Method for improving detoxification function of hepatocyte-like cells derived from human stem cells and application of method

A technology of liver-like cells and detoxification function, applied in the field of biomedicine, can solve the problems of the regulation mechanism that needs further research, low activity, inconsistency, etc., and achieves the effect of improving the detoxification function, improving the activity, and solving the problem of the detoxification function.

Active Publication Date: 2019-04-05
THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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  • Summary
  • Abstract
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  • Application Information

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Problems solved by technology

Interestingly, studies have shown that the expression levels of CYP2E1 and CYP3A4 show obvious inconsistencies at the mRNA and protein levels, and show low activity levels, which indicates that they are likely to be regulated at the post-transcriptional level (such as miRNA regulation, etc.), but specific The regulatory mechanism needs to be further studied

Method used

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  • Method for improving detoxification function of hepatocyte-like cells derived from human stem cells and application of method
  • Method for improving detoxification function of hepatocyte-like cells derived from human stem cells and application of method
  • Method for improving detoxification function of hepatocyte-like cells derived from human stem cells and application of method

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Example 1 A method for improving the detoxification function of human stem cell-derived hepatic-like cells

[0048] Transfection of miR-142-3p inhibitor to regulate CYP2E1 and CYP3A4 enzyme activity during hepatic differentiation of stem cells. However, it is necessary to determine the optimal transfection concentration of miR-142-3p inhibitor, the specific method is as follows:

[0049] 1. Prepare Matrigel-differentiation culture plates. Thaw Matrigel (Corning, 356230) slowly on ice. Take 25ml of ice-cold DMEM-F12 (without HEPES, Invitrogen 11320074), add 400μl of Matrigel (final concentration: 1.6%) and mix well, then take 0.5ml and add to each well of a 12-well plate (Sigma CLS3512). Incubate for at least 30 min at 37 °C or 2-3 h at room temperature to allow Matrigel to clot.

[0050] 2. Preparation of human ESC / iPSC cells. Human ESC / iPSC cells used for differentiation were cultured in a 37°C incubator containing 21% oxygen and 5% carbon dioxide. The medium was t...

Embodiment 2

[0067] Example 2 Inhibition of miR-142-3p in human-derived hepatic-like cells can improve the function of hepatic-like cells

[0068] The liver-like cells induced by the method of Example 1, after inhibiting miR-142-3p, greatly increased the expression levels of their mature cell-related marker genes, such as ALB, ARG1, PCK1, CPT1, CYP3A4 and CYP2E1 ( Figure 4 shown). also increased the protein expression level of albumin ( Figure 5 shown), and albumin and urea synthesis levels ( Figure 6 shown).

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Abstract

The invention belongs to the field of biological medicine, and particularly relates to a method for improving a detoxification function of hepatocyte-like cells derived from human stem cells and application of the method. The method is characterized in that the stem cells are transfected with miR-142-3p inhibitors during stem cell hepatic differentiation, and the nucleotide sequence of the miR-142-3p inhibitors is shown as SEQ ID No.1. After differentiation of the stem cells, the miR-142-3p inhibitors are transfected, after transfection, differentiation is performed and molecular and functional testing of the hepatocyte-like cells is detected, it is found that on the 8th day of stem cell hepatic differentiation, 10nM of the miR-142-3p inhibitors are transfected, and enzyme activity of CYP2E1 and CYP3A4 is the highest. The method overcomes the disadvantage of low detoxification ability of the hepatocyte-like cells derived from the human stem cells, facilitates the development of an artificial liver and the in-depth study of medical research and development by using the hepatocyte-like cells derived from the human stem cells or the artificial liver.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a method for improving the detoxification function of hepatic-like cells derived from human stem cells and an application thereof. Background technique [0002] Liver disease is a global disease with many types, wide distribution and high degree of harm. In my country, liver disease is also one of the diseases with the largest number of patients. According to the report of the World Health Organization in 2011, about 500,000 people in my country die from various liver diseases every year. At present, the most effective treatment for advanced liver disease and acute severe liver disease is whole liver transplantation, but the extreme shortage of donor liver sources limits its wide application. In recent years, researchers have proposed a variety of options to replace whole liver transplantation, such as artificial liver support system, hepatocyte transplantation, transplant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N5/10
CPCC12N5/0606C12N5/067C12N15/90C12N2501/113C12N2501/12C12N2501/237C12N2510/00
Inventor 陈叶苗肖靖芳田甘丁建强卞修武
Owner THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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