Isolation,Culture,Identification Of Tumor Stem Cells In Pancreatic Adenocarcinoma And Regulation Of Tumor Stem Cell In Pancreatic Adenocarcinoma By Gemcitabine And Hypoxic Microenvironment

Objective:1.To obtain SP cells from human pancreatic cancer cell lines through SP cell sorting by flow cytometry,and identify that SP cells of pancreatic cancer cells have the characteristics of cancer stem cells(CSC)and express specific mark genes.2.To study the resistance of pancreatic cancer cells to gemcitabine and the enrichment in human pancreatic CSC by gemcitabine.3.The hypoxic microenvironment in vivo mimicked the hypoxic culture in vitro,monitor the hypoxic state of pancreatic cancer cells by analysing HIF-la protein expression,to investigate the different biological traits of pancreatic cancer cells and to research the regulation of pancreatic CSC by hypoxic microenvironment.Methods:1.Obtain SP cells and non-SP cells from BxPC-3,PANC-1 and SW1990,the human pancreatic cancer cell lines,through SP cell sorting by flow cytometry.2,Investigate the different biological traits of SP cells and non-SP cells through several experimental methods:cloning formation assay,cell differentiation assay,cell cycle analysis,transwell assay for invasion and migration of cells,nude mice tumor assay.3.Detect the transcription of CD133,ABCG2,ALDH1,OCT4,SOX2,Nanog of SP cells and non-SP cells by Real-Time qPCR assay.4,Explore the content of SP cells and the expression of CD133,ABCG2,ALDH1,OCT4,SOX2,Nanog after gemcitabine selecting.5.To mimick the hypoxic microenvironment in vivo,the oxygen concentration in vitro hypoxia of 1% was determined by HIF-la protein expression.6,To study the basic biology of pancreatic cancer cells including cell morphology,cell growth curve,cell cycle distribution in hypoxic microenvironment.7.To monitor EMT of pancreatic cancer cells through E-cadherin and Vimentin detected by Western blot assay.8.To detect the mRNA transcription of CD133,ABCG2,ALDH1,OCT4,SOX2,Nanog in hypoxic microenvironment by Real-Time qPCR assay.Results:1.BxPC-3,PANC-1 and SW1990 contained SP cell subpopulation(0.58%±0.02%,8.74%± 0.33%,3.92%±0.16%).2.SP cells shown a stronger ability in colony formation,proliferation,differentiation,invasion and migration and had a higher proportion of G0/G1-phase cells in SP cells than Non-SP cells(P<0.05),that including 72.8±4.7 to 41.9±1.6,81.6±4.8 to 49.7±3.2 of PANC-1 and SW1990.3.A higher level of mRNA transcription of CD133,ABCG2,ALDH1,OCT4,SOX2,Nanog in SP cells than Non-SP cells(P<0.05).4.A greater tolerability of SP cells compared with non-SP cells to gemcitabine,the proportion of SP cells increased after gemcitabine treating,the transcription of CD133,ABCG2,ALDH1,OCT4,SOX2,Nanog up-regulated.5.HIF-1?protein expressed more in hypoxic microenvironment.6.Cells shown an obvious morphological changes in nucleus,cytoplasm and cell membrane;cells growth slowly,the statistically significant cell cycle distribution(P<0.05)that implied more quiescent cells in hypoxic microenvironment.7.The down-regulated expression of E-cadherin and the up-regulated expression of Vimentin that implied EMT in hypoxic microenvironment were shown.8.A higher level of mRNA transcription of CD 133,ABCG2,ALDH1,OCT4,SOX2,Nanog in hypoxic microenvironment were shown.Conclusion:1.Pancreatic cancer SP cells had pancreatic CSC characteristics,flow cytometry SP sorting method is a reliable method for pancreatic cancer cells CSC separation.2.The CD133,ABCG2,ALDH1,OCT4,SOX2,Nanog genes of pancreatic cancer cell were identified as pancreatic stem cell-specific genes.3.Pancreatic cancer stem cells could be enriched after gemcitabine treating and monitored by detecting CD133,ABCG2,ALDH1,OCT4,SOX2 and Nanog expression.4.Hypoxic microenvironment had an effect on the self-renewal of pancreatic CSC.