| Object:Multidrug resistance (MDR) of Ovarian cancer cells is one of the major reasons that lead to chemothrapy falure, therefor how to overcome MDR to increase the curative effect of chemotherapy has become the focus. Rcombinant mutant human tumor necrosis factor(rmh-TNF) is a high- efficiency low-toxicity mutation developed by chinese scholar. This experiment aimed to investigate the reversing effect and its possible mechanism of rmh-TNF on Cisplatin-resistant Human Ovarian cell lines in vitro.Methods:Choosing Human multidrug-resistant Ovarian cell lines SKOV3/DDP ,Detect rmh-TNF's cell toxicity to SKOV3/DDP to select nontoxic dose and examine the vary of Cisplatin-resistant by MTT assay. The expression of BCL-2 protein and GST-πprotein were measured by flow cytometry. The expression of BCL-2 mRNA and mdr1 mRNA were studied by RT-PCR in SKOV3/DDP cells.Results:1 MTT assay results: Within the concentration of ( 50-3200 ) U/ml, rmh-TNF can inhibit proliferation of SKOV3/DDP cells in vitro. The inhibition ration was stepping up as the concentration increasing, When rmh-TNF's concentration was low than 122.34U/ml , rmh-TNF could not obviously proliferation inhibiting by regression analysis, and the experiment choose 100 U/ml for nontoxic dose. After being treated with 100U/ml rmh-TNF for 24,48,72h,the RF are 1.19,2.06 and 2.64 .2 FCM assay results: The FI-value of GST-πprotein expression in SKOV3 was 1,while 2.33±0.30 in SKOV3/DDP, it was significant difference in the expression of GST-πprotein (P<0.01).After being treated with rmh-TNF for 24,48,72h,GST-πprotein expression in SKOV3/DDP cell became reducing as action time increasing, and the FI-value are 1.82±0.13 1.73±0.28 and 1.31±0.17 , There was significantly difference in the GST-πprotein expression between before and after rmh-TNF action. (P<0.05) ;The FI-value of BCL-2 protein expression in SKOV3 was 1,while 2.62±0.15 in SKOV3/DDP, their BCL-2 protein expression was significant difference (P<0.01). After being treated with rmh-TNF for 24h,48h,72h,BCL-2 protein expression in SKOV3/DDP cell became reducing as action time increasing, and the FI-value are 2.24±0.20,1.71±0.19 and 1.30±0.15, There was significant difference of BCL-2 protein expression between before and after rmh-TNF action. (P<0.05) 3 RT-PCR detection results: Before being treated with rmh-TNF , The gene mdr1 mRNA was expressed in SKOV3/DDP cell, and hardly espressed in SKOV3 cell.After being treated by rmh-TNF for 24,48,72h mdr1 mRNA expression in SKOV3/DDP cell became reduing as action time increasing, the weakest expression was in the group with 72h. The mdr1/β-actin value are 0.66±0.05,0.52±0.04 and 0.45±0.03,rmh-TNF could inhibiting the expression of mdr1 mRNA in a certain extent. There was significant difference of mdr1 mRNA expression between before and after rmh-TNF action. (P<0.05); The value of BCL-2 mRNA expression in SKOV3 was 0.09±0.15,while 0.47±0.01 in SKOV3/DDP, their BCL-2 mRNA expression was significant difference (P<0.01). After being treated with rmh-TNF for 24h,48h,72h BCL-2 mRNA expression in SKOV3/DDP cell became reduing as action time increasing,the weakest expression was in the group with 72h. The mdr1/β-actin value are 2.24±0.20,1.71±0.19,1.30±0.15, rmh-TNF could inhibiting the expression of BCL-2 mRNA in a certain extent. There was significantly difference of BCL-2 mRNA expression between before and after rmh-TNF action.Conclusion: 1 Within the concentration of (50-3200)U/ml, rmh-TNF might inhibit proliferation of SKOV3/DDP cells in vitro 2 Nontoxic doses rmh-TNF could partly reverse the Cisplatin-resistant of SKOV3/DDP cells. 3 The mechanisnm of reverse might conclude three sides:①Within a certain drug concentration , rmh-TNF might reverse multidrug-resistance by inhibiting expression of mdr1 mRNA.②rmh-TNF could reduce the apoptosis-inhibition action by inhibition the expression of BCL-2 mRNA and BCL-2 protein to promote cells apoptosis.③Within a certain drug concentration, rmh-TNF might reduce the drainage of celltoxicitive drug's metabolizable producetion by inhibiting expression of GST-πprotein. |
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