超声辐照联合微泡转染短发卡状重组质粒诱导裸鼠宫颈癌移植瘤凋亡的研究

Survivin-shRNA transfection facilitated by ultrasound associated with microbubbles induced apoptosis of cervix cancer transplanted tumors in nude mice

目的探讨超声辐照联合微泡对靶向存活素的短发卡状重组质粒(survivin-shRNA)的转染增效作用及其安全性,分析其凋亡诱导效应和增殖抑制作用。方法将荷人宫颈癌(Hela)裸鼠18只随机分为3组,每组6只。1组:质粒+超声辐照组,注入质粒溶液后予以超声辐照;2组:质粒+微泡+超声辐照组,注入质粒及微泡复合物后辐照;对照组:不予任何处理。对组织样本行冰冻切片、组织学检查、转染率检测。采用免疫组化SABC法检测移植瘤增殖细胞核抗原(PCNA)及survivin蛋白在各组肿瘤标本中的表达,测定凋亡指数(AI)和增殖指数(PI)。结果2组的移植瘤内荧光表达率显著高于对照组、1组(P<0.01);裸鼠其他器官组织中未观察到基因表达,且未观察到明显的组织损伤;PCNA及survivin蛋白表达下降,AI明显升高,PI明显减少,与对照组及1组比较差异均有统计学意义(P<0.05)。结论超声辐照联合微泡能显著增强基因转染效率,无明显副作用,联合Survivin-shRNA能明显诱导裸鼠体内肿瘤凋亡,抑制细胞增殖。

Objective To study the transfection efficiency and safety for recombinant plasmid of short hairpin RNA and survivin （Survivin-shRNA） by ultrasound associated with microbubble and to investigate the induction of apoptosis and suppression of proliferation. Methods The human cervical carcinoma cells （Hela） were induced subcutaneously in both flanks of female nude mice. A total of 18 nude mice were randomly classified into 3 groups （6 mice each group） ： group-1 control group： the plasmid solution was injected followed by ultrasound ; group-2 ： mixtures of plasmid and microbubbles were injected by ultrasound. The frozen section, histological examination and transfection efficiency were evaluated. The protein expressions of survivin and proliferating cell nuclear antigen （PCNA） were investigated by immunohistochemistry SABC. Furthermore, the proliferative index （PI） and apoptotie index （AI） were measured respectively in each group of tumor specimens. Results In transplanted tumors, highest transfection efficiency was observed in group-2, which was significantly higher than those in any other groups （ P 〈 0.01 ）. No increase in expression was observed in other tissues. No tissue damage was seen histologically. Compared with those in group-C and group-1, protein expression of survivin and PCNA were down-regulated markedly. PI decreased significantly, whereas AI increased remarkably （P 〈 0.05 ）. Conclusions The ultrasound associated with microbubble can enhance efficiency of gene transfection significantly without causing any obvious adverse effects. The method associated with specific survivin-shRNA can significantly induce apoptosis and inhibit proliferation of transplanted tumors in nude mice.