| Background and objectives: Cholangiocarcinoma is a malignant tumor of the hepatobiliary system,and its incidence rate is only inferior to hepatocellular carcinoma in hepatobiliary system tumors.At present,surgical treatment is the most effective treatment.However,due to the atypical symptoms,deep lesions and high degree of malignancy,cholangiocarcinoma is generally in the non early stage of diagnosis,so only a few patients have the opportunity of radical resection.The chemotherapy which is based on cisplatin is recommended by clinical guidelines,but cholangiocarcinoma has natural or acquired cisplatin resistance.Therefore,it is of great significance to explore the mechanism of cisplatin resistance in cholangiocarcinoma.But there are few studies on the mechanism of cisplatin resistance in cholangiocarcinoma.At present,it is believed that the drug resistance mechanism of cholangiocarcinoma is related to mitochondrial dynamics,and the changes of cytoskeleton are closely related to mitochondrial dynamics.This study aims to explore the mechanism of cisplatin resistance in cholangiocarcinoma through mitochondrial dynamics and its relationship with cytoskeleton,so as to provide an effective reference for the treatment of cholangiocarcinoma.Methods: Human cholangiocarcinoma cell line RBE was used as the research object.The concentration gradient of cisplatin was 0 ug / ml,25 ug / ml,50 ug / ml and 100 ug / ml.Among them,0 ug / ml was the normal control group,and RBE cells were treated for different time to obtain RBE cells with certain changes in drug resistance;MTT assay was used to detect the cell survival rate,and Western blot was used to detect the mitotic fusion related proteins Mfn1,Mfn2,OPA1 and FIS1;The mitochondrion and cytoplasmic proteins were separated by using mitochondrial separation kit,and the mitochondrial fusion related proteins were detected by Western blot;Western blot was used to detect cytoskeleton related proteins in total proteins;The mitochondrial and cytoplasmic proteins were separated by mitochondrial separation kit,the cytoskeleton related proteins were detected by Western blot;Mitochondria were stained with mitochondrial specific marker dye,and the changes were observed by confocal laser scanning;Cytoskeleton was stained with cytoskeleton specific dye,and the changes were observed by confocal laser scanning.The sensitivity of cholangiocarcinoma cells to cisplatin was observed by using cytoskeleton inhibitors.Results:(1)After RBE cells were treated with different concentrations of cisplatin(0 ug / ml,25 ug / ml,50 ug / ml,100 ug / ml)for different time(6 hours,12 hours,20 hours),the changes of mitochondria were more obvious in 12 hours and 20 hours drug groups.(2)After RBE cells were treated with different concentrations of cisplatin(0 ug / ml,25 ug / ml,50 ug / ml,100 ug / ml)for 20 hours,the changes of mitochondria were observed under electron microscope.(3)The expression of mitochondrial fusion related proteins: In the total cell protein,the levels of Mfn1,Mfn2 and OPA1 of RBE cells in the 6-hour drug group were not significantly different from those in the control group,while the FIS1 protein level was up-regulated;There was no significant difference in Mfn1,Mfn2,OPA1,FIS1 protein levels in RBE cells of 12 hours treatment group compared with control group;The protein levels of Mfn1,Mfn2 and OPA1 of RBE cells in the drug group were not significantly different from those in the control group,but the protein level of Drp1 was down-regulated and the level of FIS1 protein was up-regulated;The levels of Mfn2 and Drp1 were down-regulated in the mitochondrial protein which in RBE cells treated with drugs for 20 hours;In the cytoplasmic protein,drp1 protein level was down-regulated.(4)The expression of cytoskeleton related proteins: There was no significant difference in the levels of F-actin and non-muscle Heavy Myosin Chain ?B protein in RBE cells between the 6 hours drug group and the control group;There was no significant difference in the level of F-actin protein in RBE cells between the 12 hours drug group and the control group,the protein levels of non-muscle Heavy Myosin Chain ?A and non-muscle Heavy Myosin Chain ?B were down-regulated;There was no significant difference in the level of F-actin protein in RBE cells between the 20 hours drug group and the control group,the protein level of non-muscle Heavy Myosin Chain ?B was down regulated,the protein level of non-muscle Heavy Myosin Chain ?A was down regulated in 100 ug / ml drug group;In the mitochondrial protein which in RBE cells treated with drugs for 20 hours,there was no significant difference in the level of F-actin protein between the drug group and the control group,the protein levels of non-muscle Heavy Myosin Chain ?A,non-muscle Heavy Myosin Chain ?B were down regulated;In cytoplasmic proteins,there was no significant difference in the level of F-actin protein between the drug group and the control group,the protein levels of non-muscle Heavy Myosin Chain ?A and non-muscle Heavy Myosin Chain ?B were down regulated.(5)After RBE cells were treated with different concentrations of cisplatin(0 ug / ml,25 ug / ml,50 ug / ml,100 ug / ml)for different time(6 hours,12 hours,20 hours),the cytoskeleton changed.(6)After RBE cells were treated with different concentrations of Blebbistatin,the cytoskeleton and mitochondria of RBE cells were changed.(7)After RBE cells were treated with Blebbistatin?Latrunculin A?Latrunculin B,the resistance of RBE cells to cisplatin was enhanced;After RBE cells were treated with CCCP,the susceptibility of RBE cells to cisplatin was enhanced.Conclusion: After treatment with cisplatin on human cholangiocarcinoma cell line RBE,the mitochondria and cytoskeleton were changed.The changes of mitochondrial dynamics and cytoskeleton in cholangiocarcinoma cells may be related to its cisplatin resistance.When the drug is used to treat cholangiocarcinoma cells to make the cytoskeleton change,it may cause mitochondrial dynamics changes and affect the cisplatin resistance. |
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