Survivin—Fk质粒的构建及诱导树突状细胞疫苗抗胃癌研究

Construction of Survivin-Fk plasmid and investigation of inducing dendritic cells to anti.gastric cancer

目的 观察Survivin-FK（Sur-Fk）基因治疗小鼠胃癌的效果.方法 将FK-cDNA插入pcDNA3.1质粒的多克隆位点,用Survivin基因启动子替代pcDNA3.1质粒的巨细胞病毒（CMV）启动子,构建成Survivin-Fk重组质粒.用脂质体法将Sur-Fk基因转染到胃癌细胞SGC-7901;用逆转录-聚合酶链反应（RT-PCR）和Westem blot检测转染的效果,并进行转基因瘤细胞的成瘤性实验,实验分4组,每组20只小鼠,小鼠生存时间分为小于40、70、100 d 3个阶段,观察肿瘤的生长情况及小鼠的生存时间并测量肿瘤大小;并且切取肿瘤组织,经过处理后,用流式细胞仪分析肿瘤中树突状细胞（DC）细胞表面MHC-Ⅰ/Ⅱ表达.结果 构建得到的重组质粒Sur-Fk质粒,用ApaI和EcoRI酶切鉴定,琼脂糖电泳鉴定,得到相应的特异片段,提示Sur-Fk质粒重组质粒构建成功;转基因瘤细胞的成瘤实验中,SGC-7901-SUR-FK组的肿瘤大小为（0.30±0.13）cm3,小鼠的生存时间40、70、100 d的分别存活2、3、15只小鼠,在肿瘤大小和小鼠的生存时间方面,SGC-7901-SUR-FK组与SGC-7901组和SGC-7901-FK组之间差异有统计学意义;SGC-7901-SUR-FK组肿瘤周围DC上MHC-Ⅰ/Ⅱ类分子的平均表达率分别上调93.6%和91.4%,与SGC-7901组和SGC-7901-FK组及空白组表达率比较差异有统计学意义（P＜0.05）.结论 Survivin启动子可启动下游FK基因特异性表达于SGC-7901肿瘤细胞,从而对DC有强烈的趋化作用,引起肿瘤周围DC的MHC-Ⅰ/Ⅱ类分子的表达上调,显著提高基因治疗胃癌效果.

Objective To detect the effects of Survivin-Fk （Sur-Fk） gene on mice gastric cancer.Methods FK-cDNA was inserted into pcDNA3.1 plasmid of multiple clone site,and cytomegalovirus （CMV） promoter of cDNA3.1 plasmid was substituted with Survivin gene,and recombinant Sur-Fk plasmid was constructed.Sur-Fk gene was transfected into SGC-7901 with Liposome.The transfection efficiency was detected by using reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting.Transgenic experimentation of neoplasma format was also carried out.There were four groups （n = 20each）.There were three stages of survival time：less than 40 days,70 days,100 days.The growth of tumor,murine survival time and size of time were measured.The expression of MHC- Ⅰ / Ⅱ on the surface of dendritic cells （DCs） in tumor tissues was detected by using flow cytometry.Results The recombinant Sur-Fk plasmid was identified with ApaI and EcoRI,and by using AG Electrophoresis.The specific fragment was obtained.In the transgenic experimentation of neoplasma format,the size of murine tumor was （0.30±0.13） cm3,the number of mice with survival time of 40,70,100 days was 2,3,15 in SGC-7901-SUR-FK group.There was significant difference between SGC-7901-SUR-FK group and SGC-7901group,or plasmid group,or SGC-7901-FK group.The expression of MHC- Ⅰ / Ⅱ in SGC-7901-SUR-FKgroup was up-regulated by 93.6% and 91.4% respectively,and there was significant difference between SGC-7901-SUR-FK group and SGC-7901 group,or SGC-7901-FK group,or control group.Conclusion Promoter of Survivin can act as a targeted therapy tool,priming downstream gene of FK to be expressed specifically in the tumor cells.The promoter can generate intense chemotaxis to DCs,leading to up-regulation of MHC- Ⅰ/Ⅱ in the DCs around the tumor,finally significantly promoting the therapeutic effects of gene therapy on gastric cancer.