摘要目的 应用RNA干扰技术,观察稳定转染Her2基因的短发夹状RNA(shRNA)对不同恶性程度的卵巢上皮性癌(卵巢癌)SKOV3和SKOV3.ipl细胞Her2基因表达及细胞生物学特性的影响.方法 将表达Her2的质粒pHer2 shRNA分别转染SKOV3和SKOV3.ipl细胞,经抗性筛选获得稳定转染的细胞系.RT-PCR技术、蛋白印迹法检测转染前后细胞Her2 mRNA和蛋白的表达,体外侵袭实验观察转染前后细胞侵袭能力的改变,裸鼠接种肿瘤细胞观察转染前后细胞成瘤能力的改变.结果 转染前后SKOV3.ipl、SKOV3细胞的Her2 mRNA表达量分别为(99.9±1.3)%和(42.4±2.5)%、(68.0±3.1)%和(40.8±2.0)%.Her2蛋白的表达量分别为(98.2±1.7)%和(40.7±2.1)%、(72.1±3.4)%和(36.4±1.5)%,稳定转染后,两种细胞的Her2基因表达均明显下降,分别比较,差异均有统计学意义(P<0.05).稳定转染pHer2 shRNA后,SKOV3.ipl细胞的穿膜细胞数由(36.2±9.7)个下降为(15.7±7.2)个,SKOV3的穿膜细胞数由(7.6±1.1)个下降为(1.8±0.8)个,细胞侵袭能力均显著下降,分别比较,差异均有统计学意义(P<0.05).稳定转染pHer2 shRNA后,SKOV3.ip1和SKOV3细胞的成瘤重量分别由(1.55±0.31)g下降为(0.69±0.20)g、(1.14±0.10)g下降为(0.38±0.03)g,两种细胞的成瘤能力均显著下降,分别比较,差异均有统计学意义(P<0.05).结论 (1)针对Her2基因的RNA干扰技术可以显著降低卵巢癌细胞Her2基因的表达水平.(2)经RNA干扰作用降低Her2基因表达后,卵巢癌细胞的生物学特性改变,恶性度降低.

abstractsObjective To observe the effects of short hairpin RNA (shRNA) targeting Her2 on its gene expression when the shRNA was stably transfected into human ovarian cell lines, SKOV3 and SKOV3. ipl, which have different extent of malignancy and investigate the changes of the biological characters of the two cell lines after the stable transfection. Methods The plasmids expressing shRNA targeting Her2 gene were transfected into SKOV3 and SKOV3. ipl cells. The stably transfected cells were gained by antibiotic screening. The expression of Her2 before and after the transfection was detected by RT- PCR and western blot. The transwell experiment was used to observe the invasion ability of the cancer cells before and after the transfection, and the parent and the transfected cells were injected into nude mice to observe the tumor growth. Results After the stable transfection with Her2 shRNA, mRNA and protein levels of Her2 gene in SKOV3 and SKOV3. ipl cells were remarkably reduced. The expression of mRNA were (68.0±3. 1) %, (40. 8±2. 0) %, (99. 9±1.3) %, (42. 4±2. 5) %. The expression of protein were (72. 1±3.4) %, (36. 4±1.5) %, (98.2±1.7) %, (40. 7±2. 1) %. The invasion ability into basilar membrane of the transfected cells was greatly reduced compared with the parent cells. The invasion cell numbers were 7.6±1.1, 1.8±0. 8, 36. 2±9.7, 15.7±7. 2. The growth rate of the planted tumors was lower in transfected groups than that of the parent groups. Conclusions (1) The expression of Her2 gene in SKOV3 and SKOV3. ipl cells was remarkably reduced by RNA interference targeting Her2. (2) The biological characters of SKOV3 and SKOV3. ipl cells are changed when the expression of Her2 gene is reduced by RNA interference.