Method for inducing human placental mesenchymal stem cells to differentiate into hepatocytes in vitro and composition containing schisandrin B

A technique for schisandra B and stem cells, which is applied in the field of stem cell biology, can solve the problem that the induction method is not effective, and achieve the effects of clear ingredients, convenient extraction and improved expression level.

Active Publication Date: 2021-11-05
ZHUJIANG HOSPITAL SOUTHERN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, the differentiation technology of stem cells into hepatocytes has been continuously developed, among which changing the culture system is the main method to improve the efficiency of stem cell differentiation into liver, such as three-dimensional culture, adding small molecular compounds, adding cell matrix, etc. In additio

Method used

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  • Method for inducing human placental mesenchymal stem cells to differentiate into hepatocytes in vitro and composition containing schisandrin B
  • Method for inducing human placental mesenchymal stem cells to differentiate into hepatocytes in vitro and composition containing schisandrin B
  • Method for inducing human placental mesenchymal stem cells to differentiate into hepatocytes in vitro and composition containing schisandrin B

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Induction of Human Placental Mesenchymal Stem Cells (PDMSCs) Differentiated into Hepatocytes in Vitro

[0039] A basic method for inducing human placental mesenchymal stem cells to differentiate into hepatocytes in vitro, comprising the following three steps:

[0040] Pretreatment stage: human placental mesenchymal stem cells were induced in human mesenchymal stem cell serum-free medium containing 20ng / ml EGF (epidermal growth factor) and 10ng / ml bFGF (basic fibroblast growth factor) Cultivate for 2-3 days. Specifically:

[0041] 1) The PDMSCs cells of the P3 generation were selected and digested with a mixed liquid containing 0.25% Trypsin and 0.04% EDTA, and 1×10 4 cells / cm 2 The density was seeded in a six-well plate, and human mesenchymal stem cells were cultured in a serum-free environment at 37°C, 5% CO 2 , cultivated in a saturated humidity incubator for 24 hours. The human mesenchymal stem cell serum-free medium is prepared from human mesenchymal...

Embodiment 2

[0053] Example 2: Optimization of conditions for inducing human placental mesenchymal stem cells to differentiate into hepatocytes in vitro

[0054] In order to determine that Schisandrin B (Schisandrin B) can promote the differentiation of PDMSCs to hepatocytes, promote the expression of cytochrome enzymes, and improve the drug metabolism function of hepatocytes, the addition of Schisandrin B to the culture medium at different induction stages was designed for comparison, and the final addition was determined. Timing and dosage of Schisandrin B.

[0055] The cytotoxicity of Schisandrin B was determined by CCK-8, specifically:

[0056] 1) The cells were treated with Schisandrin B at concentrations of 0, 1, 10, 25, 50, 100, and 200 μM, cultured at 37° C. in a 5% CO2 incubator for 3 days, and the cell morphology was observed under a microscope.

[0057] 2) Remove the culture medium, add the culture medium containing 10% CCK-8 reagent under the condition of avoiding light, incub...

Embodiment 3

[0079] Example 3: Identification of Induced Differentiation Hepatocytes

[0080] According to the experimental results of Example 1 and Example 2, the optimized induction scheme was obtained, and the induced hepatocytes were further identified, and the expression of liver cell markers was identified by fluorescent quantitative PCR and cellular immunofluorescence methods, and glycogen staining, albumin Secretion test, urea synthesis test, etc. to identify liver cell function.

[0081] 1. Optimized induction program

[0082]

[0083] 2. Experimental grouping

[0084] Blank control group (MSC group): use uninduced PDMSC as blank control

[0085] Control group (HLC-A): no addition of Schisandrin B to induce PDMSC differentiation into hepatocytes was used as the control group

[0086]Experimental group (HLC-B): Add 10 μM Schisandrin B at the beginning of hepatocyte differentiation stage to induce PDMSC to differentiate into hepatocytes as the experimental group

[0087] 3. F...

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Abstract

The invention discloses a method for inducing human placenta mesenchymal stem cells to differentiate into hepatocytes in vitro. Schisandrin B is added in a hepatocellular-like induction stage, the effect of inducing the human placenta mesenchymal stem cells to differentiate into the hepatocytes with functions can be effectively improved, the used stem cells are the human placenta mesenchymal stem cells, and are rich in source, convenient to extract, clear in induction composition component and simple in induction operation, and the method has industrialization and commercialization prospects.

Description

technical field [0001] The present application relates to the field of stem cell biology, in particular to a method for inducing human placental mesenchymal stem cells to differentiate into liver cells in vitro and a composition containing schisandrin B. Background technique [0002] For end-stage liver disease, orthotopic liver transplantation is the only effective treatment, but due to severe shortage of liver donors, immune rejection after transplantation and high mortality, orthotopic liver transplantation is limited to some extent. As an alternative therapy to liver transplantation, hepatocyte transplantation has been proved to be a more feasible method in the treatment of liver failure. Compared with liver orthotopic transplantation, hepatocyte transplantation does not involve complicated operations, and is simpler to operate and less expensive, and can make up for the shortage of donor livers and other issues. However, hepatocytes cannot proliferate in large quantiti...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/0775
CPCC12N5/067C12N5/0662C12N2506/1392C12N2500/46C12N2500/62C12N2501/115C12N2501/12C12N2501/16C12N2501/237C12N2501/30C12N2500/25C12N2500/30C12N2501/11C12N2500/90
Inventor 周树勤金美先彭青高毅张莹陈琦李阳黎少梁康檐
Owner ZHUJIANG HOSPITAL SOUTHERN MEDICAL UNIV
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