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目的通过小分子干扰RNA(siRNA)对Her-2基因表达的影响,探讨其对卵巢上皮性癌(卵巢癌)细胞生物学行为的影响。方法针对Her-2基因的siRNA质粒和阴性对照质粒在脂质体介导下转染到包装病毒细胞株PT67中,嘌呤霉素筛选细胞克隆,收集其上清液并感染SKOV3细胞,经过嘌呤霉素筛选得到稳定转染的细胞株SKOV3/siRNA、SKOV3/siRNA-negative,并通过RT-PCR和免疫组化方法鉴定Her-2基因表达的抑制效果。用四甲基偶氮唑蓝法检测细胞增殖并绘制生长曲线,通过流式细胞仪检测细胞周期、细胞凋亡,并将稳定转染的细胞株接种到裸鼠皮下检测成瘤情况。结果(1)SKOV3/siRNA细胞Her-2基因的表达明显减弱。(2)SKOV3/siRNA细胞的G0/G1期细胞占68·6%,S期细胞占15·1%,SKOV3/siRNA-negative细胞的G0/G1期占55·8%,S期占23·3%。(3)SKOV3/siRNA细胞的早期凋亡率为(10·500±0·250)%,而SKOV3/siRNA-negative细胞为(0·340±0·010)%,两者比较,差异有统计学意义(P<0·01)。(4)与SKOV3/siRNA-negative细胞比较,SKOV3/siRNA细胞的生长速度减慢(P<0·05),接种于裸鼠皮下的肿瘤生长速度明显减慢(P<0·01)。结论siRNA可以有效抑制Her-2基因的表达,抑制卵巢癌细胞的生物学行为。
Objective To investigate the effect of small interfering RNA (siRNA) on the expression of Her-2 gene and the biological behavior of ovarian epithelial carcinoma (ovarian cancer). Methods siRNA plasmid and negative control plasmid targeting Her-2 gene were transfected into packaging virus cell line PT67 under liposome-mediated transfection. The cell clones were screened by puromycin, and the supernatant was collected and infected into SKOV3 cells. After puromycin The stable transfected SKOV3 / SKOV3 / siRNA-SKOV3 / siRNA-negative cells were obtained by screening, and the inhibitory effect of Her-2 gene expression was identified by RT-PCR and immunohistochemistry. Cell proliferation and growth curve were detected by MTT method. Cell cycle and apoptosis were detected by flow cytometry. The stably transfected cell lines were inoculated into the skin of nude mice for detection of tumorigenesis. Results (1) The expression of Her-2 gene in SKOV3 / siRNA cells was significantly decreased. (2) The percentage of G0 / G1 phase cells was 68.6% in SKOV3 / siRNA cells group, 15.1% in S phase cells, 55.8% in SKOV3 / siRNA-negative cells and 23.0% in S phase 3%. (3) The early apoptosis rate of SKOV3 / siRNA cells was (10 · 500 ± 0 · 250)%, while that of SKOV3 / siRNA-negative cells was (0. 340 ± 0. 010)% Significance of learning (P <0.01). (4) Compared with SKOV3 / siRNA-negative cells, the growth of SKOV3 / siRNA cells was slowed down (P <0.05), and the growth of tumors inoculated subcutaneously in nude mice was significantly slowed down (P <0.01). Conclusion siRNA can effectively inhibit the expression of Her-2 gene and inhibit the biological behavior of ovarian cancer cells.