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目的研究反义AKT2RNA逆转C6鼠脑胶质瘤细胞恶性表型。方法将逆转录病毒LXSN为载体的反义AKT2构建体脂质体复合物直接转染人脑胶质瘤细胞系TJ905和鼠脑胶质瘤细胞系C6,LXSN载体转染组为空载对照。随机筛选阳性克隆并应用蛋白印记和原位杂交鉴定转染。MTT法和TUNEL法评价细胞的增殖活性和凋亡,Transwell法分析侵袭能力,划痕实验研究细胞迁移能力,流式细胞法分析细胞周期,并进行GFAP的表达分析。结果转染ASAKT2cDNA后C6细胞AKT2表达显著抑制。与C6对照组和LXSN空载组比较,转染反义AKT2后C6细胞存活率均明显下降(P<0.01),凋亡指数增加(0.33vs13.67,P<0.01),侵袭能力和细胞迁移能力抑制,诱导细胞出现G0/G1细胞周期阻滞,上调GFAP的表达。结论反义AKT2RNA基因治疗通过抑制肿瘤细胞增殖、诱导凋亡、抑制肿瘤细胞侵袭与迁移并使G0/G1细胞周期阻滞逆转其恶性表型,AKT2可作为基因治疗胶质瘤的重要优选靶的。
Objective To investigate the anti-sense AKT2 RNA reversing the malignant phenotype of C6 glioma cells. Methods The antisense AKT2 construct liposome complex with LXSN retrovirus was directly transfected into human glioma cell line TJ905 and C6 glioma cell line, and the LXSN vector transfected group was an empty control. Positive clones were screened randomly and identified by Western blotting and in situ hybridization. MTT assay and TUNEL assay were used to assess cell proliferation and apoptosis. Transwell assay was used to evaluate invasion ability. Scratch assay was used to investigate cell migration ability. Flow cytometry was used to analyze cell cycle and GFAP expression. Results AKT2 expression was significantly inhibited in C6 cells transfected with ASAKT2 cDNA. Compared with the C6 control group and LXSN empty vector group, the survival rate of C6 cells transfected with antisense AKT2 significantly decreased (P <0.01), the apoptosis index increased (0.33 vs 13.67, P <0.01), the invasion ability and cell migration Ability to suppress, induce cell G0 / G1 cell cycle arrest, upregulation of GFAP expression. Conclusion AKT2 antisense gene therapy can be an important target for gene therapy of glioma by inhibiting tumor cell proliferation, inducing apoptosis, inhibiting tumor cell invasion and migration, and reversing the malignant phenotype of G0 / G1 cell cycle arrest .