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目的研究反义AKT2(antisenseAKT2,ASAKT2)cDNA对鼠脑胶质瘤细胞系C6的生长抑制作用。方法将ASAKT2cDNA构建体转染鼠脑胶质瘤细胞系C6,原位杂交和蛋白印迹鉴定后,应用PCNA阳性率和MTT法检测细胞增殖能力,TUNEL法计算凋亡指数。应用立体定向技术将C6细胞和转染反义AKT2cDNA的C6细胞种植到SD大鼠的右侧尾状核作为对照组和转染组;并对颅内已经形成C6胶质瘤的大鼠进行脂质体包裹的ASAKT2cDNA和空载体治疗;MRI动态监测大鼠颅内肿瘤生长情况,并检测标本AKT2和PCNA表达以及细胞的凋亡情况。结果转染ASAKT2cDNA后C6细胞AKT2表达显著抑制,增殖减慢,凋亡指数增加。反义治疗组和转染组大鼠生存时间明显延长;转染组和治疗组肿瘤标本AKT2表达下降或消失,PCNA阳性率降低,可见大量凋亡细胞,而对照组和空载组标本几乎没有凋亡细胞。结论体内外实验证明ASAKT2cDNA可以抑制肿瘤细胞增殖、诱导凋亡,AKT2可作为基因治疗胶质瘤的重要优选靶的。
Objective To study the antitumor activity of antisense AKT2 (ASAKT2) cDNA on C6 glioma cell line. Methods The ASAKT2 cDNA was transfected into C6 glioma cell line. After in situ hybridization and western blotting, PCNA positive rate and MTT assay were used to detect cell proliferation. TUNEL method was used to calculate apoptotic index. The C6 cells transfected with antisense AKT2 cDNA and C6 cells transfected with antisense AKT2 cDNA were stably transfected into the right caudate nucleus of SD rats as a control group and a transfected group by stereotactic technique. Rats with intracranial C6 glioma were also subjected to lipofectamine Plaque-encapsulated ASAKT2 cDNA and empty vector were used to treat the tumors. MRI was used to monitor the intracranial tumor growth in rats. The AKT2 and PCNA expression and the apoptosis of the cells were detected. Results The AKT2 expression of C6 cells transfected with ASAKT2 cDNA was significantly inhibited, the proliferation slowed down and the apoptosis index increased. The survival time of antisense treatment group and transfection group was obviously prolonged. The expression of AKT2 in transfection group and treatment group decreased or disappeared, the positive rate of PCNA decreased, a large number of apoptotic cells were seen, while the control group and no-load group had almost no Apoptotic cells. Conclusion In vitro and in vivo experiments demonstrated that ASAKT2 cDNA can inhibit tumor cell proliferation and induce apoptosis, and AKT2 can be an important target for gene therapy of gliomas.