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A method for inhibiting epithelial-mesenchymal transition of human amniotic membrane epithelial cells

A technology of epithelial cells and human amniotic membrane, applied in the biological field, can solve problems such as no effective inhibitors have been found

Active Publication Date: 2022-01-07
WUXI CELLULAR BIOPHARM GRP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for human amniotic epithelial cells, no effective inhibitors have been found that can be used to inhibit the epithelial-mesenchymal transition in vitro

Method used

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  • A method for inhibiting epithelial-mesenchymal transition of human amniotic membrane epithelial cells
  • A method for inhibiting epithelial-mesenchymal transition of human amniotic membrane epithelial cells
  • A method for inhibiting epithelial-mesenchymal transition of human amniotic membrane epithelial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Embodiment 1: Screening of the drug concentration of SB431542

[0103] 1.1 Proliferation curve of human amniotic epithelial cells with different concentrations of SB431542

[0104] In this example, using CCK8 as a marker of cell proliferation, the daily metabolic OD values ​​of P2 generation human amniotic epithelial cells under the influence of different concentrations of drugs were detected.

[0105] The result is as figure 1 As shown in A. From the experimental data, 50 μM SB421542 has a significant inhibitory effect on cell proliferation, while low doses of SB431542 (0 μM, 1 μM, 5 μM, 10 μM) have no inhibitory effect on cell proliferation.

[0106] 1.2 Effects of different concentrations of SB431542 on gene expression in human amniotic epithelial cells

[0107] In order to screen out the optimal drug concentration, in this embodiment, qPCR was also used to detect the effect of SB431542 on the expression levels of each gene in the P2 generation human amniotic epit...

Embodiment 2

[0110] Example 2: Effect of 5 μM SB431542 on the proliferation of human amniotic epithelial cells

[0111] In this embodiment, the number of cells with 90% confluence and the time (h) of each generation were recorded, and the time required for cell doubling of each generation was calculated. Among them, the control group is the cells without drug addition, and the experimental group is the cells treated with 5 μM SB431542.

[0112] Experimental results such as figure 2 shown. The experimental data showed that the cell doubling time of P1-P6 human amniotic epithelial cells with and without drug was basically the same. In the P7 generation, the doubling time of cells with drugs was 43.5h, and that of cells without drugs was 61.4h.

[0113] Therefore, 5 μM SB431542 can maintain the proliferation of amnion epithelial cells for 7 passages.

Embodiment 3

[0114] Example 3: Effects of 5 μM SB431542 on gene expression levels of human amniotic epithelial cells

[0115] In this embodiment, the expression levels of each gene in the P1, P3, and P7 generations of human amniotic epithelial cells in the control group and the experimental group were detected by qPCR.

[0116] The control group was cells not affected by drugs, and the experimental group was cells treated with 5 μM SB431542. The markers used were epithelial markers keratin 8 (CK8) and 19 (CK19), stem cell markers OCT4 and KLF4, and mesenchymal stem cell markers Vimentin and ACTA2 (α-SMA).

[0117] Experimental results such as image 3 shown. From the experimental data, there were significant differences in the gene expression levels of CK8, CK19, KLF4, OCT4, Vimentin and ACTA2 (α-SMA) between the control group and the experimental group (P<0.05). In particular, at the P7 generation, the expression levels of CK8, CK19, KLF4, and OCT4 in the experimental group cells were ...

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Abstract

The invention provides a method for inhibiting epithelial-mesenchymal transition of human amniotic membrane epithelial cells. Specifically, the present invention provides a method for inhibiting epithelial-mesenchymal transition (EMT) of human amniotic epithelial cells in vitro, comprising the steps of: (i) culturing human amniotic epithelial cells in a culture system in which the compound of formula I exists. In the method screened by the present invention, the compound has a significant effect on inhibiting the epithelial-mesenchymal transition of human amniotic membrane epithelial cells, and it has no effect on the cell viability and proliferation ability of the cells, and has less toxic side effects on cells; and, The present invention verifies from the level of gene expression and protein expression that the compound of formula I can maintain epithelial phenotype, stem cell characteristics and inhibit mesenchymal cell characteristics on human amniotic epithelial cells.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a method for inhibiting epithelial-mesenchymal transition of human amniotic membrane epithelial cells. Background technique [0002] The amnion is a thin membrane that lines the inside of the placenta. Human amniotic epithelial cells are cells derived from the amniotic membrane. Human amniotic epithelial cells are one of the perinatal stem cells that develop from the inner cells of the embryo. Human amniotic epithelial cells have the ability to differentiate into embryonic stem cells, and can differentiate into three germ layers in a specific environment. In addition, human amniotic epithelial cells have unique immunomodulatory functions and stem cell-like immunomodulatory properties. Human amniotic epithelial cells express a variety of surface markers of embryonic stem cells: tumor rejection antigens TRA1-60, TRA1-81, stage-specific antigens SSEA-3, SSEA4, pluripotent ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N5/073
CPCC12N5/0605C12N5/0625C12N2501/999
Inventor 李萍王静戴成祥刘必佐沈美萍李苏克
Owner WUXI CELLULAR BIOPHARM GRP LTD
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