【摘要】 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。更多还原

【Abstract】 Objective To investigate the effect of anti-sense serine /threonine protein kinase 2 (AKT2) constructs on proliferation and apoptosis of glioma cells. Methods Rat C6 glioblastoma cells were transfected with AS-AKT2 constructs,the expression of AKT2 in cells were identified by in situ hybridization and Western blotting. Proliferative activitiy of glioma cell was evaluated by MTT method and PCNA positive rate, and apoptotic cells were detected by Apoptotic Index (AI) using TUNEL method. Parental C6 cells and C6 cells trasfected with AS-AKT2 cDNA were implanted stereotactically into the right caudate nucleus of SD rats as control and transfected group. Rats with well-established cerebral gliomas were treated with AS-AKT2 cDNA and LXSN empty vector as treated and LXSN group. The dynamic MRI and histopathological changes of the tumors and the expression of AKT2 and PCNA were investigated, apoptosis in glioma cells was examined as well. Results Over-expression of AKT2 were identified in mRNA and protein level in parental C6 cells. As compared with C6 cells and LXSN transfected cells, AKT2 expression was inhibited in AS-AKT2 transfected cell,proliferative activities and PCNA positive rate were decreased in AS-AKT2 cells. Nearly no apoptotic cell could be detected in patrental C6 and LXSN transfected cells, whereas AI increased in AS-AKT2. The survival time of rats in AS- AKT2 treated group and transfected group prolonged significantly than control and empty vector group.The expression of AKT2 and PCNA were inhibited in the tumor spacimens of rats in transfected and treated groups while apoptotic cells was increased. Conclusions AKT2 pathway may exert pivotal role in proliferation and anti-apoptosis in glioma cells, AS-AKT2 can prohibit tumor cell proliferation and induce apoptosis, and thus, it can be a good candidate for gene therapy of glioma.更多还原