| Esophageal cancer(EC)is one of the high-frequency gastrointestinal tumors and has a high mortality rate in malignant tumors.The pathogenesis of esophageal cancer are mainly divided into 2 pathological types,esophageal squamous cell carcinoma(ESCC)accounts for about 4/5,and the other is esophageal adenocarcinoma(EAC).Esophageal cancer in the United States and other Western countries is mainly adenocarcinoma,and Asia and China are mainly squamous cell carcinoma.China is the country with the highest morbidity and mortality of esophageal cancer in the world.The prevalence of esophageal cancer in Linzhou Henan province,Cixian,and Shexian in Hebei province is even 10 times the world average.The global cancer surveillance report 2010-14(CONCORD-3)shows that the 5-year survival rate of esophageal cancer patients in most countries and regions is between 10-30%,while the 5-year survival rate of esophageal cancer patients in China is still low.ESCC still poses a serious threat to human health.Invasion and metastasis are the basic biological characteristics of malignant tumors,and are the main cause of death in patients with esophageal cancer.The current ESCC western medicine treatment scheme does not have a good effect on invasion and metastasis.Even if surgical resection or widespread use of systemic chemoradiotherapy,the overall survival rate of patients is still not optimistic.At the same time,there is no effective means to inhibit the invasion and metastasis of esophageal cancer.The main reason for the invasion and metastasis of esophageal cancer is that the process of ESCC cell migration and invasion is very complicated,and many mechanisms are not clear.Therefore,it is particularly important to explore the regulation process of ESCC migration and invasion.Qigefang(QGF)was derived from Qigesan,which was created by Zhongling Cheng,a well-known doctor in the qing dynasty in China.His book "Yi Xue Xin Wu" said Qigesan is mainly used for "Yege" treatment.Hebei Province is one of the highest incidence areas of esophageal cancer in China.Therefore,we have treated a large number of patients with esophageal cancer.In combination with the etiology and pathogenesis of esophageal cancer and the clinical manifestations of patients,Qigefang is widely used in the treatment of esophageal cancer.Clinical studies have shown that QGF can significantly improve the symptoms of esophageal cancer and postoperative patients,and has shown a tendency to inhibit the recurrence and metastasis of esophageal cancer.However,the specific mechanism by which QGF inhibits esophageal cancer cell invasion and migration remains unclear.Studies have shown that Growth arrest-specific 6(Gas6)and the receptor tyrosine kinase Aexelekto(Axl)are highly expressed in tumor tissues and cells such as oral squamous cell carcinoma(OSCC).The combination of Gas6 and Axl altered cell functions,including migration,proliferation,and survival,recent studies have shown that the Gas6/Axl axis affects the invasion and survival of prostate cancer cells during bone marrow metastasis.There are also reports that Gas6/Axl play a promoting role in gastric cancer,lung cancer invasion and metastasis.Studies have reported that the Gas6/Axl-PI3K/AKT pathway promotes OSCC invasion,and the Gas6/Axl-NF-?B pathway enhances OSCC cell invasion/migration capabilities.However,how Gas6/Axl mediates the process of esophageal cancer cell migration and invasion is unclear,and whether it can promote the effect by enhancing related signaling pathways remains to be further studied.The study of the previous protein chip in this study showed that QGF can reduce the expression of Gas6 in esophageal cancer cells.It has been reported that Gas6 promotes the formation of esophageal cancer.Therefore,we hypothesized that QGF inhibits the invasion and migration of esophageal cancer cells by inhibiting Gas6 protein,regulating Gas6/Axl signaling pathway and thereby inhibiting the downstream proteins PI3K/AKT and NF-?B,and affecting the occurrence of epithelial-mesenchymal transition(EMT).The purpose of this study was to investigate the effect of QGF on the regulation of Gas6/Axl and downstream signaling pathways in esophageal cancer cells,and to inhibit the invasion and migration of cells,in order to provide a theoretical basis for the treatment of esophageal cancer by QGF.In order to explore the above issues,this study selected postoperative cancerous tissues and adjacent tissues of esophageal patients,and human squamous epithelial esophageal cancer cells ECA109,TE1,and TE13 as the research objects,which were divided into three parts.Part ? Expression of Gas6 and Axl in human esophageal squamous cell carcinomaObjective: To observe the expression of Gas6 and Axl in human esophageal cancer tissues and adjacent tissues and their correlation with tumor markers.Methods: 1.Human esophageal cancer tissues and adjacent tissues randomly were select to make pathological sections.2.HE staining test confirmed the pathological diagnosis and type of esophageal cancer.3.The expression of Gas6 protein in esophageal cancer tissues and adjacent tissues were determined by immunohistochemistry.4.Immunofluorescence and laser confocal microscopy were used to determine the expression of Axl in esophageal cancer tissues and adjacent tissues.5.Tumor markers of esophageal cancer patients were determined by electrochemical luminescence method.Results: 1.Esophageal cancer tissue and adjacent tissues were observed by HE staining.Microscopic examination showed