Beclin-1 shRNA对低氧诱导的SH-SY5Y细胞自噬的影响

目的：构建Beclin-1基因短发夹干扰RNA（shRNA）慢病毒载体，感染人SH-SY5Y细胞，观察沉默Beclin-1基因后低氧对SH-SY5Y细胞自噬的影响。方法：构建特异性靶向Beclin-1基因的shRNA慢病毒表达载体和阴性对照序列慢病毒载体；再将载体转染入SH-SY5Y细胞；RT-PCR检测Beclin-1的mRNA表达；Western blot检测Beclin-1蛋白表达；CCK-8法测定Beclin-1 shRNA对SH-SY5Y细胞活力的影响。再将空白对照、阴性对照、转染型三种细胞分别以21%常氧及5%低氧培养，Western blot检测各组细胞LC3蛋白表达；电镜观察自噬小体。结果：Beclin-1 shRNA能明显抑制SH-SY5Y细胞Beclin-1的mRNA及蛋白的表达；沉默Beclin-1基因后，Beclin-1 shRNA组细胞存活率与阴性对照组相比无差异；成功建立了稳定表达Beclin-1 shRNA的SH-SY5Y细胞。5%低氧处理后，与阴性对照组相比较，Beclin-1 shRNA组细胞中LC3Ⅱ/LC3Ⅰ比值下调，细胞内自噬小体数量减少。结论：慢病毒介导的Beclin-1shRNA对SH-SY5Y细胞的活力无影响，但可以抑制低氧诱导的自噬。

Effect of Beclin-1 shRNA on hypoxia-induced autophagy in SH-SY5Y cells

Objective: To observe the effect of hypoxia on autophagy in Beclin-1-knockdown SH-SY5Y cells by constructing a stable transfected SH-SY5Y cell lines of silencing Beclin-1 gene. Methods: Beclin-1shRNA lentiviral vector and negative control lentiviral vector were constructed; the vector was transfected into SH-SY5Y cells; then the expression of Beclin-1 mRNA was detected by RT-PCR, the level of Beclin-1 protein was detected by Western blot. CCK-8 method was used to determine the effect of Beclin-1 knockdown on the viability of SH-SY5Y cells. Next, the blank control, negative control and transfected cells were cultured under 21% normoxia and 5% hypoxia conditions. The expression of LC3 protein in each group was detected by Western blot and the autophagic bodies were observed by electron microscopy. Results: Beclin-1 shRNA significantly inhibited the expression of Beclin-1 mRNA and protein in SH-SY5Y cells; after silencing Beclin 1 gene, the survival rate of Beclin-1 shRNA group cells was no different from that of negative control (NC) group. After 5% hypoxia treatment, compared with NC group, the ratio of LC3Ⅱ/LC3Ⅰand the number of autophagy bodies were all decreased in Beclin-1 shRNA group. Conclusion: Beclin-1 knockdown SH-SY5Y cell lines and negative control cell lines were successfully established. Lentivirus-mediated Beclin-1 shRNA has no effect on the viability of SH-SY5Y cells, but can inhibit hypoxia-induced autophagy.