表皮生长因子受体反义RNA抑制胶质瘤细胞生长增殖的体外试验

Inhibitory Effect of EGFR Antisense RNA on Growth of Glioma Cells in Vitro

目的：研究表皮生长因子受体基因对于胶质瘤发生发展的作用，探索以ＥＧＦＲ反义ＲＮＡ进行反义基因治疗胶质瘤的可行性。方法：将含ＥＧＦＲ反义ＲＮＡ的质粒通过脂质体介导转入Ｃ６恶性胶质瘤细胞，通过Ｓｏｕｔｈｅｒｎ印迹杂交鉴定外源基因整合情况，ＭＴＴ法测定细胞存活率，Ｎｏｒｔｈｅｒｎ印迹杂交、细胞原位ｍＲＮＡ杂交及ＥＧＦＲ免疫组化染色检测ＥＧＦＲｍＲＮＡ及蛋白表达，核仁组成区相关嗜银蛋白染色（ＡｇＮＯＲ计数）检测细胞增殖活性。结果：Ｓｏｕｔｈｅｒｎ印迹杂交表明外源性反义ＥＧＦＲ基因（互补于ＥＧＦＲ基因全长及３端部分序列）分别在Ｃ６细胞中整合（分别命名为Ｃ－ｐＲ１和Ｃ－ｐＲ３），通过Ｎｏｒｔｈｅｒｎ印迹杂交、原位杂交及细胞免疫组织化学检测均显示转染ＥＧＦＲ反义ＲＮＡ的Ｃ６细胞比转染前ＥＧＦＲｍＲＮＡ水平及ＥＧＦＲ蛋白水平有不同程度的降低。表达高水平反义ＲＮＡ的克隆在培养状态下细胞增殖活性较对照组有明显下降。结论：ＥＧＦＲ在恶性胶质瘤细胞的发生发展过程中起十分关键的作用，ＥＧＦＲ有可能成为基因治疗恶性胶质瘤的优选靶的之一。

Objective: To investigate the role of epidermal growth factor receptor gene on growth and tumorigenesis of glioma as well as to find out the feasibility of using EGFR antisense RNA for gene therapy of gliomas. Methods: Antisense EGFR cDNA was introduced into C6 glioblastoma cell line (C cells) by transfection with plasmid DNA and lipofectamine. Technics such as Southern blot,Northern blot,MTT method, immunocytochemistry study for EGFR protein,silver colloid staining for AgNORs, in situ hybridization for EGFR mRNA were used in this study. Results:Southern blot indicated that exogenous antisense EGFR gene(AS FL & AS 3)to was integrated in the correspondent genome of C6 glioma cells as C pR1 and C pR3 cells.The C6 transfectants resulted in dramatic decrease of endogenous EGF R mRNA and EGF R protein levels as demonstrated by Northern blot and in situ hybridization and immunocytochemistry study. Clones with high expression of antisense constructs also showed decreased proliferation in vitro as detected by MTT method and silver colloid staining for AgNORs. Conclusion: These results indicated that EGF R is of crucial importance in glioblastoma cell growth and tumorigenesis. EGFR may be chosen as a target for antisense gene therapy of gliomas.