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目的:利用RNA干扰技术沉默人肝L-02细胞内可诱导的同型半胱氨酸内质网应激泛素样结构域1(homocysteine-inducible,endoplasmic reticulum stress-inducible,ubiquitin-like domain member 1,HerpUD1)基因表达,探讨其对人肝L-02细胞的辐射增敏作用。方法:根据HerpUD1基因序列设计shRNA片段,构建靶向HerpUD1基因的shRNA表达载体,稳定转染L-02细胞,采用real-time PCR和Western blotting检测转染后细胞中HerpUD1 mRNA及蛋白的表达。以8 Gy X射线照射转染后细胞,Hoechst荧光染色观察细胞的凋亡情况。结果:成功构建靶向HerpUD1的真核表达干扰载体,并建立稳定转染shHerpUD1的L-02细胞株。在稳定转染的L-02细胞中,shHerpUD1-1420干扰片段干扰效果最好,与pGPU6-NC组(阴性对照组)相比,shHerpUD1-1420组HerpUD1 mRNA表达显著下降[(0.15±0.05)vs(1.00±0.08),P<0.05],其蛋白水平的表达也显著降低[(0.19±0.01)vs(1.62±0.08),P<0.01]。转染后细胞经8 Gy X射线照射后24 h,L-02-shHerpUD1-1420组细胞凋亡率显著高于L-02-NC组(阴性对照组)[(10.23±3.37)%vs(6.89±1.49)%,P<0.01],而L-02-NC组与L-02-Neo组相比则无显著差异(P>0.05)。结论:HerpUD1基因表达沉默显著增加X射线辐射引起的L-02细胞凋亡,具有辐射增敏作用。
OBJECTIVE: To silence the inducible homocysteine-inducible (endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 in human L-02 cells by RNA interference technique 1 , HerpUD1) gene expression in human liver L-02 cell radiation sensitization. Methods: shRNA fragments were designed according to the sequence of HerpUD1 gene. ShRNA expression vector targeting HerpUD1 gene was constructed and stably transfected into L-02 cells. The expression of HerpUD1 mRNA and protein was detected by real-time PCR and Western blotting. The cells were transfected with 8 Gy X-ray and the apoptosis was observed by Hoechst staining. Results: The eukaryotic expression vector targeting HerpUD1 was successfully constructed and the L-02 cell line stably transfected with shHerpUD1 was established. The interference effect of shHerpUD1-1420 was best in stable transfected L-02 cells, and the expression of HerpUD1 mRNA was significantly decreased in shHerpUD1-1420 group compared with pGPU6-NC group (negative control group) [(0.15 ± 0.05) vs (1.00 ± 0.08), P <0.05], and the protein expression was also significantly lower in patients with diabetes mellitus (0.19 ± 0.01 vs 1.62 ± 0.08, P <0.01). The apoptotic rate of L-02-shHerpUD1-1420 group was significantly higher than that of L-02-NC group (negative control group) at the 24th hour after transfection with 8 Gy X-ray irradiation [(10.23 ± 3.37)% vs ± 1.49)%, P <0.01]. However, there was no significant difference between L-02-NC group and L-02-Neo group (P> 0.05). Conclusion: The silencing of HerpUD1 gene significantly increases the apoptosis of L-02 cells induced by X-ray and has the radiosensitization effect.