肿瘤厌氧靶向双自杀基因治疗系统的构建的论文.docVIP

　　肿瘤厌氧靶向双自杀基因治疗系统的构建的论文 【摘要】 目的利用婴儿双歧杆菌对实体瘤低氧区的靶向效应，构建肿瘤厌氧靶向双自杀基因治疗系统ptrkh2pst /cd和ptrkh2pst /uprt。方法用pcr的方法从质粒pgex/cd和pgex/uprt中扩增出cd基因和uprt基因，双酶切cd基因、uprt基因和质粒ptrkh2pst，分别连接后重组于大肠杆菌中。之后用电转化的方法将重组质粒转入婴儿双歧杆菌中。用rtpcr检测该系统mrna水平的表达，sdspage检测该系统在蛋白质水平的表达。在黑色素瘤b16f10细胞上检测该系统的体外肿瘤细胞杀伤效果。结果成功地将cd基因和uprt基因转入了质粒ptrkh2pst，cd基因和uprt基因的测序结果表明序列与genebank公布的序列一致。rtpcr检测到cd和uprt mrna水平的明显表达。在含有cd基因的婴儿双歧杆菌细胞全蛋白中发现了cd蛋白质的表达，在含有uprt基因的婴儿双歧杆菌上清液中发现了uprt蛋白质的表达。黑色素瘤细胞的低存活率证明了ptrkh2pst/cd、ptrkh2pst/uprt自杀基因治疗系统对黑色素瘤的显著杀伤作用。结论肿瘤厌氧靶向双自杀基因治疗系统ptrkh2pst/cd、ptrkh2pst/uprt构建成功并显示出杀伤肿瘤细胞的作用。 【关键词】 婴儿双歧杆菌； 自杀基因；cd基因；uprt基因； 肿瘤靶向性 construction of anaerobic infant bifidobacterial mediated ptrkh2pst/cd and ptrkh2pst/uprt dual suicide system for tumortargetting gene therapy ye peng1, li zhuhua2, yang yanbiao3, ent of pathophysiology, edical sciences and forensic medicine, sichuan university, chengdu 610041, china；2. department of pathophysiology, luzhou medical college；3. the first peoples hospital of lanzhou corresponding author：ail: into tumorhypoxiatargeting carrier of infant bifidobacterium.methodscd gene and uprt gene plified from pgex/cd and pgex/uprt by pcr. the cd gene, uprt gene id ptrkh2pst, then constructed tids of ptrkh2pst/cd and ptrkh2pst/uprt using electroporation. the expression of cd and uprt in bifidobacterium on melanoma b16f10 cells easured using methyl thiazolyl tetrazolium (mtt) assay.resultsthe cd gene and uprt gene id ptrkh2pst. the sequencing results of cd gene and uprt gene in dual suicide system are consistent rna and protein level of cd and uprt . the survival rate of melanoma b16f10 cells treated 1.7%~13.3%, 81.3%~91.7%,26.7%~58.3% and 78.3%~86.7%, respectively.conclusioncd gene and uprt gene . this dual suicide gene therapy system shoor cells. key infantis；suicide gene；cd gene；uprt gene； tumortargeting 1材料与方法 1.1材料质粒ptrkh2pst由复旦大学生命科学院黄教授赠予，婴儿双歧杆菌2001株由四川大学华西口腔医学院赠予，质粒纯化试剂盒和pcr产物纯化试剂盒购自omega co.，pcr反应试剂盒和dna marker购自promega co.(usa