Experimental Study Of Pancreatic Cancer Treated By PE38KDEL Gene Mediated By Adeno-associated Virus Under The Control Of HTERT Promoter

Objective:The human pancreatic cancer is one of the common clinical highly malignant, resectability rate is very low, the5-year survival rate is less than5%; for unresectable cases, the chemotherapy and radiotherapy, and can not improve the survival of patients period but with obvious side effects. Therefore, to improve the therapeutic effect and explore gene therapy for pancreatic cancer is important. Suicide gene targeting vector is a bottleneck restricting gene therapy. In this study, we constructed the gene vector of PE38KDEL controlled by hTERT promoter and mediated by adeno-associated virus.To observe is effcet of inhibite protein synthesis and induce of apoptosis on human pancreatic cancer cell in vitro and inhibite. xenograft tumor growth in vivo.Methods:l.The construction of PE38KDEL gene vector regulated by hTERT promoter and mediated by adeno-associated virus; human genomic DNA was extracted, the hTERT promoter was by PCR and inserted into the MLU I monoclonal sites of vector plasmid pAAV-hrGFP before the CMV promoter and named the plasmid of pAAV-hTERT-hrGFP, selected the plasmid pAAV-hTERT-hrGFP with hTERT promoter fragment was inserted forward direction by sequencing.The PE38KDEL fragment was gene synthesised and then insert the tail Xba I and Mun I sites, to constructed the plasmid pAAV-hTERT-PE38KDEL-hrGFP.Then the vector plasmid pAAV-hTERT-PE38KDEL-hrGFP and helper plasmid pAAV-RC pHelper were transfected HEK293cells using the calcium phosphate coprecipitation method, chloroform Processing-PEG/NaCl precipitation-chloroform extraction "three-step method for concentrating and purifying rAAV, the purity of the SDS-PAGE method to detect viruses, electron microscopy virus morphology and ELISA viral titer was determined.2In vitro:the experiment were divided into three groups:blank control group, empty virus Groups and pAAV-hTERT-PE38KDEL-hrGFP group.The MiaPaC2pancreatic cancer cells, and human embryonic lung fibroblast cells WI-38were transfected with rAAV of1×106v.p/cell,then observed the expression of green fluorescent to confirm tumor-specific of hTERT promoter.The effects of virus on protein synthesis, apoptosis, cell proliferation to human pancreatic cancer cells were detected by Leucine incorporation, caspase activity assay,TUNEL method, MTT method respectively.The changes of Bcl-2, Mcl-1, Bcl-XL were detected by Western Blot to preliminary explore induced gene mechanism of apoptosis by PE38KDEL.3, In vivo experiments:The tumor xenografts in nude mice model.were established by seeded5.0×106logarithmic growth phase MiaPaCa2human pancreatic cancer cells subcutaneously in15nude mice..The virus was delivered by tail vein injection or intratumoral injection method to observe the impact of the virus on tumor growth.The tumor-bearing nude mice were sacrificed30days after virus injection, the tumor specimens were removed to prepare conventional HE staining to observed the pathological necrosis of tumor cells in each group; through TUNEL method to detect apoptosis in tumor cells; the by Western blot detected PE38KDEL the expression.Results:1The correctness of plasmid pAAV-hTERT-hrGFP and pAAV-hTERT-PE83KEL-hrGFP were identified by restriction enzyme digestion and sequencing and the result were corret;The morphology of packaged rAAV were detected by electron microscopy.The high purity of the viras was detected by SDS-PAGE,the titer of virus was detected by ELISA assay,and the titer reached1.2×1012v.p/ml.2, In vitro experiments:the human pancreatic cancer cells MiaPaCa2and human embryonic lung fibroblast WI-38cells wred transfected by constructed virus and the expression of green fluorescent protein was observed by fluorescence microscope The expressive rate of Fluorescent protein was44.2%in MiaPaCa2human pancreatic cells and less than<5%in WI-38human embryonic lung fibroblasts. After transfection of virus for24hours,90%protein synthesis is suppressed, accompanied by cell decreased activity. The apoptosis rates of MiaPaCa2cells were:25.2%,37.4%,58.3%after transfection24h,48h,72h detected by MTT method. Caspase-3,-8,-9, were activated by activity assay and Westernblot72hours after transfection, along with the anti-Apoptotic protein Mcl-1is significantly reduced.3.In vivo experiments:From the graph of tumor growth, we found that there is a significant difference between the experimental group could significantly inhibit the growth of tumor cells were not found between the blank control group and the group of viruses without load. Under the microscope experimental group cells apparent apoptosis and apoptosis area of the region is greater than the control group. In addition, the of PE38KDEL was detected byConclusion1.The expressive gene vector of PED8KEDL regulated by hTERT promoter and mediated by adeno-associated virus-was successfully constructed, laid the foundation for invro and invivo study..2. In vitro experiments showed that hTERT promoter-specific regulated the PE38KDEL and GFP-specific expression in the telomerase positive MiaPaCa2human pancreatic cancer cells; rAAV-hTERT-PE38KDEL-hrGFP can inhibit MiaPaCa2human pancreatic carcinoma cell protein synthesis and induction of its withered death, apoptosis induced activation of caspase-3,-8,-9and inhibit the anti-apoptotic protein Mcl-1.3. In vivo experiment, rAAV-hTERT-PE38KDEL-hrGFP inhibited tumor growth significantly,and the mechanism for the inhibition of protein synthesis and induction of apoptosis were by activated caspase-3,-8,9and inhibitied the anti-apoptosis protein of Mlu-, the expression of PE38KDEL protein.was detected in MiaPaCa2human pancreatic cancer cells.