The Effect Of Primary Culture Of Colon Cancer-associated Fibroblasts On The Proliferation And Invasion Of Colon Cancer Cell Line

[Objective]To investigate primary culture of the colon cancer-associated fibroblast(CAF) and the effect of purified colon CAF on the proliferation and invasion of colon cancer cell line,in order to study the role of cancer cells micro-environment in the development of colorectal cancer,and further to the prevention and treatment in seeking new ways.[Methods]The changes of biological characteristics were determined by electron microscopy and immunohistochemistry to identify CAF and NF of primary culture.GroupA:HCT-116 cells cultured with CAF and NF conditioned medium, using serum-free medium to culture HCT-116 cells as control,assaying the proliferation of HCT-116 cell line by MTT method;GroupB:Using Transwells to establish a co-cultivated model of CAF/NF and HCT-116 cells,also with 10% complete medium cultured HCT-116 cells as control,to observe the morphological changes of HCT-116 cells,analysis the proliferation cycle of HCT-116 cells by flow cytometry;GroupC:Again adopting Matrigel simulated reconstruction of the basement membrane,in order to observe the invasion characteristics of HCT-116.[Results]Purified cells of colon CAF are more heteromorphism,vigorous growth than NF cells,moreover loss of the contact and density inhibition.CAF and NF cells were expressed negative with cytokeratin(AE1/AE3),positive with vimentin, Positive withα-SMA in CAF,negative with NF,Ultrastructure of myofilaments with CAF,while NF addition to mitochondria-rich structure no significant myofilaments;Indirect co-culture model prompt morphological changes on HCT-116 was not obvious.MTT results showed that by adding the conditioned medium of CAF proliferative activity of HCT-116 was stronger than cultured by NF conditioned medium or cultured by the serum-free medium.24h,48h,72h,the proliferative activity of HCT-116 was cultured by CAF conditioned medium group,compared with the other two groups the differences were statistically significant(P＜0.05).Flow cytometry analysis showed that HOT-116 was co-cultured by CAF,G2 phase and S phase cell percentage than NF co-culture group and in serum-free medium group were significantly increased,differences were statistically significant(P＜0.05);Invasion assay HCT-116 cells under the action of CAF the invasion ability significantly enhanced,as compared with other groups,differences were statistically significant (P＜0.05).[Conclusions]1.The expeiment have the model of purified primary cultured colon cancer-associated fibroblasts and normal colonic mucosa fibroblasts;2.Colon CAF have the characteristics promote colon cancer cell lines HCT-116 proliferation and invasion.3.CAF conditioned medium may be some soluble substances can promote the proliferation and invasion of HOT-116 cells.