| PART ONE The establishment of the human multi-drug-resistant bile duct carcinoma cellline(QBC939/ADM) and its biological characteristicsObjective: To establish the human multi-drug-resistant bile duct carcinoma cell line (QBC939/ADM) which can multiply and grow steadily in the culture fluid and to illustrate its biological characteristics.The culture fluid contains DMEM and adriamycin(concentration of adriamycin is1μg/ml).Methods: The human multi-drug-resistant bile duct carcinoma cell line (QBC939/ADM) was established by exposuring gradually increased with high and low alternated concentration of ADM persistently. The human cholangiocarocinoma cell line QBC939 and QBC939/ADM were to be effected by adriamycin(ADM),mitomycin(MMC),vindesine(VDS)and then detected respectively by MTT assay.Growth cycle of above-mentioned each group cells were performed by flow cytometry (FCM). To observe content of ADM in the human cholangiocarocinoma cell line QBC939 and QBC939/ADM by flow cytometry (FCM). The levels of their P-glycoprotein (P-gp) were detected by immunohistochemistry(IHC).Results: The inhibitive rate of ADM, MMC, VDS on the human multi-drug-resistant bile duct carcinoma cell line (QBC939/ADM) was rather lower than the human cholangiocarocinoma cell line (QBC939). on nothing intervention, the numbers of QBC939/ADM (S 43.69±3.26%,G2/M 11.12±0.31%)in S and G2/M phase were increased than QBC939 (S 32.05±3.26 %,G2/M 6.15±0.31 %) and decreased in G0/G1. (QBC939/ADM 45.19±3.58 %, QBC939 61.79±4.18 %).The content of ADM in the QBC939/ADM Group was much lower than in the QBC939 Group(P<0.05). The results of the IHC showed P-gp in the QBC939/ADM Group over-expressed.Conclusion: The human multi-drug-resistant bile duct carcinoma cell line (QBC939/ADM) can multiply and grow steadily in culture fluid which contains DMEM and adriamycin, concentration of adriamycin is1μg/ml. And its sensibility to the chems descend obviously. That may have something to do with the over-expression of the P-gp.PART TOW To study the growth-inhibited effect of verapamil connecting with chemotherapeutics on the human cholangiocarocinoma cell line QBC939Objective: To investigate the growth-inhibited effect of verapamil connecting with chemotherapeutics on the human cholangiocarocinoma cell line QBC939 and to illustrate its initial mechanism.Methods: The human cholangiocarocinoma cell line QBC939 to verapamil(VER),adriamycin(ADM),mitomycin(MMC),vindesine(VDS)and VER building in ADM,MMC,VDS individually were detected respectively by MTT assay. Growth cycle and apoptosis of above-mentioned each group cells were performed by flow cytometry (FCM). The levels of their P-glycoprotein (P-gp) were detected by immunohistochemistry. To observe content of ADM in the human cholangiocarocinoma cell line QBC939 by flow cytometry (FCM).Results: The inhibitive rate of the human cholangiocarocinoma cell line QBC939 to ADM(25.00μg/ml),MMC(2.00μg/ml),VDS(0.50μg/ml)were 48.84%,44.19%,45.76% ,but The toxicity of VER(2.50μg/ml) building in ADM ,MMC,VDS individually was very strong with an inhibitive rate 64.37%,52.68%,65.90% ,respectively. The apoptosis rate of the cells increased to 20.66%,14.18%,21.91%from 11.37%,7.25%,9.04% after VER was combined. The influence of growth cycle was not obvious on VER to ADM,MMC groups. However, cells of G2/M phase rised on VER to VDS group. The levels of P-glycoprotein (P-gp) on the human cholangiocarocinoma cell line QBC939 was 38.00%. The content of ADM in the VER/ ADM Group was higher than in the ADM Group.Conclusion: VER was able to enhance the relatively sensitivity of chemotherapeutics to the human cholangiocarocinoma cell.PART THERR To study verapamil reversing multi-drug resistance in the human cholangiocarocinoma cell line QBC939/ADMObjective: To investigate the reversal effect of verapamil on the human cholangiocarocinoma cell line QBC939/ADM and to illustrate its initial mechanism.Methods: The human cholangiocarocinoma cells line QBC939 and QBC939/ADM to verapami(lVER),adriamycin(ADM),mitomycin(MMC),vindesine(VDS)and VER building in ADM,MMC,VDS individually were detected respectively by MTT assay. Growth cycle and apoptosis of above-mentioned each group cells were performed by flow cytometry (FCM). The levels of their P-glycoprotein (P-gp) were detected by immunohistochemistry. To observe content of ADM in the human cholangiocarocinoma cell line QBC939 and QBC939/ADM by flow cytometry (FCM).Results: The inhibitive rate of the human cholangiocarocinoma cell line QBC939 and QBC939/ADM to VER,ADM,MMC,VDS were (1.25±0.08)%,(83.52±2.56)%,(82.08±2.58)%,(86.49±2.54)% and(1.51±0.15)%,(46.38.±1.32)%,(44.71±1.39)%,(48.15±1.31)% ,but The toxicity of VER building in ADM ,MMC,VDS individually was very strong with an inhibitive rate(89.68±3.61)%,(86.31±3.69)%,(97.63±3.72)% and(78.67±2.38)%,(70.04±2.41)%,(79.62±2.40)%,respectively. The apoptosis rate of the cells increased to (37.43±3.31)%,(26.50±3.39)%,(42.07±3.42)% and(26.52±3.30)%,(17.19±2.21)%,(29.91±3.34)% from (28.11±2.26)%,(18.32±2.28)%,(24.13±2.24)%and (11.37±2.22)%,(7.53±1.19)%,(9.02±1.23)%. Between of them were significant different(P<0.05). on nothing intervention, the numbers of QBC939/ADM (S 43.69±3.26%,G2/M 11.12±0.31%)in S and G2/M phase were increased than QBC939 (S 32.05±3.26 %,G2/M 6.15±0.31 %) and decreased in G0/G1. (QBC939/ADM 45.19±3.58 %, QBC939 61.79±4.18 %) .after QBC939 and QBC939/ADM cells line were used by ADM,MMC,VDS. Growth cycle of two groups was significant different. after VER was combined. The influence of growth cycle was obvious in two groups. However, cells of G2/M phase rised on VER to VDS group, obviously. The content of ADM in the QBC939 Group was higher than in the QBC939/ADM Group. The contents of ADM in the QBC939 Group was 260.48,It only was 101.33 in the QBC939/ADM Group. The levels of P-glycoprotein (P-gp) on the human cholangiocarocinoma cell line QBC939 was 38.00% and was 72.00% in QBC939/ADM cells line.Conclusion: VER can reverse the multi-drug-resistant of human bile duct carcinoma cell line (QBC939/ADM), VER was able to enhance the relatively sensitivity of chemotherapeutics to the human cholangiocarocinoma cell. That may have something to do with the over-expression of the P-gp. |
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