Effect Of Chemotherapy With Cisplatin And Rapamycin Against Hela Cells In Vitro And Inhibition Of Hela Cell Growth And The Expression Of HIF-1α And VEGF

Background:Cervical carcinoma is a very common malignant tu-mor In female carcinoma,the incidence of cervical carcinoma is secon-d only to mammary cancer.it is a disease that is very serious to the female.Although,to a great extent,cervical carcinoma is to be prevented,It inclines to have a relapse and transfer.To the cervical carcinomt is obvious that the chemotherapy is very important.Today,cisplatin sillgi-ve first place to chemotherapy combining with the 5FU,bolaimeisu etc.But effect is dissatisfing,and toxico-side effect is obvious,so it is i-mpending seek to new method curing cervix carcinoma.Recent studies reveal that rapamycin may be to reduce the HIF-1αand VEGF,some author discover that HIF-1αmay be the key translator-factor to adapt t-he hypoxia condition for most cells.HIF-1αwas over expression in multiple malignant neoplasm. HIF-1acan modulate expression of multiple target gene like vascular endothelial growth factor(VEGF),erythropoietin(EPO) and sustain energy metabolism of neoplasm,contribute angiogenesis,promote proliferation and metastasis of neoplasm. VEGF as imp-ortant in endothelial growth and tumorigenesis.but we have not find the report to the killing effect and mechanism about rapamycin comb-ined with Cisplatin.Objective:To evaluate the effect of combined use of cisplatin and rapamycin against Hela cells in vitro. To study inhibitive effect of cisplatin and rapamycin on the expression of HIF-laand VEGF and proliferation of cervical cancer Hela cell line in vitro. This study will help to identify HIF-1aas a new target for gynecologic tumor gene therapy.Methods: 1.Cell culture:Revive hela cells freezed in liquid nitrogen withroutine method,and inoculated in RPMI-1640 blended with 10% foetus Cattle blood serum cultured in culture bottle.then put the bottle in the incubator with the condition of 37 C5% C02.We observed the growth of cells every day.When 70% of the cells confluence,We digested them with 0.25% Trypsin and then made them into cell suspension. There were four groups in this experiment which was cisplatin, cisplatin, cisplatin and rapamycin,control group.2.The inhibitory effects of rapamycin and cisplatin, used alone or in combination,on the proliferation of Hela cells cells were measured with MTT assay and median-effect plot analys.3.The mRNA of the HIF-1a and VEGF weredetected before and after 72h rapamycin combined with Cisplatin by reverse transcription-polymerase chain reaction.4.Using SPSS 11.5 software to assay the data.Results:1.The proliferation of hela cells were measured with MTT The i-nhibitory rates on proliferation of Hela cells intervened by rapamycin, The inhibitory rates are dose-dependent, as the Graph 1,IC5o of rapa-mycin is 100.50ng/ml;The inhibitory rates on proliferation of Hela ce-lls intervened by rapamycin,The inhibitory rates are dose-dependent,too, as the Graph 2,ICso of Cisplatin is 1.25mg/ml;Rapamycin and cispatin showed synergistic effects in the chemotherapy of Hela cells (q> 1.15, King'S Formul),as the Graph 3,as mentioned above Combined use of r apamycin and cisplatin sign ificantly improves the chemotherapeutic e-ffect against Hela cells.2.The mRNA and protein expression of HIF-1a and VEGF in cer-vical cancer Hela cells was significantly downregulated by rapamycin group,cisplatin group,rapamycin combined with cisplat group in comp-ared with control group all(p<0.05);The mRNA and protein expression of HIF-la and VEGF was significantly down regulated by the comb-ined group compared with rapamycin group or cisplatin (p<0.05);The mRNA and protein expression of HIF-1a and VEGF was not obviousl y down regulated by rapamycin compared with cisplat group(P>0.05).T he results indicate, Combined use of rapamycin and cisplatin si-gnific antly improves the chemotherapeutic effect against Hela cells.Conclusion:1.Cell culture in vitro suggested that rapamycin only or Combine-d with Cisplatin inhibit cell proliferation of hela cell the effect of in hibition raised greatly,Showing dose dependency.2.we get the conclusion by RT-PCR that rapamycin combined wi-h Cisplatin could down-regulated the gene expression of HIF-1a and VEGF,then inhibiting cells proliferation.3.we get the conclusion by Western Blot that rapamycin combine-d with Cisplatin could down-regulated the protein expression of HIF-1 a and VEGF,then inhibiting cells proliferation.4.This experiment conformed rapamycin as immunosuppressive ag-nt has the action of anticancer.It has the function of synergism when combined,and increase the sensitivity of Cisplatin.Rapamycin combined with Cisplatin abundanced the chemtherpy of cervical carcinoma.