针对Her-2基因的小分子干扰RNA对卵巢上皮性癌细胞生物学行为影响的研究

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。

Effects of small interfering RNA specific for Her-2 gene on biological behavior of ovarian carcinoma cell

OBJECTIVE: To explore the effects of small interfering RNA (siRNA) specific for Her-2 gene on biological behavior of ovarian carcinoma cell. METHODS: Her-2 siRNA recombinant plasmid and negative control plasmid were transfected into packing cell line PT67 by liposome, and PT67 was selected by puromycin later. SKOV3 was infected by the virus supernatant of stably transfected PT67 cell lines, and the stably transfected SKOV3 cell lines (SKOV3/siRNA, SKOV3/siRNA-negative) established by selection with puromycin were investigated in terms of the reduction levels of Her-2 mRNA and p185 by RT-PCR and immunohistochemistry. Cell proliferation was assayed with methyl thiazolyl tetrazolium, and cell cycle distribution and cell apoptosis were assayed with flow cytometry. The tumor growth of the null mice was analyzed after injection of SKOV3/siRNA and SKOV3/siRNA-negative into the skin. RESULTS: (1) The stable SKOV3 cell lines with a persistent silence of Her-2 gene were established. (2) The percentages of SKOV3/siRNA in G(0)/G(1) phase and S phase were 68.6%, 15.1% respectively; while the percentages of SKOV3/siRNA-negative in G(0)/G(1) phase and S phase were 55.8%, 23.3%. (3) The percentage of SKOV3/siRNA in early apoptosis was (10.500 +/- 0.250)%, while the percentage of SKOV3/siRNA-negative was (0.340 +/- 0.010)% (P < 0.01). (4) Compared with SKOV3/siRNA-negative, the proliferation of SKOV3/siRNA was delayed obviously (P < 0.05), and the growth of the corresponding implanted tumor slowed down significantly (P < 0.01). CONCLUSION: siRNA can inhibit the expression of Her-2 gene effectively, which restrains the biological behavior of ovarian carcinoma cell.