动物造模 z-bio 2022-08-09 523

　　OBJECTIVE: To inhibit the expression of autophagy-regulating gene Beclin 1 by RNA interference technology, and to detect the effect of Beclin 1 expression on the proliferation and apoptosis of naked mole rat skin fibroblasts and the expression of p53, BAX, Bcl2 and other genes.

　　Methods: The expression of Beclin 1 in naked mole rat fibroblasts after starvation and H2O2 stimulation were detected respectively, and then the designed interfering RNA of Beclin 1 gene and negative control were used to transiently transfect naked mole rat fibroblasts respectively. Real-time PCR and Western blot were used to detect the effect of silencing, CCK-1 assay was used to detect the cell proliferation activity after silencing, flow cytometry was used to detect cell apoptosis, and Western blot was used to detect the expression levels of related genes and proteins.

　　RESULTS: Both starvation and H2O2 stimulation could induce changes in the expression level of Beclin 1. The transfection efficiency of naked mole rat skin fibroblasts by gene expresso transfection reagent can reach more than 90%. The results of real-time PCR and Western blot show that the designed Beclin 1 siRNA can effectively reduce the expression of Beclin 1. After Beclin 1 gene silencing, the proliferation inhibition rate of naked mole rat skin fibroblasts was significantly higher than that of the control group, and the early and late apoptosis rates of cells were significantly increased, while p53, BAX, Bcl2, LC3B, p-AKT, The expression levels of mTOR, etc. decreased.

　　Conclusion: The expression of Beclin 1 changes significantly in the process of naked mole rat fibroblasts resisting starvation and H2O2 stimulation. Simultaneously inhibiting the expression of Beclin 1 can inhibit the proliferation of naked mole rat cells and promote their apoptosis. Autophagy, proliferation and apoptosis of naked mole rats play a regulatory role.