that all esophageal cancer groups were compared with adjacent tissues,squamous cell carcinomas are degenerate and swollen,with irregularity and significant atypia.The nuclei become larger and deeper stained.2.The immunohistochemical results of Gas6 in esophageal cancer and adjacent tissues show: Gas6 expression in esophageal cancer tissue and adjacent tissues increased,and the expression of Gas6 in ESCC group was significantly higher than that in NC group.Gas6 expression in esophageal cancer patients was positively correlated with tumor markers CEA,SCC and CYFRA21-1.3.Immunofluorescence and laser confocal observation of esophageal cancer and adjacent tissues showed that Axl immunofluorescence in ESCC and adjacent tissues The expression was increased,and the expression of Axl in the ESCC group was significantly higher than that in the adjacent tissues of the NC group.Axl expression was positively correlated with tumor markers CEA,SCC and CYFRA21-1 in patients with esophageal cancer.Summary: The expression and activity of Gas6 and Axl were significantly increased in the pathological tissues of human esophageal squamous cell carcinoma.However,the expression of adjacent tissues was not obvious or less.The expression of Gas6 and Axl in esophageal cancer patients was positively correlated with the expression of tumor markers CEA,SCC and CYFRA21-1.Part ? Qigefang inhibits the migration ability of esophageal cancer cells by regulating Gas6/AxlObjective: To take esophageal cancer Eca109 and TE1 cells as experimental objects,perform cell colocalization on Gas6/Axl,and verify the regulatory effect of QGF on Gas6/Axl complex,affecting downstream signaling pathways such as Phosphoinositide 3-kinases(PI3K),protein kinase B(AKT)and nuclear factor-kappa B(NF-?B),thereby inhibiting esophageal cancer The ability of cells to migrate and invade.Methods: 1.CCK-8 test was used to analyze and analyze the effect of QGF on the cytotoxicity of esophageal cancer cells,and the experimental intervention concentration related to cell migration and invasion was selected.2.Perkin-Elmer high-content cell imaging system and Harmony image analysis software were used to detect the movement capacity and distance of esophageal cancer cells.3.Immunofluorescence and laser confocal microscopy were used to detect the localization and binding of Gas6,Axl and Gas6/Axl in esophageal cancer cells,and the regulatory effect of QGF intervention.4.Immunofluorescence and laser confocal microscopy were used to detect the localization and expression of PI3 K,AKT and NF-?B in esophageal cancer cells,and the effects of QGF on PI3 K,AKT and NF-?B nucleation.5.Western blotting to detect the protein expression of Gas6,Axl,PI3 K,AKT,NF-?B,and Matrix Metalloproteinase-2(MMP2)and Matrix Metalloproteinase-9(MMP9)in esophageal cancer cells.And changes after QGF intervention.6.QGF to the migration ability of esophageal cancer cells was determined in vitro scratch method.7.The effect of QGF on the invasion ability of esophageal cancer cells was determined in the transwell method.Results: 1.QGF experimental intervention drug concentration determination.CCK-8 analysis QGF concentration selection experiment proves that after the intervention of QGF on esophageal cancer cells ECA109 and TE1,400?g/ml showed inhibition of cell proliferation(cytotoxic effect),while 200?g / ml and below did not;culture time 48 hours showed inhibition of cell proliferation(cytotoxicity),but not 24 hours.Therefore,it is determined that ?200 ?g/ml is a suitable intervention concentration for 24 hours.2.QGF reduces the speed and distance of esophageal cancer cells.High content cell imaging experiments show that QGF's stimulation can significantly reduce the speed of movement of esophageal cancer cells ECA109 and TE1,and can reduce the effective distance of esophageal cancer cells,and these inhibitory effects show a significant concentration-dependent trend.3.QGF regulates the expression of Gas6 and Axl in the cell membrane and the binding of Gas6/Axl complex.Immunofluorescence and laser confocal results showed that Gas6 and Axl were mainly expressed in the cell membrane and formed complexes in the membrane of esophageal cancer cells;QGF could significantly inhibit Gas6 and Axl in the cell membrane and binding after esophageal cancer cells intervened,and the separated trend,and with the increase in the concentration of intervention,the regulatory effect becomes more and more obvious.4.QGF inhibits the expression of PI3K/AKT and NF-?B.The experiment detected esophageal cancer cells PI3K/AKT and NF-?B by immunofluorescence.The results showed that compared with the control group,the QGF group can significantly inhibit the expression of PI3 K and AKT,reduce the entry of NF-?B into the nucleus,and these inhibitory effects show A certain concentration-dependent trend.5.QGF inhibits the protein expression of Gas6/Axl downstream signaling pathway.The results of Western blotting showed that QGF stimulation significantly reduced the expression of Gas6 and Axl in esophageal cancer cells and showed a concentration-dependent trend.QGF stimulation significantly reduced the protein expression of P-PI3 K,P-AKT and P-NF-?B,and showed a concentration-dependent trend.QGF stimulation significantly reduced the protein expression of MMP2 and MMP9,and increased with increasing concentration.The same concentration-dependent trend was found in esophageal cancer cells ECA109 and TE1.6.QGF inhibits the migration and invasion of esophageal cancer cells.The