The Effects Of 5-ALA-PDT And HTERT ASODN Transfection Combination On Survival Of Human Ovarian Carcinoma Cells In Vitro

Ovarian cancer is one of the three most cancer in women's reproductive system neoplasia.And 70%patients are at advanced stage when diagnosed,owning to the lack of unique symptoms and sensible screening methods.Photodynamic therapy is newly developed treatment for tumor,and has being tested in the clinic for use in oncology.There is approximately 90%malignant tumors have over expressed telomerase activity.The telomerase activity was effective inhibited by the antisense oligonuletotide of hTERT.The aim of the research is to investigate the effects of sigle 5-ALA-PDT or antisense oligonuletide against hTERT and combined with them on the survival of human ovarian cancer in vitro.PART 1 Effects of photodynamic therapy with 5-aminolevulinic acid on the survival of human ovarian carcinoma 3AO cells in vitroObjective:To investigate the effects of 5-aminolevulinic acid(5-ALA) mediated photodynamic therapy(PDT) on human ovarian carcinoma 3AO cells in vitro.Methods:Morphologic changes were examined by microscope;the inhibition effects of 5-aminolevulinic acid mediated photodynamic(5-ALA-PDT) on 3AO cells were examined by MTT assay;Cell cycle of 3AO cells was observed by flow cytometry with PI staining;the death modality of the 3AO cells induced by 5-ALA-PDT was determined by flow cytometry with Annexin V-PI staining.;The expression of apoptosis related genes caspase-3 was detected with RT-PCR.Result:Typical morphologic transform of apoptosis appeared after 5-ALA-PDT.The inhibition rates of cells gradually increased with the increase of the 5-ALA concentration and laser irradiation dose(P＜0.01).5-ALA-PDT cause G_o/G₁ phase arrest of 3AO cell cycle(P＜0.05).The apoptotic and secondary necrotic rate of the ovarian carcinoma 3AO cells after photodynamic therapy significantly increased,and they were higher than those of the controls respectively(P＜0.01).The expression of easpase-3 gene was enhanced.Conclusion:5-ALA has a photodynamic effect on ovarian carcinoma 3AO cells;apoptosis might be one of the mechanisms of 5-ALA-PDT on ovarian carcinoma.PART 2 Effect of telomerase antisense oligonucleotides on the survival of human ovarian carcinoma cells in vitroObjective:To investigate the inhibitory effect of antisense oligodeoxynucleotide targeted against telemerase catalytic subunit gene hTERT on the growth of ovarian cancer cellsMethods:Antisense and sense oligonucleotide against human telomerase reverse transcriptase was synthesized and was transfected into ovarian cancer 3AO cells by lipofectin 2000;the expression of target gene hTERT mRNA was detected by RT-PCR;the proliferation and growth were investigated by MTT;the apoptotisis percentage was analyzed by flow cytometry.Result:The level of hTERT mRNA was decreased statistically in ASODN group,which is lower than the control(P＜0.05).The inhibition rates of cells gradually increased with the increased of the concentration of ASODN(P＜0.05).The efficiency depends on dose and period of administration.At 72h,2.0umol/L ASODN had the highest activity.hTERT ASODN can effectively induce cell apoptosis.Conclusions:Telomerase antisense hTERT inhibit the proliferation of ovarian carcinoma cells,probably depending on the way which induces apoptosis. PART 3 The synergism on suppression to the ovarian carcinoma cell multipication by application of 5-ALA-PDT and the hTERT ASODN transfection combinationObjective:To investigate the synergism on suppression to the ovarian carcinoma cell multipication by application of 5-ALA-PDT and the hTERT ASODN transfection combinationMethods:By application of 5-ALA-PDT and hTERT ASODN transfection combination,the proliferation and growth were investigated by MTT;the apoptosis percentage was analyzed by flow cytometry.Results:By application of the two methods,the very good synergism on suppression of ovarian carcinoma cells was caused,which was higher than that by use any one of methods above.The cells percentage of apoptosis was remarkably advanced(P＜0.05),contrast with use each one of methods separately.Conclusions:The synergism on suppression to the ovarian carcinoma cell multipication caused by application of 5-ALA-PDT and hTERT ASODN transfection combination.The synergism can effectively induce cell apoptosis.