| Objective: To isolate gastric cancer-associated fibroblasts(CAFs),normal fibroblasts(NFs)and primary gastric cancer cells,compare the difference of proliferation and the effect of clone formation on primary gastric cancer cells between CAFs and NFs,then explore the difference of transcriptomics.Methods:(1)Gastric cancer tissues and adjacent normal tissues(which were more than 5cm away from the cancer and pathological examination was negative)were got from the patients with gastric cancer,then digested using collagenase and cultured in the pre-coated culture flask.CAFs and NFs were identified using immunocytochemical staining to detecte the expression of smooth muscle actin ?(?-SMA)and fibroblast activation protein(FAP),the mRNA expression of ?-SMA was measured using real-time quantitative PCR(RT-qPCR).Primary gastric cancer cells were identified using HE staining,soft agar clone formation and immunocytochemical staining to detecte the expression of carcino-embryonic antigen(CEA),cytokeratin 18(CK-18).The proliferation and the growth curve of CAFs and NFs were measured using CCK8.The clone formation ability of primary gastric cancer cells was detected using crystal violet staining after co-culturing primary gastric cancer cells with CAFs(or NFs).CAFs and primary gastric cancer cells was infected with Helicobacter pylori at a multiplicity of infection(MOI)of 100 or 200 respectively,then the adhesion of bacteria was observed by microscopy.(2)Three pairs of CAFs and NFs as biological repetition were selected from patients with similar clinical phenotypes and diagnoised with gastric adenocarcinoma for transcriptome sequences,and the gene expression considered significantly different when the ratio of CAFs / NFs was more than twice.Combine any two of the biological repetition and make it a summary,then use of omicsbean online software for bioinformatics analysis.Results:(1)8 sample of CAFs,5 sampleof NFs and 3 sample of primary gastric cancer were successfully isolated.Immunocytochemical staining showed that the expression of ?-SMA,FAP strongly positive in CAFs,weakly positive or negative in NFs,no expression of CK-18 in both of them;RT-qPCR results showed that the expression of ?-SMA's mRNA was significantly higher than NFs,the difference was statistically significant(P<0.05),suggesting that CAFs and NFs successfully isolated.The growth curve showed that the growth of CAFs was significantly higher than that of NFs after 3 days' culture,and the difference reached to maximum at 6th day(P<0.05).(2)The results of HE staining showed that primary gastric cancer cells were pleomorphic nuclei and cytoplasmic ratio increased,deformity,the number of nucleoli increased,immunocytochemical staining showed that the expression of CEA and CK-18 in primary gastric cancer cells were strongly positive,soft agar clone formation showed that primary gastric cancer cells could grow in vitro.These results indicated that the primary gastric cancer cells had malignant characteristics and were successfully identified.After co-culturing primary gastric cancer cells with CAFs(or NFs),the number of clones of primary gastric cancer cells number in CAFs(70.4±8.6),was significantly higher than that of NFs(35.2±5.7)and negative control(33.1±6.6)(P<0.05),while there was no difference between the NFs and negative control(P>0.05).Besides,it was found that H.pylori could not only adhered to gastric cancer cells,but also to CAFs.(3)The results of transcriptomics showed that a total of 147 genes were differentially expressed which 76 genes up and 71 down in CAFs after combining the biological repetition together.A total of 1727 biological processes,84 cell components,153 molecular function GO terms,and 10 pathways were significantly changed through cluster analysis,GO enrichment and KEGG enrichment.Conclusion: 1 CAFs,NFs and primary gastric cancer cells were successfully isolated from clinical gastric cancer tissues.2 CAFs grew significantly faster than NFs in vitro,and were more favorable for the formation of primary gastric cancer cells 3 There were 147 differentially expressed genes found in CAFs,which may which may play an important role in the development and progression of gastric cancer. |
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