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A method for improving the detoxification function of human stem cell-derived hepatic-like cells and its application

A technology of liver-like cells and detoxification function, which is applied in the field of biomedicine, can solve the problems of low activity, inconsistency, and the regulation mechanism needs to be further studied, and achieve the effect of improving activity, improving detoxification function, and solving the problem of detoxification function

Active Publication Date: 2021-03-12
THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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  • Summary
  • Abstract
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  • Claims
  • 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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  • A method for improving the detoxification function of human stem cell-derived hepatic-like cells and its application
  • A method for improving the detoxification function of human stem cell-derived hepatic-like cells and its application
  • A method for improving the detoxification function of human stem cell-derived hepatic-like cells and its application

Examples

Experimental program
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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 its albumin ( Figure 5 shown), and albumin and urea synthesis levels ( Image 6 shown).

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Abstract

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. It is characterized in that the miR-142-3p inhibitor is transfected during the hepatic differentiation of stem cells, and the nucleotide sequence of the miR-142-3p inhibitor is shown in SEQ ID No.1. After differentiation of stem cells, transfection of miR-142-3p inhibitor, after transfection differentiation and molecular and functional detection of liver-like cells, it was found that 10nM miR-142-3p inhibitor was transfected on the 8th day of stem cell hepatic differentiation , the enzyme activities of CYP2E1 and CYP3A4 are the highest. The method overcomes the disadvantage of low detoxification ability of hepatic cells derived from human stem cells, and is beneficial to the development of artificial livers and the in-depth research of pharmaceutical research and development using hepatic cells derived from human stem cells or artificial livers.

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 Patents(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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