In Vitro Co Culture Of HBMSCs And HUVECs Regulate Invasion,epithelial Mesenchymal Transition Of Panc-1 Pancreatic Cancer Cells

Pancreatic cancer is one of the most malignant tumors of the digestive system with rapid development and poor prognosis.Now the pathogeny and pathophysiology of pancreatic cancer need further study.In recent years,the three-dimensional co culture technology of tumor cells has developed rapidly.This model can simulate the proliferation and invasion behavior of tumor cells in the real three-dimensional spatial structure of human body.The three-dimensional culture system,can be formed in vitro,has gradually become a powerful tool to study the malignant phenotype of tumor cells,the signal transmission of cells and extracellular matrix,and the interaction of cancer cells and stromal cells.Pancreatic cancer has a high degree of malignancy and early invasion and metastasis.The structure of pancreatic cancer is complex and there are many kinds of cells.Common two-dimensional cell culture is not sufficient to reveal the pathophysiological mechanism of tumor progression.Therefore,it is of great significance to make use of the new three-dimensional co culture technique to prepare the new model of pancreatic cancer cells in vitro,and to study the interaction between cells in cancer tissues to reveal the pathophysiological mechanism of the progression of pancreatic cancer.Objective: The aim of this study was to construct the microtissue containing multiple cells in vitro by three-dimensional co culture.Investigate the effects of co culture of hBMSCs and HUVECs regulate invasion,epithelial mesenchymal transition of Panc-1 pancreatic cancer cells.Methods:1.Construction of three dimensional microtissue model of pancreatic cancer by SIS hydrogel and cells.The first group: pure Panc-1 cells;the second group was Panc-1 cells combined with HUVECs;the third group was Panc-1 cells combined with hBMSCs;the fourth group:Panc-1 cells combined HUVECs and hBMSCs.Live/dead cell viability assays of cells in hydrogels.2.CCK-8 analysis proliferation of cells in three dimensional culture of pancreatic cancer.3.Transwell experimental study on the effect of three-dimensional culture of pancreatic cancer microtissue on the invasion of Panc-1 cells.4.Real-time quantitative PCR analysis of MMP-9,E-cadherin and Snail.5.Determination of MMP-9,E-cadherin and Snail protein by Western blot.Results:1.Three-dimensional co culture model preparation and cell proliferation results.Live/dead result show cells can grow well in the hydrogel.The hBMSCs group and the HUVECs group batter than Panc-1group(P<0.05).There was no statistical difference between HUVECs group and hBMSCs group.2.The results of invasion of Panc-1 cells in pancreatic cancer.Compared with the Panc-1 group,the three different cell co culture can promoted the invasion of Panc-1 cells in different degrees.The hBMSCs group showed more cells invasion than Panc-1group(P<0.05).When added the h BMSCs and HUVECs simultaneously,the effects of decrease apoptosis more significant(P<0.05).3.RT-qPCR detection of gene results.Real time quantitative PCR assay showed that compared with Panc-1 group as control,three kinds of co culture of different cells promoted Snail and MMP-9 gene expression in Panc-1 cells to varying degrees.The most significant effects were added the stem cells and endothelial cells simultaneously(P<0.05).Compared with the Panc-1 group,the expression of E-cadherin gene in Panc-1 cells was inhibited by three different cells co cultured in different degrees.The most significant effects were added the stem cells and endothelial cells simultaneously(P<0.05).4.The results of Protein.Western Bolt results showed that,compared with the Panc-1 group,the expression of Snail and MMP-9 protein in Panc-1 cells was promoted by three different cell cultures in different degrees.The most significant effects were added the stem cells and endothelial cells simultaneously(P<0.05).Compared with the Panc-1 group,the expression of E-cadherin protein in Panc-1 cells was inhibited by three different cells co culture in different degrees.The most significant effects were added the stem cells and endothelial cells simultaneously(P<0.05).Conclusions: Panc-1 pancreatic cancer cells were co cultured with hBMSCs and HUVECs in SIS hydrogel.The complicated microtissue of the pancreatic cancer was successfully constructed.hBMSCs play an important role in the invasion of pancreatic cancer by regulating MMP-9 and epithelial mesenchymal transition.