突变型p27基因治疗裸鼠大肠癌移植瘤的研究.DOCVIP

临床医学论文-突变型p27基因治疗裸鼠大肠癌移植瘤的研究 ???????????????????? 作者：杜勇，徐少勇，杨建业，张丽 [摘? 要]? 目的：研究外源性突变型p27(p27 mt)基因对大肠癌的体内抑制作用。方法：应用细胞移植法把人大肠癌SW480细胞接种于BALB/c裸鼠皮下，构建大肠癌裸鼠模型，将腺病毒介导的突变型p27（Ad-p27 mt）及LacZ注射到瘤体内，绘制肿瘤生长曲线，计算肿瘤生长抑制率，并进行细胞凋亡及MMP－9的相关检测。结果：Adp27 mt基因治疗组肿瘤生长明显受到抑制，MMP9的表达也明显较低，与LacZ、空白对照组比较，有显著性差异（P＜0.01）。结论：Adp27 mt基因对大肠癌裸鼠的体内肿瘤有明显的抑制作用，并能抑制MMP9。 　　[关键词]? 腺病毒；p27 mt；大肠癌；裸鼠；MMP9 　　Abstract:Objective To investigate the inhibitory effect of adenovirus mediated p27mt gene tramsfer on xenograft of in colorectal carcinoma nude mice. Methods The xenograft model of colorectal carcinoma in nude mice was established by inoculating the SW480 cells of human colorectal carcinoma. Adp27mt and LacZ was injected into the tumors respectively. The growth curves and the growth inhibitory rates of tumors were analyzed and compared with control group.Apoptosis and matrix metalloproteinase9 (MMP9) were also detected .Results Adp27mt markedly suppressed the growth of tumors and promoted apoptosis of SW480 cells, which had a significant difference compared with control group(P0.01),and MMP9 expression was lower in Adp27mt group than Lacz group and control group. Conclusions Adp27mt has obviously inhibitory effect on the tumor growth of colorectal carcinoma in nude mice in vivo. 　　Key words:Adenovirus p27mt;Colorectal carcinoma;Nude mice;MMP9 　　正常情况下p27基因在G0/G1期表达增高，当细胞进入S期则表达下降。p27基因高表达抑制细胞增殖是一个普遍现象。将外源性p27基因导入细胞内造成p27蛋白高表达，可使细胞DNA合成强烈受抑制，细胞中止于G1期而停止增殖[1]。把苏氨酸187换成蛋氨酸187，称为突变型p27 mt，其比体内天然存在的p27有更强的抗肿瘤作用。 　　1? 材料和方法 　　1.1? 材料 　　SPF级BALB/c裸鼠购自湖北省实验动物中心，雌雄兼用，大肠癌SW480细胞购自第四军医大学细胞库，独立送回风动物饲养笼具(IVC笼具，苏杭动物实验器材公司)，pORF9hp27 mt质粒购自Invivogen 公司，腺病毒骨架质粒pAdeasy1，质粒pShuttleCMV,pBluesciptⅡSK(+)，Ad293细胞，大肠杆菌BJ5183和XL10－gold购自Stratagene公司，DH5α由郧阳医学院临床医学研究所保存；AgeⅠ，NheⅠ，KpnⅠ，NotⅠ，PacⅠ和PmeⅠ购自New England Biolabs公司，Hind Ⅲ,EcoRⅠ，λDNA HindⅢ marker,200 bp DNALadderdNTP，Taq酶和T4 DNA 连接酶购自华美生物公司,hp27 mt 引物由北京赛百盛生物公司设计合成；DMEM购自Gibco公司，CsCl购自Sigma公司，胎牛血清购自杭州四季青生物工程材料有限公司。所用试剂AntiMMP9为鼠抗人单克隆浓缩型抗体,浓缩型免疫组化SP试剂盒、DAB 显色试剂盒、胃蛋白