Lu Na, Li Cheng-Zhang, Luo Xiao-Qiu, Yang Kun-Li
Department of Physiology and Neurobiology, Xinxiang Medical University, Xinxiang 453003, China.
Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2018 May 8;34(5):464-469. doi: 10.12047/j.cjap.5661.2018.105.
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.
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.
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.
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.
通过构建稳定转染的沉默Beclin-1基因的SH-SY5Y细胞系，观察缺氧对Beclin-1基因敲低的SH-SY5Y细胞自噬的影响。
构建Beclin-1shRNA慢病毒载体和阴性对照慢病毒载体；将载体转染至SH-SY5Y细胞；然后通过RT-PCR检测Beclin-1 mRNA的表达，通过蛋白质免疫印迹法检测Beclin-1蛋白水平。采用CCK-8法测定Beclin-1基因敲低对SH-SY5Y细胞活力的影响。接下来，将空白对照、阴性对照和转染细胞分别在21%常氧和5%缺氧条件下培养。通过蛋白质免疫印迹法检测各组LC3蛋白的表达，并通过电子显微镜观察自噬体。
Beclin-1 shRNA显著抑制SH-SY5Y细胞中Beclin-1 mRNA和蛋白的表达；沉默Beclin 1基因后，Beclin-1 shRNA组细胞的存活率与阴性对照（NC）组无差异。5%缺氧处理后，与NC组相比，Beclin-1 shRNA组中LC3Ⅱ/LC3Ⅰ的比值和自噬体数量均减少。
成功建立了Beclin-1基因敲低的SH-SY5Y细胞系和阴性对照细胞系。慢病毒介导的Beclin-1 shRNA对SH-SY5Y细胞的活力无影响，但可抑制缺氧诱导的自噬。
