Study On Inhibitory Action Of The Apoptosis And The Chemotherapy Sensitivity Of Signal Transduction PI3K/AKT/PTEN/mTOR In Cholangiocarcinoma

Demic concordance and unification in allomeric function is through the inter-identification, intercommunication and interaction between the cells intracellular and intercellular. These inter-actions between the cells intracellular and intercellular construct the extremely complicate signal network system through the controlling and regulation on the multi-layers. We define the cell signal transduction as what the cells accept the outsiders signal extracellular and transduct it to the inner. It's the premise for the normal vital movement to modulate delicately and abnormal signal transduction could lead to the pathologic course.Many investigations demonstrated that it could provoke the loss of control of the cell growth and even provoke the tumor generation or tumor apoptosis if certain link which controls the cell proliferation and differentiation could generate dysfunction. Among so many signal transduction pathways mediated the tumor cell apoptosis, the PI3K/Akt/PTEN/mTOR signal transduction pathway shows importance to the regulation of the apoptosis. The activation of this signal transduction pathway could inhibit the cell apoptosis induced by much stimulus, promote the progression of the cell cycle and promote the cell survival and proliferation and also participate in the vasiformation. It takes an important part in the tumor formation and participates in tumor invasion and transfusion. It shows the important pole in the generation and development of the malignant tumor through inducing the tumor cells survival, differentiation and vasiformation.Cholangiocarcinoma is mainly named as the extrahepatic bile duct malignant tumor. The prognosis of it isn't idealization. It's scarce in epidemiology and is about 0.01%～0.5% in biopsy, about 3% in the total count of the malignant tumor. It's about 0.07%～0.3% in domestic biopsy. The incident rate is increasing progressively. The treatment of the cholangiocarcinoma is troublesome in the clinical. It is important to explore the regulation of PI3K/Akt/PTEN/mTOR signal transduction pathway and the cross-link between other pathways which could increase the recognition of the functionary of this pathway and conduce to grasp the malignant behavior. And we could look for the new anti-tumor medicine through the pathway. So it will do good to the therapy of the cholangiocarcinoma.With the leading under professor Zou, our group of the topic has investigated deeply on the cholangioearcinoma bionomics. We concluded that the mutation, absence and the hyper-express of the oncogene such as k-ras, c-erbB-2 and c-myc and the anti-oncogene such as PTEN, DPC4/Smad, p15, p16 and the related gene of the apoptosis such as bcl-2 and bax, were close to the cholangioearcinoma. It's a great breakthrough in the research of the molecular cytogenetics in the cancer of extrahepatic biliary duct. We based on the prophase research, put our emphasis on the PI3K/Akt/PTEN/mTOR signal transduction pathway, and make some investigate on the apoptosis of the cholangioearcinoma and the fuction of the sensitization of the chemotherapy. We try to develop a new rationale and the theory of the experiment to the diagnosis and the therapy of the cancer of extrahepatic biliary duct. Restricted to the time, we are limited and superficialis level, and we will make the further step in the future. PART ONE The relationship between the PTEN-PI3K/AKT signal transduction pathway and mTOR signal transduction pathwayPAPER ONE Expression of PTEN and Akt, mTOR Gene Protein in cholangiocarcinomaObjective:To explore the express and relationship of the mTOR and PTEN in the cholangiocarcinoma, reveal the effect of the PI3K/PTEN/AKT/mTOR signal transduction in the prognosis of the cholangiocarcinoma.Method:Analyze the express of the gene mTOR, Aktand PTEN by the immunohistochemistry and RT-PCR method.Result:Compared to the ordinary tissue, the expression of the gene Akt, mTOR in the cholangiocarcinoma increase, but the expression of the gene PTEN decrease. At the same time, the correlation of the gene Akt, mTOR and gene PTEN is significant.Conclusion:The expression of the gene