Zhang Qin-li, Niu Pi-ye, Niu Qiao
Department of Occupational Health, School of Public Health, Shanxi Medical University, Taiyuan 030001 China.
Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2008 Feb;26(2):65-71.
To find the optimal design of small interfering RNA compounds, transfection concentration and transfection time to reduce the Al-induced apoptosis in SH-SY5Y cells.
Three siRNA sequences on bak gene were designed and transfected into SH-SY5Y cells, which were treated at various concentrations of aluminum. Cell viability was detected by CCK-8 kit on different siRNA sequences, various transfection concentrations, and diverse transfection courses. Transfection efficiency was determined by fluorescent staining of CY3, and interference efficiency was measured by QRT-PCR. Besides, immunohistochemical staining was used to express Bak protein content. Finally, apoptotic rate and necrotic rate in Al treated SH-SY5Y cells transfecting by the selected bak siRNA 1 were detected.
Based on the viability of siRNA sequences, siRNA 1 was selected as the optimal siRNA sequences. The optimal transfection concentration was 10 nmol/L, and the optimal time course was 24 h after transfection. The transfection efficiency was above 90% and the interference efficiency with bak gene was 57.76%. Furthermore, there was significant transfection effect on Bak protein. The apoptotic rate in Al treated SH-SY5Y cells were significantly decreased by bak siRNA 1 transfection.
Apoptosis is one of the major cell death pathways in SH-SY5Y cells induced by aluminum. When chemically synthesized siRNA is inducted to neural cells, it can significantly reduce bak gene level, decrease Bak protein expression and apoptotic rate, which may serve as the basis for preventing neural cells apoptosis and inhibiting the development of neurodegenerative diseases.
寻找小干扰RNA化合物的最佳设计、转染浓度和转染时间，以减少铝诱导的SH-SY5Y细胞凋亡。
设计针对bak基因的三条siRNA序列，并将其转染至SH-SY5Y细胞，用不同浓度的铝处理这些细胞。采用CCK-8试剂盒检测不同siRNA序列、不同转染浓度和不同转染时间下的细胞活力。通过CY3荧光染色测定转染效率，采用QRT-PCR检测干扰效率。此外，用免疫组化染色检测Bak蛋白含量。最后，检测经所选bak siRNA 1转染的铝处理SH-SY5Y细胞的凋亡率和坏死率。
根据siRNA序列的活力，选择siRNA 1作为最佳siRNA序列。最佳转染浓度为10 nmol/L，最佳转染时间为转染后24小时。转染效率高于90%，对bak基因的干扰效率为57.76%。此外，对Bak蛋白有显著的转染效果。经bak siRNA 1转染后，铝处理的SH-SY5Y细胞的凋亡率显著降低。
凋亡是铝诱导的SH-SY5Y细胞主要的细胞死亡途径之一。当化学合成的siRNA导入神经细胞时，可显著降低bak基因水平，减少Bak蛋白表达和凋亡率，这可能为预防神经细胞凋亡和抑制神经退行性疾病的发展提供依据。
