Expression Of CD24CD44 Positive Cells In Pancreatic Cancer Cells After Infection With Specific Smo Lentivirus And Its Biological Characteristics

Objectives:1.Flow cytometry was used to analyze the expression of CD24CD44positive cells in BxPC-3,SW1990 and Panc-1 of pancreatic cancer cell lines,Comparison of the three groups of cells,flow sorting the highest positive rate of a group of cells,for the follow-up experiment to prepare;2.Construction of lentiviral vector which carrying Smo interference gene,Transfection of CD24CD44 positive cells of pancreatic cancer cells and detection of their infectivity;3.To analyze the expression of Hh signaling pathway and the biological characteristics of CD24CD44 cells transfected by lentivirus in pancreatic cancer cells.Methods:1.Flow cytometry was used to analyze the expression of CD24CD44 positive cells in 3 kinds of pancreatic cancer cells.Choose the highest positive rate of a group of pancreatic cancer cells,The CD24CD44 positive cells were selected as the research object for the following experiments;2.Design and synthesis of lentiviral vector carrying Smo interference fragment,including over expression group,inhibition group and negative group,for subsequent experimental study.3.After transfection of pancreatic cancer cells by Flow cytometrywith Smo lentiviral vector,RT-PCR and Western blot were used to detect the gene expression and protein expression of Hedgehog signaling pathway,and the cell biological characteristics were detected.Results:1.The expression rates of CD24CD44 positive cells in pancreatic cancer cell lines BxPC-3,SW1990 and Panc-1 were respectively BxPC-3:11.75±1.6264%,Panc-1:28.05±2.4749%,SW1990:57.70±2.2627%;2.Successful design and production of the virus,(the virus is the amount of 1X10~6 TU,add Polybrene dilution),cell growth and fluorescence effect is the best in this condition,so the follow-up experiment to select these infection conditions and infection parameters.3.After transfection of SW1990 CD24+CD44+cells with Smo lentiviral vector.RT-PCR was used to detect the Hedgehog signaling pathway members Shh,Smo,Ptch1,Gli1,Gli2,and Bcl2 in five groups.There was no significant difference in the expression of Shh,Gli2 and Bcl2 between the blank group,negative group and the virus group(P>0.05no statistical significance);The expression of Smo in the pathway group was 2.78times higher in the LV-SMO15570-2 group than in the blank group,and was reduced to0.24times in the SMO-RNAi37304-1 group,nearly 1/4 times.The channel member Gli1was reduced to 0.41 of the blank group in the SMO-RNAi37304-1 group,nearly 1/2times.(P<0.05Statistically significant)Combined with the results of Western blot,the data are basically the same.Detection of cell biological characteristics:Cell morphology:blank group,negative virus group,LV-SMO15570-2 overexpression group showed no significant changes.LV-SMO-RNAi37304-1(control group),was transfected into SW1990 CD24+CD44+cells,the cells loosely connected,changes in cell morphology,pseudopodwere significantly reduced.Detection of surface markers:the results showed that the expression of CD24 and CD44 in pancreatic cancer cell line SW1990CD24+CD44+was significantly reduced after transfection with LV-SMO-RNAi37304-1(inhibitor)lentiviral vector.Proliferation assay:The OD value of determination of five gruop;Blank gruop:0.705±0.108?0.969±0.067?1.938±0.690?2.222±0.385?3.136±0.926;CON238:0.724±0.038?1.003±0.048?1.482±0.394?1.838±0.219?3.417±0.688;CON077:0.819±0.147?1.015±0.244?1.253±0.326?1.893±0.355?3.549±0.399;LV-SMO15570-2:0.830±0.158?0.773±0.033?1.209±0.540?1.400±0.228?2.373±0.426;LV-SMO-RNAi37304-1:0.681±0.146?0.800±0.010?1.402±0.080?1.973±0.346?3.033±0.842?(P<0.05Statistically significant)The ability of cell migration:the wound healing area of LV-SMO-RNAi37304-1(inhibitor)group was 25.92±5.19%,which was significantly lower than that of SW1990CD24+CD44+cells by 67.61±5.02%(P<0.05Statistically significant).Conclusion:Compared with SW1990,BxPC-3,and Panc-1,the expression rates of CD24CD44 positive cells in 3 pancreatic cancer cell lines were highest in SW1990,which had the biological characteristics of tumor stem cells,After the flow separation as the experimental target cells.2.According to the Hh signal pathway key genes Smo design siRNA interference fragment,Lentiviral vector carrying Smo interference fragment was successfully constructed;After transfection of SW1990CD24CD44 positive cells,the expression of Smo was analyzed.The results showed that the interference fragment of key gene Smo was successful.3.CD24+CD44+cells in pancreatic cancer cells with stronger tumorigenicity;fluorescence quantitative RT-PCR results and Western blot assay results showed that overexpression group and inhibition group compared to the control group the expression of Smo was significantly changed,while the altered expression of Smo,the expression of Gli1 in inhibit group have also been reduced by intervention.In inhibition group of tumor cells in biological characteristics were changed significantly,showed growth inhibition and biological activity decreased,which confirmed that the Hh signal pathway to maintain an important role in the process of the malignant biological characteristics in pancreatic cancer cells.Objective to provide reliable experimental evidence for the treatment of pancreatic cancer by targeting the Hh signaling pathway.