| Esophageal cancer is a common malignant tumor in china. Most patients have been progressing to advanced stage at the time of diagnosis. All the efforts of conventional treatments including operation, radiation and chemotherapy has little influence on the advanced disease with metastatic and recurrence, even exerting great side effect to patients. In searching for a new way to the treatment of such a malignant disease, the gene therapy has been introduced and displayed its promising. One of the landmark discoveries is application of the suicide gene to cancer treayments, the prototypic example of which is the herpes simplex virus thymidine kinase (HSV-tk) gene. The basic mechanisms of HSV-tk to antitumor include: (1) "suicide effect": HSV-tk phosphorylates the pro-drug ganciclovir (GCV) into a toxic nucleotide analogue, which inhibits acticity of the DNA ploymerase and becomes incorporated into the cellular DNA as a terminal factor to block cell proliferation, finally leading to cell death. (2) "bystander effect": Even though there are 10% or less tumor cells transfected with HSV-tk gene,the rest of tumor cells will be killed after treated with GCV. The mechanism of the bystander effect in vivo remains elusive, although some studies have suggested that gap junction formation between neighboring cells is an important contributing factor.The unique advantages of bystander effect have greatly improved the therapeutic efficacy of HSV-tk gene, and HSV-tk/GCV suicide gene system has been proved to be effective in many experimental studies in vivo or in vitro, and even in clinical application, suggesting that it is becoming more effective and more poromising inclinical application. Some results from clinical phases I and II have demonstrated the good clinical effects, but its use in human esophageal cancer has not been reported so far. In the present study, we constructed a recombinant eukaryotic expression vector containing HSV-tk cDNA, which would be transfected into esophageal cancer cell Eca-109 by liposome, and also investigated the expression of the vector in transfected esophageal cancer cell lines by RT-PCR. 1. Method1.1 Construction of eukaryotic expression vector containing the HSV-tk gene: The plasmid pBabe-tk was transferred into competent E.coli JM109, positivecoloniess were picked up, and plasmid DNA was prepared, After digesting plasmid pBabe-tk with BamHI, a fragment of the llOObp tk gene was recovered by agarose gel eleclrophoresis, extracted and then purified. Similarly, digesting eukaryotic expression vector pcDNA3 with BamHI and extracting linear vector pcDNA3 were carried out. In order to prevent from circling itself, the 5 '-PO4 (phosphoric acid) was removed from the linear vector pcDNA3 with calf intestine alkaline phosphatase, subsequently the vector was purified by phenol/chloroform extration method. The tk gene fragments and linear vector fragments were ligated by T4 DNA ligase, and incubated at 16癈 overnight. After the products of ligation were transferred to the competent E.coli, coloniess from LA solid plates were selected for preparing plasmid to identify.1.2 Characteristics of recombinant vectorThe tk gene fragment was confirmed to insert into the linear vectors by digesting recombinant vectors with BamHI. The recombinant vector must be characterized to prove whether it is forward ligated, because the ligation was performed after a single but not double restriction endonuclease digestion. According to different fragments produced after digestion with EcoRV, direction of the insert was identified. The forward recombinant vector was named pcDNA3-tk. pcDNA3-tk, which was sequenced with reverse primer Sp6 for further confirming.1 .3 Transfection of esophageal cancer Eca- 1 09 cellsEsophageal cancer Eca-109 cells were flourished approach 90% convergeantion for transfection and seeded at 1 10cells/well in 24-well plates. The esophageal cancer cells were transfected with pcDNA3-tk and pcDNA3 reapectively usinglipofactamine as recommended by the manufacturer, co...
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