| Purpose: Lung cancer is one of the malignant tumors with the highest mortality rate,and its onset is insidious.Most of the patients are diagnosed with advanced stage,and the resection rate is low.At present,the clinical use of chemotherapy drugs,but with the progress of treatment,there is a progressive increase in resistance.In the process of drug resistance,the epithelial mesenchymal phenomenon of tumor cells appeared.Epithelial mesenchymal transition refers to the process of epithelial cells into mesenchymal cells with migratory ability.It is an important step in the process of tumor invasion and metastasis.In recent years,the study on the abnormal activation of PI3K/Akt signaling pathway in malignant tumor tissues,people began to pay attention to its relationship with the occurrence and transformation of tumor.With the deepening of research,it was found that the PI3K/Akt pathway is closely related to the occurrence and development of EMT,which may be one of the key factors in the process of EMT signaling.The motherland medicine thinks the occurrence of lung cancer with lung and spleen deficiency is closely related to treatment when Spleen Qi,mainly Pei Tu Sheng Jin.Buzhong Yiqi decoction is a representative prescription peitushengjin method,with Jianpi Yiqi resistant,effectively alleviate the side effects of chemotherapy.Clinical Buzhong Yiqi Decoction Combined with cisplatin and chemotherapy in treatment of non-small cell lung cancer has achieved good results.In this study,TGF-?1 was used to induce epithelial mesenchymal transition of human lung adenocarcinoma cell line A549,We selected the medicine serum of Buzhong Yiqi Decoction to interfere with this process,The changes of drug resistance and the mesenchymal related indexes of the cells were detected at the cellular and molecular level,The interference of the serum in the process of intracellular PI3K/Akt signal transduction pathway was also studied.This study aimed to investigate the mechanism of alleviating lung cancer drug resistance of Buzhong Yiqi Decoction which alleviate the epithelial mesenchymal transition of lung cancer cells,and investigate the function of Buzhong Yiqi Decoction in the process.Provide an experimental basis for the effective biological targets for Chinese medicine Buzhong Yiqi Decoction intervention of the drug resistance of lung cancer.At the same time as the rich peitushengjin combined treatment of non-small cell lung cancer and disease resistant treatment theory provides new ideas and methods.Material and method: 1.Experimental study on the relationship between epithelial mesenchymal transition and drug resistance in 1 human lung adenocarcinoma cells Using immunohistochemical staining,Western blot and Realtime PCR methods,detect the expression of epithelial-mesenchymal marker Vimentin and transcription factor Twist1 in the A549 and A549/DDP cells.Comparison of cell migration differences by cell scratch method of two groups;and then use the TGF-?1 to induce epithelial mesenchymal transition in A549 cells,and compare the changes of mesenchymal transition related factors(GST-? and Topo??)between A549 cells and primary A549 cell in protein and gene levels.2.Buzhong Yiqi Decoction intervention in human lung adenocarcinoma cells EMT reversal of drug resistance The preparation of 10% different concentration of Buzhong Yiqi Decoction and its function in the TGF-?1 induced A549 cells,Using MTT method to select the best drug concentration,Using immunohistochemical method,Western blot and Realtime PCR methods to detect the drug resistance and the mesenchymal related factors(MRP1,Topo??,ZEB1,Twist1)on the expression level of gene and protein.The experimental groups are blank serum group,blank serum+TGF-?1 group,and the best concentration of Buzhong Yiqi Decoction group + TGF-?1.3.Study on the mechanism of Buzhong Yiqi Decoction intervention of human lung adenocarcinoma cell EMT Applicate Buzhong Yiqi Decoction on A549 cells which are induced by TGF-?1,The experimental groups are blank serum group,blank serum+TGF-?1 