摘要
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 effect of anti-sense serine/threonine protein kinase 2 (AKT2) constructs on phenotypic invert of C6 glioma cells.Methods Anti-sense AKT2 constructs in pLXSN retrovirus vectors were transfected to rat glioma cell line C6,and empty pLXSN vector transfected as controls.The positive clones were randomly selected and identified by Western blot and in situ hybridization.MTT and TUNEL methods were used to evaluate cell proliferation and apoptosis.Tumor invasion was examined by Transwell method,and tumor cell migration by wound-healing method.Flow cytometry was used for cell cycle analysis,and the GFAP expression was also detected for evaluation of cell differentiation by Western blot.Results After C6 cells were transfected with antisense AKT2 constructs,insitu hybridization and Western blot revealed that the expression of AKT2 was significantly down-regulated.As compared to control and LXSN transfected cells,in antisense AKT2 transfected glioma cells,the proliferation rate was decreased (P< 0.01),the apoptotic index increased (0.33 vs 13.67, P< 0.01),and the invasive and migratory capabilities were inhibited as well.Cell cycle analysis indicated G_0/G_1 arrest,and the GFAP expression was up-regulated.Conclusion Antisense AKT2 can invert malignant phenotype through growth suppression,apoptotic induction,invasion and migration inhibition and G_0/G_1 arrest.It was suggested that AKT2 could be used as a good candidate for gene therapy of gliomas.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2005年第7期852-854,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目(30100050)
天津市自然科学基金资助项目(003703011)
