动物实验 z-bo 2020-08-21 756

　　Objective: To use RNA interference technology to inhibit the expression of the autophagy regulatory gene Beclin1, and to detect the effect of Beclin1 expression on the proliferation and apoptosis of nude mouse skin fibroblasts and the effects of genes such as p53, BAX and Bcl2.

　　Method: Detect the expression of Beclin1 in fibroblasts of naked gerbils under conditions of starvation, H2O2 stimulation, and then use the designed Beclin1 gene interfering RNA and negative control to transiently mobilize sea buckthorn fibroblasts for transfection. Real-time PCR and Western blotting were used to detect the silencing effect, then CCK-1 experiment was used to detect cell proliferation activity, flow cytometry was used to detect cell apoptosis and Western blotting, and then the expression levels of related gene proteins were detected.

　　Result: Both starvation and H2O2 stimulation can cause changes in Beclin1 expression levels. The transfection efficiency of gene expression transfection reagent on nude mouse nevus skin fibroblasts reaches more than 90%. The results of real-time fluorescent quantitative PCR and western blotting show that the designed Beclin1 siRNA can effectively reduce the expression of Beclin1. After Beclin1 gene silencing, the growth inhibition rate of skin fibroblasts in nude mice was significantly higher than that of the control group, and the early and late apoptosis rates of cells were significantly increased. At the same time, the expression of p53, BAX, Bcl2, LC3B, p-AKT and mTOR equivalents decreased.

　　Conclusion: The expression of Beclin1 changes significantly in the process of resisting H2O2 stimulated starfish and mole rat fibroblasts. At the same time, inhibiting the expression of Beclin1 can inhibit the growth of nude mole rat rat cells, thereby promoting cell apoptosis. This indicates that the Beclin1 gene affects nude mice. Le mice play a regulatory role in autophagy, proliferation and apoptosis.