Akt, mTOR and PTEN in the cholangiocarcinoma are increase and decrease and the negtive correlation between two is significant. It reveals the gene Akt, mTOR and PTEN should play the important role of the prognosis of the cholangiocarcinoma. PAPER TWO: The relationship between the PTEN-PI3K/AKT signal transduction pathway and mTOR signal transduction pathwayPAPER TWO Investigation of the inhibition of the cell growthand down-regulation of the mTOR in the cholangiocarcinomaQBC939 cells transfected plasmid PTEN in vitroObjective:To investigate the effects of the tumor suppressor gene PTEN growing inhibition and down-regulating mTOR in cholangiocarcinoma QBC939 cells.Method:QBC939 cells were transfected with plasmids wild-type PTEN and C124S-PTEN in vitro. After transfection, we detected the express of the PTEN and phosphorylation of AKT and mTOR by western blotting. And Flow cytometry was used to analyze apoptosis and cell cycle of the transfected cells.Results:Compared with the control, the expression of phosphorylation AKT were decreased when transfected with the wild-type PTEN and mTOR were down-regulated respectively.Conclusion:PTEN phosphorylated AKT to active it. Actived AKT decreased the mTOR which led the tumor cells apoptosis and regulated the cell cycle. PAPER THREE: The construction and identify of pcDNA3.1-mTOR expression plasmidThe construction of the eukaryotic express plasmid of the gene mTOR andverificating it.Method1. Amplification the total length of gene mTOR's cDNA;2. Gathering the no-loading plasmid pcDNA3.1 and amplification mTOR, ambiocatalyst them with Hind III and BamH I. Recollection by and emendation and unification;3. Inoculation in LB nutrient medium, ambiocatalyst and unification with PCR;4. DNA sequencing. RESULT1. We could draw the conclusion from the MTT that it exists the obviously difference when associated with rapamycin in 3 experiment group than without rapamycin. IC50 increased obviously in the former. The difference has the obviously significance(p<0.01).2. It shows from the flow cytometry that the apoptosis rate in the 3 group affiliated with rapamycin to the cholangiocarcinoma cells increased obviously and raised up obviously than without rapamycin. The difference has the significant statistics difference(p<0.01). CONCLUSION1. Rapamycin which is the specific inhibitor of mTOR could effectively elevated the sensibility of chemotherapeutics: 5-FU, MMC, CELECOXIB resulting in the QBC939 cells inhibition in vitro. It could inhibit the activity through suppress the mTOR signal transduction pathway and consequently strengthen the therapeutic effect to the cholangiocarcinoma cells.2. We could draw the conclusion that the it could play the role in the inhibition of the apoptosis of the cholangiocarcinoma cells throught inhibition the mTOR signal transduction pathway. PAPER FOUR: Investigation of the inhibition of the cell growthand in the cholangiocarcinoma QBC939 cells transfectedplasmid mTOR in vitroObjective:To investigate the cell growing depressant effect of the mTOR signal transduction pathway and down-regulating anti-oncogene PTEN in cholangiocarcinoma QBC939 cells. Method:1. QBC939 cells were transfected with plasmids eukaryotic expression plasmid pcDNA3.1-mTOR and no-loading plasmid in vitro.2. After transfection, we detected the express of the mTOR by western blotting.3. Flow cytometry was used to analyze apoptosis and cell cycle of the transfected cells.4. Detect the expression of the protein PTEN after transfection. Results:1. When transfected with the pcDNA3.1 -mTOR, we identified it successfully.2. The group transfeced with the pcDNA3.1-mTOR show that the growth velocity increase more rapidly than the group transfected with the no-loading pcDNA3.1 and without transfected.3. The expression of the protein PTEN decreased obviously after having been transfected the mTOR. It shows the obvious difference between two group. 