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目的采用RNA干扰技术沉默CCS(copper chaperone for SOD1)基因,构建相关小干扰RNA(siRNA),探索出针对CCS的高效siRNA序列。方法合成用于人脐静脉内皮细胞(HUVEC)细胞中沉默CCS基因的siRNA。应用脂质体转染的方法在HUVEC细胞中对CCS基因进行RNA沉默。蛋白免疫印迹Western blotting检测沉默前后CCS蛋白表达变化的情况,甲基四唑蓝法MTT检测转染前后细胞活力。最后用单因素方差分析对数据进行统计学分析,以确定有效的siRNA序列。结果转染前后细胞形态无肉眼可见变化,转染后细胞活力分别为98.5%和98.8%。CCS蛋白沉默率分别为63.7%和61.4%。结论采用siCCS-2和siCCS-3序列转染条件对HUVEC细胞活力损伤小,CCS沉默效率高,实验条件稳定,重复性好。为我们继续研究沉默CCS后抑制血管内皮细胞的生长增殖、血管形成提供了稳定的实验基础。
Objective To silence CCS (copper chaperone for SOD1) gene by RNA interference and construct small interfering RNA (siRNA) to explore efficient siRNA sequences targeting CCS. Methods siRNA for silencing CCS gene in human umbilical vein endothelial cells (HUVEC) cells was synthesized. The CCS gene was silenced in HUVEC cells by liposome transfection. Western blotting was used to detect the changes of CCS protein expression before and after silencing. MTT assay was used to detect cell viability before and after transfection. Finally, the data were statistically analyzed using one-way ANOVA to determine valid siRNA sequences. Results There was no macroscopic change in cell morphology before and after transfection. The cell viability after transfection was 98.5% and 98.8% respectively. CCS protein silencing rates were 63.7% and 61.4%. CONCLUSION: The transfection conditions of siCCS-2 and siCCS-3 sequences have little effect on the cell viability of HUVECs, and the CCS silencing efficiency is high. The experimental conditions are stable and the repeatability is good. As we continue to study the inhibition of vascular endothelial growth and proliferation after vascular endothelial cell proliferation, angiogenesis provides a stable experimental basis.