell differentiation, more specifically, to a method for inducing human bone marrow mesenchymal stem cells to become liver cells by using extracellular matrix. Background technique [0002] Liver failure is very harmful and has a high mortality rate, and there is no effective treatment method. At present, various treatment methods including comprehensive medical treatment, artificial liver treatment, antiviral treatment, and liver transplantation treatment have certain defects. Bone marrow mesenchymal stem cells (BMSCs) are a type of pluripotent stem cells with self-renewal and multidirectional differentiation potential. More and more animal and clinical studies have shown that BMSCs have good efficacy and safety in the treatment of end-stage liver diseases sex. The mechanism is that BMSCs can repair the liver by differentiating into hepatocytes and exerting immune regulation. [0003] At present, there ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 高志良何宏亮彭亮何帆龚逸鸿
Owner THE THIRD AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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