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
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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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