| Department of general surgery, Anhui Provincial Hospital Affiliated to Anhui Medical University, Hefei 23001, China Cholangiocarcinoma is the most common malignant tumor in the liver and biliary system, which is second only the liver cell carcinoma.Due to lack of specific symptoms and signs, its unique biological characteristics and its anatomic features, it is very difficult to diagnose patients with cholangiocarcinoma in the early stage and resect the tumour radically .Along with its sensitivity to chemotherapy and radiotherapy, the prognosis is still very poor. In recent years cholangiocarcinoma in the crowd to the incidence rate of 5% evry year increase in gastrointestinal tumor is the fastest increase in the tumor, malignant tumors of the hepatobiliary system of 10% to 15%, so it is regarded as crucial to study the carcinogenesis mechanism of cholangiocarcinoma and find new efffective therapeutic approaches of them. The complex prescription danshen root is frequently used as promoting blood flow drug in Chinese traditional medicine, the essential component is tanshinone, neotanshinone and tanshinol. In recently years investigation was indicated that tanshinone have the pharmacologic function expand the aeteria coronaria, degrade blood apparent viscosity, antiplatelet, anti-infection and improve the heart, lung,brain, hepatic's ischemical reperfusion injuty and adjust the immune response. TanshinoneⅡA which is the liposolube constituent of dansen have the function acess cell-toxic function, induce cell differentiation to poly-tumor cells. At present tanshinoneⅡA was investigated the depressant effent to lung cancer, ovarian cancer, cancer of liver, gastric carcinoma and promyelocytic leukemia.This experiment carries on the related tanshinoneⅡA conduction function on human cholangiocarcinoma cell line HCCC-9810 is helpful to inveinvaginate the function that tanshinoneⅡA depressant effect to cholangiocarcinoma and the possible mechanism effection for exploring new according to prevention and cure cholangiocarcinoma.Objective To investigate the effect of tanshinoneⅡA on human cholangiocarcinoma HCCC-9810 cell line in vitro and its probable mechanisms.Methods HCCC-9810 cells were cultured in vitro and seeded into culture plates. At the state of culturing, observe the morphology of cell in culture flask by invert microscope every day. Following exposure to tanshinoneⅡA at different concentrations in different times,then HCCC-9810 cells were harvested and investigated. Cell growth inhibition was evaluated by MTT assay,cell quantification and colony-forming test. The morphologic changes were estimated by fluorescence-microscope and electronic microscope, cell cycle changes of HCCC-9810 cell line induced by tanshinoneⅡA were measured by means of flow cytometry, DNA ladder to assay the DNA degradation, RT-PCR technique was used to investigate the transcriptional changes of gene bcl-2/ bax/survivin. Furthermore, immunocytochemistry was used to examine the changes of their protein expression.Results (1)The growth inhibition of HCCC-9810 cell line induced by tanshinoneⅡA was enhanced in a time-dependent and dose-dependent manner and the half inhibiting concentration rate was 6.25μg/ml. The colony-forming rates were 73.6% and 22.9% respectively. (2) Apoptosis was detected by fluorescence-microscope /eletronic microscope/flow cytometry/DNA ladder determinations, some morphologic features of apoptosis, including cell nuclear condensation and apoptotic bodies can be seen by transmission electron microscopy after tanshinoneⅡA 72h.(3)The apoptotic rates increased in a dose dependent manner detected by flow cytometry, pre-G1 peak also was detected after expoure to 10μg/ml tanshinoneⅡA 72h. (4) Immunocytochemistry and RT-PCR showed that tanshinoneⅡA induced up regulation of bax and down regulation of bcl-2 and survivin. These results suggest that bcl-2,bax and survivin were involved in the process of tanshinoneⅡA induced apoptosis in human cholangiocarcinoma cell. .Conclusions :(1) TanshinoneⅡA can significantly inhibit the proliferation of HCCC-9810 in vitro in a dose-dependent and time-dependent manner.(2) TanshinoneⅡA could induce apoptosis in cholangiocarcinoma cell line HCCC-9810 in vitro, bcl-2,bax,survivin were involved in the process of tanshinoneⅡA induced apoptosis. |
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