| Objective:To investigate the impact of hypoxia environment has on the stemness of pancreatic cancer cells and how the exosomes derived from those cells will affect angiogenensis,so as to reveal the mechanism of how the pancreatic cancer cells respond to the microenvironment and important interactions between hypoxia niche and pancreatic cancer stem cell development,thereby we can provide new perspectives for future clinical treatment regarding pancreatic cancer invasion and metastasis.Methods:(1)CCK-8 assay was used to detect the proliferation rate of pancreatic cancer cells under normoxia and hypoxia condition,and the proliferation rate of pancreatic cancer cells under hypoxia with gemcitabine treatment.(2)Flow Cytometry(FCM)and fluorescence activated cell sorting(FACS)analysis was used to detect the expression of cancer stem cell marker CD133 in pancreatic cancer cell lines.Flow cytometry was used to detect the percentage of the CD133 positive subpopulation of pancreatic cancer PANC-1 and AsPC-1 cell lines under normoxia and hypoxia cell culture condition.The subpopulation of CD133 positive pancreatic cancer cell PANC-l cell line was selected by flow cytometry indirect immunofluorescence labeling.The ratio of CD133+subpopulation of PANC-1cells in pancreatic cancer under normoxia and hypoxia was determined.(3)Western-blot method was used to detect the expression of stem cell marker CD133 of pancreatic cancer cell under different hypoxia time points.(4)Ultracentrifugation was used to isolate and purify the exosomes secreted by pancreatic cancer PANC-1 cells under normoxia and hypoxia condition.(5)Transmitted electron microscopy(TEM),Dynamic Light Scattering(DLS)and western-blot were used to characterize exosomes isolated under normoxia and hypoxic conditions.(6)The tube formation Assay and sprouting assay were used to detect the release of exosomes from pancreatic cancer PANC-1 cells and PANC-1 CD133 positive cells on angiogenesis under different oxygen condition.Results:(1)The results of CCK-8 test showed that the proliferation rate of pancreatic cancer cell lines PaTu8988t,BxPC-3,PANC-1,AsPC-1 and SW1990 decreased under hypoxic conditions;Chemotherapy resistance of the pancreatic cancer cell line under hypoxic condition was significantly enhanced,in the meantime,the apoptotic rate of those cells was reduced.(2)Flow cytometry results showed that the proportion of CD133~+cells in pancreatic cancer PANC-1 and AsPC-1 cells increased significantly under hypoxic conditions,and the proportion of CD133~+in PANC-1 cells was higher.(3)Western-blot results showed that the protein expression of stem cell marker CD133~+molecule in pancreatic cancer PANC-1 cells increased with the prolongation of hypoxia time,and the expression change of Nanog was not very obvious.(4)Dynamic light scattering results showed that,compared with normoxia condition,the size of exosomes isolated from PANC-1 cells increased under hypoxic conditions,and the protein content increased.The result of HUVEC tube formation assay and sprouting assay showed that the secretion of exosomes by pancreatic cancer cells under hypoxia had an enhanced effect on angiogenesis,and the exosomes released from CD133+cells under hypoxic conditions seemed to have a stronger role in enhancing blood vessel formations.Conclusion:(1)Under the condition of hypoxia,the drug resistance of pancreatic cancer cells is improved,the stemness of the cancer cell is enhanced,and the proportion of CD133 positive cancer stem cells increases.(2)The size of exosomes secreted by pancreatic cancer cells induced by hypoxia increased,and the protein content of secret exosome increased.(3)Hypoxia-induced exosome secreted by pancreatic cancer stem cells through the exosome pathway promotes angiogenesis,thereby promoting invasion and metastasis of pancreatic cancer.The experiment provides a new clue and theoretical basis for revealing the early invasion and metastasis to support the clinical treatment of pancreatic cancer in the future. |
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