A Study On Changes In Normal Colonic ²⁰e²⁰pithelial Cells And Mesenchymal Stem Cells Co-cultured With Colon Cancer Cells

Objective: By constructing an in vitro co-culture model and simulating an in vitro tumor microenvironment,the role of colorectal cancer microenvironment in inducing normal colonic epithelial cells and mesenchymal stem cells was explored.Methods: We select the colon cancer cell line(SW480),normal colon epithelial cells(NCM460)and human umbilical cord mesenchymal stem cells(h UC-MSCs)to establish a co-culture model in vitro;NCM460 cells were seeded on the lower chamber of Transwell in each group,and the cells in the upper chamber were grouped as follows: A: control group(without cell inoculation),B: h UC-MSCs inoculation only,C: SW480 only,D: mixed of h UC-MSCs and SW480.We observed the possible changes in morphology of NCM460 after co-cultivation,and detected the migration ability of NCM460 through wound healing and Transwell migrating assay;Meanwhile,the change of markers of epithelial-mesenchymal transition(EMT)after co-culture.Next,SW480 and h UC-MSCs cells were co-culture model to investigate the induction effect of SW480 on h UC-MSCs;In the group of 7 days of co-cultivation,we created three groups,and seeded h UC-MSCs in the lower chamber of Transwell,SW480 cells were inoculated in the upper chamber,and each group seeded cells according to specified proportion(SW480: h UC-MSCs=0:1,1:1,2:1),and their morphological changes,and protein expression of TP53 and α-SMA were tested.Second,we tested the protein expression of TP53 and α-SMA of h UC-MSCs after co-cultivation for 14 days.Experiment is divided into four groups,each group plant cells according to the proportion(SW480: h UC-MSCs=0:1,1:1,2:1,10 :1).Results: 1.After co-cultivation,no significant changes in cell morphology were found,but the migration ability of normal colon epithelial cells NCM460 was enhanced.The differences between the groups as following: h UC-MSCs SW480NCM460 group> h UC-MSCs NCM460 group> SW480 NCM460 group;2.EMT markers contain Vimentin and E-Cadherin protein.Vimentin protein of NCM460 was up-regulated and E-Cadherin protein was down-regulated in NCM460 after co-culture with SW480 and/or h UC-MSCs;3.The medium supernatant of h UC-MSCs group and h UC-MSCs+SW480 group contained abundant IL-6/IL-8 factor;4.The expression level of α-SMA protein of h UC-MSCs did not change significantly after 7 days co-cultivation;However,after 14 days co-cultivation,the α-SMA protein expression of h UC-MSCs in the10:1 co-cultivation group is increased.;5.After 7 days co-cultivation,the expression level of TP53 protein of h UC-MSCs in each group did not change significantly(P>0.05);After 14 days co-cultivation,the TP53 protein expression level of h UC-MSCs is significantly reduced(P<0.05).Conclusion: After co-cultivation,h UC-MSCs or SW480 may promote NCM460 to a certain degree of EMT.SW480 can induce the down-regulation of the TP53 protein expression level of h UC-MSCs,and may increase α-SMA protein expression of h UC-MSCs.