4. Flow cytometry experiment shows the apoptosis rate of the group transfected was (4.3% ±0.8)%. Contrasting to it, the group without transfected was (8.2% ±1.3)%. It has the statistics significance. Conclusion:1. It could increase the growth of the cholangiocarcinoma cells after transfected the mTOR successful. It provides the evidence to the spreading the mTOR specific inhibiter in the clinical.2. The expression of the protein PTEN increased after transfected the mTOR in the QBC939. It show that mTOR could feedback regulate the expression of the protein PTEN through the PI3K/AKT/PTEN. PART THREE: The study of the rapamycin and LY294002 affecting the apoptosis inhibitory and sensitization of the chemotherapeutics to the cholangiocarcinomaPAPER FIVE: The study of the specific inhibitor Ly294002 of the signaling pathway PI3K/AKT increasing the sensitivity of the cholangiocarcinoma in vitroObjective:To explore the specific inhibitor LY294002 of the signaling transduction pathway PI3K/AKT increasing the sensitivity of the regular chemotherapeutic agent of the cholangiocarcinoma.Method:By MTT detect the inhibition of the cholangiocarcinoma when single applied with 5-FU, MMC, CELECOXIB contrast to associated with LY294002. At the same condition, detect the apoptosis ratio by the flow cytometer.Result:The phosphorylation inhibitor LY294002 can increase the sensitivity of the 5-FU, MMC, and increase the apoptosis ratio in vitro.Conclusion:The phosphorylation inhibitor LY294002 can enhance significantly the sensitivity of the cyto-inhibition of the chemotherapeutic agent. Inhibit the PI3K/AKT signaling pathway could make the synergistic effect to the treatment of cholangiocarcinoma. PAPER SIX: The study of the specific inhibitor of the signaling pathway mTOR inhibiting the growth of the cholangiocarcinoma in vitroObjective:To explore the specific inhibitor of the signaling pathway mTOR of its increasing the sensitivity of the regular chemotherapeutic agent of the cholangiocarcinoma.Method:1. We explored the sensitivity of the chemotherapeutics with the intervention of the specific inhibiter of mTOR of the cholangiocarcinoma.2. At the same condition, detect the apoptosis ratio by the flow cytometry.Result:1. The group association with the rapamycin show that the IC50 increase obviously than the group single with the chemotherapeutics from the MTT experiment.2. From the flow cytometry, the apoptosis of the cholangiocarcinoma cellsincreased obviously in the group association with the rapamycin than single with the chemotherapeutics. The difference has statistics significance (p<0.01) .Conclusion:1. The specific inhibitor of the signaling pathway mTOR can enhance significantly the sensitivity of the cyto-inhibition of the chemotherapeutic agent.2. Inhibit the mTOR signaling pathway could make the synergistic effect to the treatment of cholangiocarcinoma. SummaryThis topic put the importance on the signal transduction pathway. We investigated the close relationship between the PI3K/AKT/PTEN signal pathway and mTOR signal pathway and study the important action of the mTOR and anti-oncogene PTEN. We explore the effect of the specific phosphorylation inhibiter of PI3K to the PTEN and the specific inhibiter of the mTOR to the signal transduction pathway. We draw the conclusion as below:1. We are sure that the PI3K/AKT/PTEN signal transduction pathway and mTOR signal transduction pathway is close in promoting the cell survival and having the intimate positive feedback and degenerative relationship in promoting the cells survival.2. We put our emphasis on the close relationship between the anti-oncogene PTEN and mTOR from the protein level and mRNA level. The expression of the mTOR would decrease with the transfection of the plasmid PTEN, and the expression of the PTEN would decrease with the transfection of the plasmid mTOR.3. We are sure from the experiment that the specific inhibitor of PI3K could increase the sensitivity of the chemotherapeutics and induce the apoptosis of the cholangiocarcinama cells. It shows the important role of the adjustment of the apoptosis depressant effect of the cholangiocarcinoma of the PI3K/AKT/PTEN/mTOR signal transduction pathway.4. Our experiment provided the theory what the mTOR specific inhibiter rapamycin could be resist the tumor. We try to look for the new target to increase the effect of the chemotherapeutics for the cholangiocarcinoma.5. We success to construct the eukaryon plasmid vector leading in the domestic which give the powerful assistance in the future research in the PI3K/AKT/PTEN/mTOR signal transduction pathway.