group,inhibitor LY294002+TGF-?1 group and the best concentration of Buzhong Yiqi Decoction group+TGF-?1.The expression of PI3K/Akt pathway related factors p-Akt was detected by immunohistochemistry and Western blot.The expression of EMT related factors(Twist1,ZEB1)were detected by immunohistochemistry,Western blot and Realtime PCR.Results: 1.Compared to A549 and A549/DDP cells,A549/DDP cell migration speed(P<0.05).The immunohistochemical results showed that more Vimentin and transcription factor Twist1 protein expression in A549/DDP(P<0.05),the expression of Vimentin and Twist1 Realtime PCR for detecting A549/DDP gene level was also higher(P<0.05).2.Compared with A549 cells and TGF-?1 induced A549 cells,immunohistochemistry results showed that,compared with A549 cells,TGF-?1 induced resistance GST-? protein expression in A549 cells increased(P<0.05).The protein expression of Topo?? was decreased(P<0.05).Realtime PCR detection,compared with A549 cells,the expression on gene level of GST-? in A549 cells induced by TGF-?1 increased(P<0.05),the expression of Topo?? decreased(P<0.05).3.Screening the medicated serum of Buzhong Yiqi Decoction by MTT,,The results show that after 48 hours of treatment with the drug concentration increased,Buzhong Yiqi Decoction on inhibition of TGF-?1 induced A549 cells was significantly increased,But with the Buzhong Yiqi Decoction concentration on cell toxicity is enhanced,so the middle concentration of Buzhong Yiqi Decoction is the best(5.67g·kg-1 concentration is the best).4.Detecting the expressions of drug resistance and interstitial related factor(MRP1,Topo??,ZEB1,Twist1)with immunohistochemical method,Western blot and Realtime PCR,the expression of MRP1,ZEB1,Twist1 of blank serum,inhibitors of LY294002+ TGF-?1 group and Buzhong Yiqi Decoction containing serum+TGF-?1 group which are compared with the A549+TGF-?1 blank serum group,are decreased(P<0.05).The expression of inhibitors of LY294002+TGF-?1 group and Buzhong Yiqi Decoction containing serum+TGF-?1 group which were compared with the blank serum group,are increased(P<0.05).Topo?? is the opposite.5.In this study,the effect of the serum containing Yiqi Yiqi Decoction on A549 epithelial mesenchymal transition was detected,the expressions of p-Akt,Twist1 and ZEB1 with immunohistochemical method and Western blot,the levels of blank serum,inhibitors of LY294002+TGF-?1 group and Buzhong Yiqi Decoction containing serum+TGF-?1 group which are compared with the A549+TGF-?1 blank serum group,are decreased(P<0.05).The expression of p-Akt,Twist1 and ZEB1 in protein levels of inhibitors of LY294002+TGF-?1 group and Buzhong Yiqi Decoction containing serum+TGF-?1 group which were compared with the blank serum group,are increased(P<0.05).Realtime PCR was used to detect the expression of Twist1 and ZEB1 genes,and the results were consistent with the results of histochemical analysis.Conclusion: 1.Compared with A549/DDP,the expression of EMT related factors Vimentin and Twist in A549 were higher in protein and gene level in A549/DDP.2.TGF-beta 1 induced a higher expression drug resistance related factor of A549,GST-?,at protein and gene levels,whereas Topo?? was lower at protein and gene levels.3.With the optimal concentration of Buzhong Yiqi Decoction on TGF-?1 induced A549 cells,the EMT related factors Vimentin,Twist and drug resistance factors of GST-? and Topo?? decrease in protein and gene expression level in A549 cells,indicating the best concentration of Buzhong Yiqi Decoction by the intervention of A549 epithelial mesenchymal transition the process of drug intervention cell.4.With the optimal concentration of Buzhong Yiqi Decoction on TGF-?1 induced A549 cells,the expression of pathway protein p-Akt decreased;and at the same time,the expression of EMT related factors Twist?ZEB1 in protein and gene levels decreased,indicating the best concentration of Buzhong Yiqi Decoction intervention on epithelial mesenchymal transition of resistance pathway is through the intervention of PI3K/Akt signal pathway. |
PDF Full Text Request