Experimental Study Of Norcantharidin On Proliferative Growth And Apoptosis Of Human Cholangiocarcinoma Cell Line QBC939

Objective:To study the growth-inhibited effect of norcantharidin(NCTD)on the human cholangiocarcinoma cell line QBC939 and to illustrate its initial mechanism.Methods:The cell grown well was used to experiment after QBC939 was revived.It was divided into control and experiment groups to culture.The experiment groups cell was treated with different concentration of NCTD.The control group cell was treated with DMEM.After a piece of time QBC939 was detected as follows:(1)MTT methods:To inoculate QBC939 cell into 96 wells cultivation board and culture for about 24h.Then the experimental groups cell was cultured with 0.125,0.75,2.5,10,120μg/ml NCTD,and the control group cell was cultured with DMEM for 48h.To add 25μl MTT per well in cultured QBC939 cell about 4-8h,and dissolved with 100μl DMSO after the cell was dyed for 10min.Then to detect inhaling light degree in detection instrument.The same way was used afer QBC939 was co-cultured with NCTD for 12h,24h,36h,48h,72h.(2)Detection of cell cycle and apoptosis:To inoculate QBC939 cell into 6 wells cultivation board and culture for about 24h.Collecting the cell that had been treated by different concentration of NCTD for 48h,washed with PBS,fixed with alcohol,dyed with PI,placed it for 20min without light,and analysed cell cycle and DNA content with flow cytometry.(3)Detection of caspase-3 protein by immunohistochemistry:To inoculate QBC939 cell into 6 wells cultivation board and culture for about 24h.Then the experimental groups cell was cultured with 0.125,0.75,2.5,10μg/ml NCTD,and the control group cell was cultured with DMEM for 48h.The remains of procedures was operated as instruction of reagent box.The data was collected with BI2000 image analysis system.Results:(1)MTT methods:NCTD displayed inhibitory effect on growth of QBC939 from 0.125,0.75,2.5,10,120μg/ml after 48h(F=2.63,P=0.007).The survival rates were(94±10)%,(77+12)%,(47±5)%,(26±4)%,(15±3)%.It was in a dose-and time-effective dependent manner.Dose-effect curve was drew and IC50 value is 3.66±1.14(μg/ml);The results shows that the survival rate descended following the increase of time(F=2.40,P= 0.0127).(2)Detection of cell cycle and apoptosis:The levels of cell apoptosis rate had statistic significance(P＜0.001)and the rate of cell apoptosis increased following concentration increase of NCTD.The percentage of G2-M phase rised compared with control group after QBC939 cell was co-cultured with 2.5μg/ml NCTD about 48h.There was significant difference between control group and 2.5μg/ml NCTD group(P＜0.05).(3)Detection of caspase-3 protein by immunohistochemistry:The population means of value of gray scale was not all equality among different concentrations(F=223.103,P＜0.001).The other two groups at random had statistic significance except between 0.125μg/ml and 0.75μg/ml group(P＜0.05).The expression of the protein caspase-3 elevated following the increase of NCTD concentration when the concentrations were 0.75,2.5,10μg/ml.Conclusion:Study proves that NCTD can suppress proliferation of QBC939 cell line,and the mechanism might be related to the induction of cell apoptosis and blockade of cell cycle.