| Objective: Cervical carcinoma is a very common malignant tumor In female carcinoma, the incidence of cervical carcinoma is second only to mammary cancer. Operation were adapted to morning period but with macrotrauma and radiotherapy is a most cardinal therpy means but the function of endocrine were impaired after radiotherapy, the two means all lead to degrade the quality of life, especially the younger chemotherapy were applied to late-period or recrudescent patient. The traditional chemotherapeutics have different toxico-side effect and drug fast, and then lead to failure in the final. But with the incidence of cervical carcinomor promote younger staff and the increase of adenocarcinoma, cancer patitent require higher quality of life after treated. So it is extremely important to search some chemotherapeutics with hypotoxicity and low medicine reaction or using then to increasing the sensitivity of chemotherapeutics. In this study, we investigate the effect of chloromazine only or combined with cisplatin to hela cells, and seek to new method curing cervix carcinoma .Methods 1 Cell culture:Revive hela cells freezed in liquid nitrogen with routine 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 oC,5% 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.2 MTT estimating the growth suppression of cell: hela cells cultured in vitro were treated with Chloromazine only,Cisplatin only,Chloromazine combined with Cisplatin, then detected the inhibition of cells by MTT. Fluorescence Activated Cell Sorter(FACS) to observe proliferation inhibtion and apoptosls rate by Chloromazine only or combined with Cisplatin. Expression of bcl-2 and Bax were detected before and after Chloromazine only or Combined with Cisplatin by flow cytometry. The mRNA of bcl-2 and Bax were detected before and after 2h, 4h Chloromazine combined with Cisplatin by reverse transcription-polymerase chain reaction. Analyses the light quantum of DNA electrophoresis strip , as the product of amplification by gel scanner .β-actin, as inner standard, the proportionality of bcl-2 and Bax/β-actin is relative express to bc1-2 and Bax.3 Isobolanalysis analysis Iteraction Index to explain the action after combind.if Iteraction Index>1, they are rivalry, Iteraction Index<1, they are cooperation , if=1, their action are addition. Results1 Chloromazine , Cisplatin of three different concentrations (1×10-4,1×10-5,1×10-6mol/L)inhibited the growth of hela cells after added to drug for 24, 48, 72 hours , compaired with control array, the OD numerus of treated array have different descent, they have significant difference when compared. (P <0.05). Further more,with the increase of concentration and prolong of action time, the OD numerus fall-off accordingly. Showing the time and dose dependency.2 Chloromazine (1×10-6mol/L ) combind with Cisplatin of three different concetrations inhibited the growth of cells after added to drug for 24, 48, 72hours, compaired with control array,the OD numerus of treated array have different descent, they have significant difference when compared (P <0.05). Showing the time and dose dependency. The inhibition rate was more significant than that of applied alone (P <0.05). we presume they synergism each other..3 Cell cycle and apoptosis in hela cells by FCM display Chloromazine only or combined with Cisplatin after 24 hours, with the accrescence of concentration, cell in G0/G1 are increasingly raise. S and G2/M are reduce accordingly. showing the time and dose dependency. And they have displaied hypodiploid apoptotic peak. The ratio of apopatosis after Chloromazine array is 32.97%; the ratio of apopatosis after comband array are 54.71% , 65.11%.4 The expression of 2 kinds of protein in hela cells after 24 hours by FCM (FI) display :the FI of bcl-2 will decrease with the increased concentration of Cisplatin but the FI of Bax will increase with the increased concentration of Cisplatin. compaired with control array, FI of two kinds of protein have significant difference when compared (P <0.05).5 The result of RT-PCR shows: The mRNA expression of bc1-2 and Bax changed before and after using Chloromazine combined with Cisplatin. The expression level of bc1-2 decreased with the extension of processing time and the expression level of Bax increased with the extension of processing time. The proportionality of bcl-2 and Bax/β-actin is relative express to bc1-2 and Bax, they have significant difference when compared (P <0.05).6 The fomular of Isobolanalysis analysis Iteraction Index display: the Iteraction Index <1 when inhibition come to 50%, it certificate Chloromazine can assist Cisplatin on cell inhibit.Conclusions1 Cell culture in vitro suggested that Chloromazine only or Combined with Cisplatin inhibit cell proliferation of hela cells and induce to apoptosis, the effect of inhibition raised greatly. Showing the time and dose dependency. The inhibition rate of combind were more significant than applied chloromazine alone(P<0.05). It justify that chloromazine increase the sensitivity of cisplatin.2 It conformed that Chloromazine combind with Cisplatin could hold up hela cell in G1 stage and induced apoptosis.3 We get to the conclusion by FCM that Chloromazine combind with Cisplatin could down-regulated the expression of bcl-2 and up-regulated the expression of Bax. Further more, by RT-PCR, we can conclude that Chloromazine combind with Cisplatin could down-regulated the expression of bcl-2 and up-regulated the expression of Bax,then inhibiting cells proliferation and induced apoptosis.4 This experiment conformed Chloromazine as a traditional Psychotolytic has the action of anticancer. It has the function of synergism when combined, and increase the sensitivity of Cisplatin. Chloromazine combined with Cisplatin abundanced the chemtherpy of cervical carcinoma.
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