含hTERT启动子、PE38KDEL片段质粒构建及其对人胰腺癌细胞凋亡的影响  

Construction of recombinant plasmid containing hTERT promoter and PE38KDEL gene and its anti-apoptosis effect on human pancreatic cancer cell MiaPaCa2

作　　者：李宝玉[1] 孙晋津[1] 陈剑秋[1] 

机构地区：[1]天津医科大学第二医院,天津300211

出　　处：《山东医药》2010年第34期4-6,共3页Shandong Medical Journal

基　　金：教育部博士点基金资助项目(20060062008);天津市自然科学基金自助项目(07JCYBJC10300)

摘　　要：目的构建含hTERT启动子和PE38KDEL片段的重组表达质粒,并观察其对人胰腺癌MiaPaCa2细胞凋亡的影响。方法采用PCR法构建含hTERT启动子和PE38KDEL片段的重组表达质粒,并用双酶切法鉴定;将MiaPaCa2细胞分为5组,A组为空白对照组,仅加入含10%FBS的DMEM;B、C、D、E组分别转染pIRES2-EGFP、phTERT-IRES2-EGFP、pPE38KDEL-IRES2-EGFP、phTERTp-PE38KEDL-IRES2-EGFP,采用TUNEL法检测MiaPaCa2细胞的凋亡指数。结果重组质粒的PCR产物及酶切后片段的测序结果与GeneBank报道序列一致。A组转染24、48 h时MiaPaCa2细胞凋亡率分别为17.12%±1.66%、24.86%±2.80%,B组分别为18.14%±1.61%、26.47%±2.71%,C组为19.08%±3.84%、27.42%±7.26%,D组为29.46%±2.86%、38.12%±3.19%,E组为72.16%±4.79%、86.40%±7.71%。B^E组与A组比较,P均<0.05;E组与D组比较,P<0.05。结论成功构建了含hTERT启动子和PE38KDEL片段的重组表达质粒,该质粒可促进人胰腺癌MiaPaCa2细胞的凋亡。Objective To construct a recombinant plasmid containing hTERT promoter and PE38KDEL gene and observe its anti-apoptosis effects on human pancreatic cancer cell MiaPaCa2.Methods The recombinant plasmid contain hTERTpromoter and PE38KDEL gene（phTERTp-PE38KDEL-IRES2-EGFP）was constructed by PCR and double digestion methods.phTERTp-PE38KDEL-IRES2-EGFP was transfected to MiaPaCa2 cell and apoptotic index of MiaPaCa2 cell were detected by TUNEL.Results The recombinant plasmid phTERTp-PE38KDEL-IRES2-EGFP was highly expressed in human pancreatic cancer cell line MiaPaCa2 of telomerase positive,and it could selective killed MiaPaCa2 in vitro.Conclusion The recombinant plasmid phTERTp-PE38KDEL-IRES2-EGFP is constructed successfully,and it can kill human pancreatic cancer cell line MiaPaCa2 specifically.

关 键 词：胰腺癌 人端粒酶反转录酶 启动子 铜绿假单胞菌外毒素 绿色荧光蛋白 基因疗法 

分 类 号：R753.9[医药卫生—皮肤病学与性病学]