| ObjectiveTo observe the reverse effect of Brucea javanica oil emulsion on drug-resistant human ovarian carcinoma cell line A2780/ DDP.Then explored it's mechanism to provide some experimental references to the treatment of ovarian carcinoma using Brucea javanica oil emulsion.MethodsMTT assay was used to analysis the cell cytotoxics of A2780 and A2780/DDP cell line.Cell growth rate=0D valuesexperimentalgroups/ 0D valuecontrolgroup×100%.The experiments were repeated 3 times. Cell growth inhibiting curve was drew by excel software with the horizontal axis and longitudinal axis representing concentration of Brucea javanica oil emulsion and cell growth ratio respectively.The median concentration(IC50) was calculated by the software for IC50 calculation,Resistance index(RI)=IC50 (A2780/DDP)/IC50(A2780).Calculated in accordance with the above two A2780/DDP and A2780 cell lines IC50 and the RI(not reversal agents).The same method of Brucea javanica oil emulsion of the non-toxic doses of IC98.Add A2780/DDP at IC98 concentration of Brucea javanica oil emulsion,after 24h culture, inoculated into 96-well plates,MTT determination of cisplatin on the cell line A2780 / DDP's IC50 and RI'(add reversal agents). And calculate the resistance in multiples of RI/RI'The cell cycle was determined by flow cytometry.The experiments were divided four groups:Blank control group(control group),Brucea javanica oil emulsion group(traditional chinese medicine group),DDP group(western medicine group)and DDP plus Brucea javanica oil emulsion group(traditional chinese medicine + western medicine group).Three parallel samples were installed for each group,1×105 cells were inoculated into culture flask of 25 cm2.Collecting the treated cells of each group, then fixed and stained.At last,cell apoptosis was detected by flow cytometry and the results were analyzed by the multicycle software.Immunoflurescence method were applied to detect the expression of Bcl-2 of Blank control group,Brucea javanica oil emulsion group,DDP group and DDP plus Brucea javanica oil emulsion group.All the performance was carried out according to the kit instruction.Western blot method were applied to detect the expression of MRP of Blank control group,Brucea javanica oil emulsion group,DDP group and DDP plus Brucea javanica oil emulsion group.All the performance was carried out according to the kit instruction. Results1.The growth inhibiting of Brucea javanica oil emulsion to A2780/DDP cells of Human ovarian Carcinoma was enhanced with the increase of Brucea javanica oil concentration.2.Add A2780/DDP at IC98 concentration of Brucea javanica oil emulsion,after 24h culture,the RI/RI' is 5.49(5.6/1.02). The reverse effect of Brucea javanica oil emulsion on drug-resistant human ovarian carcinoma cell line A2780/ DDP.3.Blank control group,Brucea javanica oil emulsion group, DDP group and DDP plus Brucea javanica oil emulsion group was 1.98%,2.11%,3.15%,10.3%respectively.The arrest effect was much more evident when Brucea javanica oil were applied combinatively than either of them was used alone.4.Immunoflurescence method were applied to detect the expression of Bcl-2.When Brucea javanica oil was applied combinatively,the expression of Bcl-2 protein was increased much more than that of either of them used alone.While the expression of Bcl-2 protein was decreased on the counterparts (P＜0.05).5.Western blot method was applied to detect the expression of MRP.When Brucea javanica oil was applied combinatively,the expression of MRP protein was increased much more than that of either of them used alone.While the expression of MRP protein was decreased on the counterparts.Conlusion1.The reverse effect of Brucea javanica oil emulsion on drug-resistant human ovarian carcinoma cell line A2780/ DDP.2.The mechanism of Brucea javanica oil emulsion on drug- resistant human ovarian carcinoma cell line A2780/ DDP may be cell apoptosis and down-regulating the expression of Bcl-2 and MRP protein.
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