scratch test results showed that compared with the control group,the concentration of QGF was 200?g/ ml,which significantly inhibited the cell migration of TE1 cells within 12 h.More importantly,we observed that the 100,200?g/ml group significantly inhibited cell migration at 24 h compared with the control group,and the trends of the two cells were the same.7.The results of the transwell experiment showed that compared with the control group,the QGF concentration was 100,200 ?g/ml,which gradually reduced the number of cells passing through the Transwell chamber and showed a concentration dependence.The two cells have the same trend,but 200 ?g/ml The effect is better than the 100 ?g/ml group.Summary: Gas6 and Axl are highly expressed in esophageal cancer cells ECA109 and TE1.QGF regulates the binding of Gas6 and Axl at the cell membrane,inhibits downstream protein expression,reduces NF-?B nucleation,and blocks the signaling pathway,thereby inhibiting the migration and invasion of esophageal cancer cells.Part ? Qigefang regulates Gas6/Axl to inhibit EMT and affects migration and invasion of esophageal cancer cellsObjective: To observe the mechanism of QGF on tumor cell migration and invasion by inhibiting the occurrence of epithelial-mesenchymal transition(EMT)in esophageal cancer by regulating Gas6/Axl.Methods: 1.Detection of Gas6 and Axl co-localization of esophageal cancer cells Eca109,TE13 by double immunofluorescence colocalization.Locate and detect the intervention effects of Qilufang.2.Western blotting to detect protein tables of EMT markers E-Ca,N-Ca and Snail1 Reach the intervention effect of QGF.3.Immunofluorescence detection QGF localized E-Ca,N-Cad and Snail markers of esophageal cancer cells.4.Ghost pen cyclic peptide cytoskeleton experiment to determine the effect of QGF on the morphology of esophageal cancer cells.5.Scratch and transwell method to determine the effect of QGF on the motility of esophageal cancer cells.Results: 1.QGF inhibited the binding of Gas6/Axl complex.The results of immunofluorescence experiments showed that the control group Gas6 and Axl in esophageal cancer cells Eca109 and TE13 mainly formed complexes on the cell membrane.Compared with the control group,QGF could significantly inhibit the binding of Gas6 and Axl on the cell membrane after intervention with esophageal cancer cells Gas6/Axl has a tendency to separate,and with the increase of the intervention concentration,the regulatory effect becomes more and more obvious;2.QGF inhibited the formation of EMT in esophageal cancer cells.Immunofluorescence and laser confocal microscopy showed that compared with the control group,with the intervention of QGF,the expression of E-ca in the cell membrane gradually increased,showing a concentration dependence.In contrast to E-ca,QGF's stimulation concentration increased,and the expression of N-ca and Snail in the cell membrane gradually decreased,showing a concentration-dependent manner.The same trend was also observed in the two esophageal cancer cells.Western blotting results show that compared with the control group,QGF intervention can significantly increase the expression of E-ca protein,significantly reduce the expression of N-ca and Snail protein,and show the same trend in both cell lines,showing a concentration dependence Sex;3.QGF can promote the rearrangement of microfilament skeleton of esophageal cancer cells.Microfilament cytoskeleton staining experiments showed that compared with the control group,after QGF stimulated esophageal cancer cells,the arrangement of microfilaments in the cells became more regular,the microfilament skeleton of the cells was more isotropic,and the cell morphology was more regular,and It showed a concentration-dependent trend;4.QGF inhibited the migration and invasion of ECA109 and TE13 cells.The scratch test results showed that QGF at different concentrations interfered with esophageal cancer cells for 24 hours.Compared with the control group,QGF significantly inhibited the cell migration ability,showing a significant concentration dependence,and the same trend was observed in both cell lines.The results of the Transwell experiment showed that compared with the blank group,as the concentration of QGF increased,the number of cells passing through the Transwell chamber gradually decreased,and the two cell trends were the same,but the effect of 200 ?g/ml was significantly better than that of the 100 ?g/ml group.Summary: QGF can significantly regulate the expression of Gas6/Axl in esophageal cancer cells Eca109 and TE13 and inhibit the formation of complexes on the cell membrane,inhibit the formation of epithelial-mesenchymal transition in tumor cells,and then affect the migration and invasion of esophageal cancer cells.Conclusions: 1.The expression and activity of Gas6 and Axl in ESCC cancer tissues were significantly increased,and Gas6 and Axl expression were positively correlated with the tumor markers CEA,SCC and CYFRA21-1 2.QGF can regulate Gas6/Axl signaling pathway and protein expression in esophageal cancer cells.3.QGF can reduce Gas6 expression,inhibit Gas6/Axl binding,and reduce NF-?B enter the nucleus,reduce the expression of downstream proteins,thereby inhibiting the migration and invasion of esophageal cancer cells.4.QGF inhibits the formation of EMT by regulating the Gas6/Axl signaling pathway and affects esophageal cancer cells migration and invasion thereby preventing esophageal cancer from metastasis. |
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