P16基因重组质粒的构建及其对人脑胶质瘤细胞的抑制作用

目的：探讨P16基因对人胶质瘤细胞的抑制作用，为胶质瘤致瘤机理的研究和基因治疗提供理论基础。方法：运用分子克隆技术构建重组人P16基因功体（pcDNA3-P16)，通过脂质体介导法将P16cDNA转染到存在P16基因纯合缺失的人胶质瘤细胞系SHG－44。结果：PCR证实含P16cDNA质粒DNA已转入SHG－44细胞，Western blot显示有P16蛋白表达。细胞生长曲线显示转杂P16基因的SHG－44细胞（SHG－44－P16）生长明显受到抑制，基抑制率达84．27％。在软琼脂上克隆形成能力下降约6倍。细胞周期分析表明，处在G1期细胞由30．70％提高到66．21％。结论：野生型P16基因导入P16基因功能缺失的肿瘤细胞，能恢复其抑制肿瘤细胞生长的功能。

CONSTRUCTION OF p16 GENE RECOMBINANT EXPRESSION VECTOR AND ITS ANTITUMORIGENIC EFFECTS ON HUMAN GLIOMA CELLS

Objective:To explore the effect of p16 gene on the growth inhibiti on of glioma cell line SHG-44 .Methods:We cloned a p16cDNA into the pcDNA3 vector at the sites of BamH Ⅰ and Xho Ⅰ to gain a p16 gene recombinant expression vector plasmid . The p16 gene recombinant plasmid was transfected int o the glioma cell line ,In these glioma cells ,the p16 gene was homozygously del eted .Results:Expression of exogenous p16 gene in SHG-44 cell was id entified by PCR and Western blot analysis .The growth rate of SHG-44 transfected with p16 gene (SHG-44-p16) was markedly suppressed . Colony formation in soft a gar was also decreased significantly .Cell cycle analysis by flow cytometry show ed that the number of cell in G1-G0 phase of SHG-44 cells was significantly inc reased while cells in S and G2 M phase was decreased compared to that of contr ol SHG-44 cells .Conclusion:Transfection of wild-type p16 gene into p1 6 gene-deleted glioma cells can restore its suppressive effect on cell growth by arrest of cell cycle at G1 phase.