| In the last few years more and more attention has been paid to the use of photodynamic therapy (PDT) as a method with potential for the selective destruction of malignant tumors without producing any damage to adjacent normal tissue. Photodynamic therapy had been successfully used in treatment of gastrointestinal tumors with encouraging results. The patients must be to avoid light exposure for more than 20 days after administration common photosensitizers, the drawback may limit its applicability in treatment of tumors. δ-aminolevulinic acid(ALA) based photodynamic therapy is a promising PDT because of reducing skin photosensitivity and side effects. The accumulation of protoporphyrin IX (PPIX) following administration of ALA is more pronounced in colon malignant cells as compared to their normal counterparts in vitro and in vivo.Colon carcinoma could be killed sufficiently after PDT, but the killing effect was not enough satisfactory. In order to enhance the efficiency of ALA-PDT, combinations of PDT with cytosine deaminase (CD)/ 5-flurocytosine (5-Fc) suicide gene have been applied to improved treatment of colon carcinoma cell. The present study was designed to investigate the effects of Chk1, Cyclin E and Cdk2 on SW480 colon carcinoma cell after PDT.MethodOn the basis of SW480 colon carcinoma cell culture, CD suicide gene was transfected into SW480 cell, and then to investigate the synergistic effects and the effects of SW480 cell cycle checkpoints after PDT. We carried out the following experiments in this study.1.CD gene was inserted into the multicloning site of eukaryotic expression vector pcDNA3.0 to construct pCMVCD plasmid. The plasmid was replicated in Escherichia coli DH5α,and purified using E.Z.N.A.Plasmid Miniprep Kit I,and identified by electrophoresis and gene sequencing. The pCMVCD plasmid was used to transfect the SW480 cells using This work was supported by the National Natural Scicence Foundation of China(No.30271481).Lipofectamine2000 cationic lipid reagent. The killing effects of 5-Fc for SW480-CD cells and 5-Fu levels in the cell culture solution were studied by MTT assay. 2. Laser confocal fluorescence microscopy,high performance liquid chromatography(HPLC) and MTT assay were applied to investigate the intracellular fluorescence intensity induced by 5-Fc,5-Fu concentration in cell culture solution of SW480-CD, and synergistic effects of PDT combination with CD/5-Fc suicide gene on SW480 colon carcinoma cell respectively. 3. Flow cytometer, in situ hybridization method, reverse transcription polymerase chain reaction (RT-PCR),western blot ,immunohistochemistry etc were be applied to study these effects of PDT combination with CD/5-Fc suicide gene on cell cycle cheakpoint regulators Chk1,Cdk2,Cyclin E of SW480 colon carcinoma cell.Results 1.The pCMVCD plasmid was replicated, purified and cleaved by restriction endonuclease and identified by electrophoresis and gene sequencing. The fragment was our need CD sequenced. The pCMVCD plasmid was used to transfect SW480 cells using Lipofectamine2000 cationic lipid reagent.Then the SW480 cells were screened by G418 presence. G418-resistant clones were selected after 2-3 weeks.2. After adding 5-Fc for 4 days, 5-Fu was examined by HPLC in SW480-CD cell culture solution, no 5-Fu in SW480 cell culture solution. After 5-Fc exposure for 24 hours, 5-Fu levels in SW480-CD cell culture solution increased significantly at high 5-Fc concentration as compared with that of lower concentration in the same time(P<0.01).In SW480-CD cell culture solution, 5-Fu levels increased accompanying with increasing concentration of 5-Fc and elongating time in a relative dimension:0.001-10mMol/ml after 5-Fc exposure for 24 hours. 3.With increasing concentration of 5-Fc(from 0.001 mMol/ml to 10 mMol/ml) and elongating 5-Fc exposure time(from 0 hour to 72 hours) ,the surviving fraction of SW480-CD cells decreased by 5-Fc. But 5-Fc could not affect SW480 cell surviving fraction.4.Fluorescent intensities of PPIX became much stronger with prolonging time of A...
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