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A method of transforming fibroblasts into immortalized cells and its application

A fibroblast, immortalization technology, used in biochemical equipment and methods, animal cells, vertebrate cells, etc.

Active Publication Date: 2021-07-02
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after years of research, it has been found that although the mechanism of immortalization is similar, the same immortalization method is not applicable to all cells

Method used

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  • A method of transforming fibroblasts into immortalized cells and its application
  • A method of transforming fibroblasts into immortalized cells and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Transformation from fibroblasts to immortalized cells

[0041] Taking a 12-well plate as an example (corning, 3335), each well was prepared with 20 μg / mL matrigel solution (BD, 354277) 1×DMEM, coated for 12 hours, and washed once with 1×PBS after removing the coating solution.

[0042] Mouse embryonic fibroblasts (prepared from C57, E13.5) or tail tip fibroblasts (prepared from one week after birth or from adult mice) were evenly seeded in each well, 2×10 per well 4 Each cell was cultured for 24 hours with basal medium (high glucose DMEM (Gibco, C12430500BT), double antibody) plus 10% fetal bovine serum (Gibco, 16000-044). Remove the culture medium and wash again with PBS.

[0043] The above-mentioned fibroblasts were added to the immortalization induction medium: (N2B27 medium: DMEM / F12 (gibco, 10565018) and Neurobasal (Gibco, 21103-049) were mixed at a ratio of 1, and N2 additive (100×, Gibco, 17502048), B27 additive (50×, Gibco, 17504044), 2% bovine seru...

Embodiment 2

[0048] Example 2: Detection of Proliferation and Senescence Genes

[0049] The experimental method is as follows: Immortalized cells SMPC (mouse embryonic fibroblasts as control) were collected. (a) RNA was extracted by kit method. Add an appropriate amount of TRIzol to the cell pellet to lyse the cells, add 1 / 5 volume of chloroform, vortex and mix well, let stand on ice for 3 minutes, centrifuge at 10000g, 4°C for 15 minutes. Transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of 75% ethanol and transfer it to the adsorption column, centrifuge at 10,000 g for 15 seconds, discard the liquid in the collection tube, wash with Wash Buffer I once, and add 10 μL DNase I+70 μL Buffer RDD, incubate at room temperature for 15 minutes to digest DNA, then wash once with 350 μL Wash Buffer I, once with 500 μL Wash Buffer II, and elute RNA with RNase-Free Water. Concentrations were determined with a UV / Vis spectrophotometer. (b) Reverse transcription of RNA i...

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Abstract

The invention relates to a method for inducing fibroblasts to transform into immortalized cells and its application. The method comprises the following steps: cultivating fibroblasts in culture medium, adding Myosin inhibitor to the culture medium, and continuing to cultivate until immortalized cells are obtained. The present invention also relates to a medium for inducing transformation of fibroblasts into immortalized cells. The method of the present invention only needs a single small molecule or single factor treatment, simple operation, good repeatability, and can be performed efficiently in vivo and in vitro, does not involve transgenic operation, and the obtained immortalized cells are safe, and are suitable for understanding the law of cell growth , Exploring the causes of cell aging and solving the problem of organ transplantation can also lay a certain foundation for treating tumors and controlling the proliferation of tumor cells, which is of great significance.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for transforming fibroblasts into immortalized cells and its application. Background technique [0002] Somatic cells derived from normal tissues can grow and divide under normal in vitro culture conditions, but after a limited number of cell passages, they will stop proliferating, senescence and die, which limits the application of cell culture technology. Cell immortalization refers to the fact that cells escape from the crisis of proliferation and aging due to their own gene changes or various external stimuli during in vitro culture, avoiding the aging and death process of normal cells, and can be subcultured for a long time and unlimited Division and proliferation. [0003] A large number of studies have been carried out on the mechanism and method of cell immortalization, and it has been confirmed that radioactive factors, telomerase activation, viral gene transfectio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077
CPCC12N5/0656C12N2501/999C12N2510/04
Inventor 周琪李伟何正泉王柳
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI