Colorectal cancer cells induce the formation of cancer-associated fibroblasts by activating the ERK signaling pathway in fibroblasts / 南方医科大学学报

OBJECTIVE@#To investigate the mechanism by which conditioned medium of colorectal cancer cells promotes the formation of cancer-associated fibroblasts (CAFs).@*METHODS@#Normal human colorectal fibroblasts (CCD-18Co cells) in logarithmic growth phase were treated with the conditioned media of colorectal cancer HCT116 cells (HCT116-CM) or Caco-2 cells (Caco-2-CM) alone or in combination with 300 nmol/L ERK inhibitor SCH772984. The expression levels of CAFs-related molecular markers were detected in the treated cells with real-time quantitative PCR (RT- qPCR) and immunofluorescence assay, and the changes in cell proliferation, colony formation and migration were assessed with RTCA, colony formation and wound healing assays; Western blotting was performed to detect the activated signaling pathways in the fibroblasts and the changes in CAFs formation after blocking of the signaling pathway.@*RESULTS@#HCT116-CM and Caco-2-CM significantly upregulated mRNA expression levels of CAFs markers (including α-SMA, FAP, FN and TGF-β) in CCD-18Co cells, and strongly promoted fibroblast transformation into CAFs (P < 0.05). The two conditioned media also promoted the proliferation, colony formation and migration of CCD-18Co cells (P < 0.05) and significantly increased the levels of α-SMA protein and ERK phosphorylation in the cells (P < 0.05). The ERK inhibitor SCH772984 obviously inhibited the expression of α-SMA and the transformation of CCD-18Co cells into CAFs induced by the conditioned medium of colorectal cancer cells (P < 0.05).@*CONCLUSION@#Colorectal cancer cells may induce the formation of colorectal CAFs by activating the ERK pathway in the fibroblasts.

Construction and biological identification of the transfectant cells expressing ??TCR with specific CDR3 sequence / 中国免疫学杂志

Objective:To construct of transfectant cells expressing ??TCR with specific CDR3 sequence.Methods:Specific CDR3 region sequence of DBS4.3,a known ??T cell clone,was inserted into ?9 and ?2 chain to substitute original CDR3 sequence using overlapping PCR method.After the full-length ?9 and ?2 chains were ligated with expression vector pREP7and pREP9 respectively,they were co-trasfected into a cell line of human Jurkat cells with TCR ? chain gene-defective mutant J.RT3-T3.5.When the transfectant cells expressing specific ??TCR were stimulated by antigen,production of IL-2 was detected by ELISA and Realtime PCR.Results:By ELISA and Realtime PCR,it was exhibited that the transfectant cells expressing DBS4.3 specific ??TCR secreted IL-2 under stimulation of iso-butylamine and anti ??TCR antibody.Conclusion:The transfectant Jurkat cells expressing ??TCR with specific CDR3 sequence are successfully constructed.It provides a platform for the research of recognition mechanism of ??TCR.