Effect Of Elimination Of Blood Stasis Method On Proliferation Of Lung Cancer Stem Cells By Controlling Wnt Pathway Key Protein?-catenin And CyclinD1

Objective: To study the effect of Elimination of blood stasis method on the proliferation of lung cancer stem cells(LSCs)by regulating the key proteins of ?-catenin and Cyclin D1 of Wnt pathway,and to elucidate the mechanism of Elimination of blood stasis method in the treatment of lung cancer.Methods:(1)The sorting of human lung adenocarcinoma cell line A549 cells(Side population cells referred to as SP cells)and their stem cell characterization: FACS was used to study the expression of human lung adenocarcinoma Apoptosis of SP cells and non-SP cells in A549 cells were detected by MTT assay.Cell cycle was observed by MTT assay.Flow cytometry analysis of cell cycle and differentiation and tumor formation in nude mice were performed to analyze SP cells Subgroups,non-SP cell subsets and total cells in vivo,proliferating ability,cell differentiation ability and in vivo tumorigenic ability.(2)Effect of Elimination of blood stasis method on proliferation of Lung Cancer Stem Cells by controlling Wnt pathway key protein?-catenin and Cyclin D1: The human lung adenocarcinoma cell line A549 was cultured by flow cytometry,and the stem cells(11.6g / kg,5.8g / kg,2.9g / kg)in the high,medium and low dose groups of the oral liquidand blank control group that the cells were intervened with the serum containing the oral liquid.The lung cancer stem cells of each group were treated with left axillary subcutaneous tumor in nude mice.The tumor weight and tumor inhibition rate were measured after tumor formation.The key points in lung cancer stem cells were detected by Western blot Protein ?-catenin and Cyclin D1 expression levels.Results:(1)FACS sorting results showed that human lung adenocarcinoma cell line A549 contained approximately(1.79 ± 0.12)% SP cells.In vitro clone formation assay and MTT assay and the results showed that SP cell proliferation ability was stronger(P <0.05).The results of cell differentiation showed that SP cells and non-SP cells were cultured in vitro.The cell cycle of SP cells was significantly higher than that of non-SP cells(P <0.05)(1.49 ± 0.05)% and(0.16 ± 0.04)%(P <0.05).The tumorigenic ability of SP cells was significantly stronger than that of non-SP cells.(2)Compared with the blank control group,the tumor weight in the high and middle dose group were significantly lower(P <0.01).The expression of?-catenin and Cyclin D1 in lung cancer stem cells of high dose and middle dose group were significantly decreased(P<0.01,P<0.05).Conclusion:Human lung adenocarcinoma cell line A549 has the characteristics of stem cells by FACS.SP cells are used as a surface marker without looking for specific surface markers,but by using the characteristics of the fluorescent dye efflux,Enrichment of tumor stem cells,can be used as an ideal and simple way to study tumor stem cells.One of the molecular mechanisms of Elimination of blood stasis method therapy for lung cancer may inhibit the abnormal activation of lung cancer stem cells by inhibiting the expression of?-catenin and Cyclin D1,and affect the proliferation of lung cancer stem cells,thereby inhibiting lung cancer.