反义AKT2 RNA抑制U251胶质瘤细胞生长的体内外研究

目的 研究反义AKT2RNA对U251人脑胶质瘤细胞在体内外的生长抑制效用。方法 将逆转录病毒pLXSN为载体的反义AKT2构建体转染U251人脑胶质瘤细胞系，应用蛋白印记确定基因转染前后AKT2的表达水平。流式细胞法与Matrigel基质生长实验评价肿瘤细胞转染前后的增殖活性。进一步应用裸鼠皮下荷瘤模型观察脂质体介导pLXSN、pLXSN-AS-AKT2基因治疗对U251细胞生长抑制作用。在28d的观察期内定期测量皮下肿瘤体积，对肿瘤标本应用免疫组化的方法进行AKT2和胶质纤维酸性蛋白（GFAP）表达比较。结果 脂质体介导pLXSN-AS-AKT2可显著抑制U251细胞AKT2表达。与对照组和pLXSN转染组比较，细胞周期分析结果表明AK—AKT2转染组进入S期的细胞数减少了8．5％～8．9％，而进入G0＋G1期细胞则增加了7．9％～8．6％。Matrigel基质生长实验显示对照组和pLXSN转染组细胞呈正常形态贴壁生长，而AS～AKT2转染组细胞不能贴壁生长，呈团块状簇集生长。裸鼠皮下荷瘤模型实验显示pLXSN-AS-AKT2显著抑制皮下肿瘤生长，组织病理学分析显示AS-AKT2转染组AKT2表达下降而GFAP表达上调。结论 体内外实验证明反义AKT2方法在抗胶质瘤增殖方面作用重要，AKT2可作为基因治疗胶质瘤的优选靶标。

Growth suppressive effect of antisense AKT2 RNA on U251 gliomas: an in vitro and in vivo study

Objective To investigate the suppressive effect of antisense serine/threonine protein kinase 2 (AS-AKT2) RNA on the growth of U251 glioma cells. Methods Human U251 glioma cells were transfected with AS-AKT2 constructs in retrovirus vector pLXSN, and the expression of AKT2 in the cells was identified by Western blotting. The proliferative activities of the tumor cells were evaluated by flow cytometry and in vitro growth in Matrigel matrix. The inhibitory effect of tumor growth was further studied in vivo. pLXSN and pLXSN-AS-AKT2 were injected into established subcutaneous U251 gliomas in nude mice mediated by lipofectamine, respectively. During the observation period of 28 d, tumor volume was measured every 3 d and the tumor mass was taken out on the 28th day. The expressions of AKT2 and GFAP in the tumors was examined by immunohistochemical staining. The cell proliferative activity were examined by PCNA immunostaining. Results Lipofectamine-mediated AS-AKT2 dramatically down-regulated AKT2 expression in U251 glioma cells. Compared with parental U251 glioma cells and the cells transfected with empty vector pLXSN, decreased AKT2 expression in the AS-AKT2 transfected cells was accompanied by 8.9 % and 8.5% decrease of S phase fraction and 7.9% -8.6% increase of GO+G1 phase. Parental cells and pLXSN-transfected cells growing in matrigel matrix showed normal appearance. While the cells transfected with AS-AKT2 were detached from the matrix or grew in a scattered clustering pattern, indicating poor cell growth activities. The tumor volume of AS-AKT2 treated group was smaller than that of the control and empty vector (pLXSN) treated groups. The effect of knocking down AKT2 expression and the up-regulation of GFAP in AS-AKT2 treated group were more significant than those in the two control groups. Conclusion Antisense AKT2 RNA may exert pivotal role in anti-proliferation effect of glioma cells, and AKT2 can be a good candidate for gene therapy of gliomas.