Method for transdifferentiation of somatic cells into mammary epithelial cells by using small-molecule compound through in-vitro induction

A technology of mammary gland epithelial cells and small molecule compounds, which is applied in the field of cell transdifferentiation, can solve the problems of lack of lactation function and limited proliferation ability of mammary gland epithelial cells, and achieve the effect of avoiding the lack of lactation function and the limited proliferation ability in vitro

Active Publication Date: 2020-04-03
GUANGXI UNIV +1
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  • Abstract
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  • Application Information

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Problems solved by technology

[0003] However, regardless of the collagenase digestion method or the tissue block culture method, there are problems with the limited proliferation of breast epithelial cells in vitro and the lack of lactation function.
However, there have been no reports of the use of any method to induce transdifferentiation of terminally differentiated somatic cells into mammary epithelial cells in any species

Method used

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  • Method for transdifferentiation of somatic cells into mammary epithelial cells by using small-molecule compound through in-vitro induction
  • Method for transdifferentiation of somatic cells into mammary epithelial cells by using small-molecule compound through in-vitro induction
  • Method for transdifferentiation of somatic cells into mammary epithelial cells by using small-molecule compound through in-vitro induction

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Experimental program
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Effect test

Embodiment 1

[0055] The method of using small molecular compounds to induce the transdifferentiation of somatic cells into mammary gland epithelial cells in vitro and the detection experiment, the specific operations are as follows:

[0056] 1. The ear margin fibroblasts (GEFs) of black goats were isolated and cultured by the tissue-adherence method to provide cell materials for subsequent induction.

[0057] Goats aged 30-60 days were selected, and the ear skin was disinfected, and then the edge tissue pieces were cut with a scalpel, washed 2-3 times in PBS buffer containing double antibodies, high-sugar DMEM+10% FBS ( Percentage by volume) stored in medium. To process the tissue block in the laboratory, alcohol disinfection was performed first, hair and cartilage were removed in PBS buffer, and after removal, the PBS buffer was washed three times. The treated tissue pieces were placed in a 1.5mL centrifuge tube, cut to an appropriate size with ophthalmic scissors, spread evenly on a 60m...

Embodiment 2

[0068] Taking the ear margin fibroblasts of Guanzhong dairy goats as the experimental object, the concentrations of the five small molecular compounds currently used were adjusted, and the concentration of BFRTV was adjusted as a whole under the condition that the base solution remained unchanged. The results show that (such as Figure 7 ), the rest of the experimental steps and experimental parameters are the same as in Example 1; after induction, the cells can still have a cell morphology similar to BFRTV, that is to say, the concentration of BFRTV within 0.5 times to 4 times can induce it into mammary gland epithelial cells.

[0069] Through screening, it was found that using the inhibitor Repsox (R induction medium) of TGFbeta R1 alone can obtain mammary gland epithelial cells (CiMECs) similar to BFRTV induction medium induction, and then the concentration of R was screened, and the results showed (such as Figure 8 ) can produce mammary gland epithelial cells within the c...

Embodiment 3

[0071] Using gene interference technology to down-regulate the expression of TGFbeta R1 in fibroblasts can also induce the transdifferentiation of fibroblasts into mammary epithelial cells.

[0072] First, we constructed the lentiviral recombinant plasmid pSicoR-Ef1a-mCh TGFBR1 shRNA, then used Lipofectamine 3000 to co-transfect it with VSVG and NRF into 293T cells for lentiviral packaging, and finally used the packaged lentivirus to infect fibroblasts. The cells infected with lentivirus were cultured in BFTV induction medium at 37°C, 95% saturated humidity, 5% CO 2 incubator. It was found that after culturing for 8 days, mammary epithelial cells similar to those induced by BFRTV medium could be formed ( Figure 10 ). It shows that inhibiting the expression of TGFbeta R1 is the key to inducing transdifferentiated mammary epithelial cells (CiMECs) in vitro.

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Abstract

The invention provides a method for transdifferentiation of somatic cells into mammary epithelial cells by using a small-molecule compound through in-vitro induction. Expression of TGFbeta R1 and related loci thereof is inhibited, and transdifferentiation of somatic cells into mammary epithelial cells through in-vitro induction is achieved. By adopting the method, the blank of a technique for transdifferentiation of fibroblast into mammary epithelial cells through induction of the small-molecule compound is made up, and a research platform for research of in-vitro research on mammary gland bioreactors, mammary development and differentiation, breast cancer and ransdifferentiation of fibroblast into other types of functional cells is provided.

Description

technical field [0001] The invention belongs to the technical field of cell transdifferentiation, and in particular relates to a method for inducing somatic cell transdifferentiation into mammary gland epithelial cells in vitro by using a small molecule compound. Background technique [0002] Mammary gland epithelial cells are an in vitro model for studying mammary gland growth and development, lactation mechanism, and verifying the effectiveness of mammary gland tissue-specific expression vectors. At present, most primary mammary epithelial cells are cultured by collagenase digestion and tissue block culture. The purer epithelial cells can be obtained by digesting mammary gland tissue with collagenase and then undergoing density gradient centrifugation. The tissue block culture method is simple in operation, saves tissue samples, and avoids the adverse effects of digestion and centrifugation on cells. However, it takes a long time for cells to grow out of the tissue block...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0631C12N2501/15C12N2500/44C12N2533/90C12N2533/54C12N2506/08C12N2506/1307C12N2501/999
Inventor 黄奔张丹丹叶升朱少倩刘权辉覃梁珊石德顺胡吉刚谢小莲
Owner GUANGXI UNIV
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