| Background and ObjectiveDespite widespread vaccination against human papilloma virus and periodic cancer screening, cervical cancer remains one of the highest ranking diseases causing mortality in women, and its incidence is getting younger. The pathogenesis of cervical cancer and new strategies to treat this disease are urgently needed.Cancer-associated fibroblasts(CAFs) is the most abundant cell type in the tumor microevironment. It has both the characteristics of smooth muscle cells and fibroblasts in morphology and biological behavior. It can secrete a variety of cytokines, inducing malignant transformation and the development of cancer epithelial cells. Although different types of mesenchymal and epithelial cells are proposed to be precursors of the myofibroblasts present in tumors, the precise cellular origins of CAFs and the molecular mechanisms by which these cells evolve into cervical myofibroblasts remain unclear.Transforming growth factor beta1(TGF-?1) is widely present in the cells. It is considered to have a central role in inducing the myofibroblastic phenotype because it is capable of up-regulating fibroblast alpha-smooth muscle actin(?-SMA), both in vitro and in vivo. Study shows more intense TGF-?1 expression was observed in cervical cancer cells than in normal cervical epithelium or cervical intraepithelial neoplasias. Over-production of TGF-?1 is one potential mechanism mediating the transformation of stromal cells to myofibroblasts in cervical carcinogenesis. It is not known if myofibroblastic depend on TGF-?1 signaling to induce their unique phenotypes.Our study is to isolate and identify CAFs and normal fibroblasts(NFs),and investigate CAFs biological characteristics.we also investigate the role of TGF-?1 in the differentiation of cervical cancer-associated fibroblast. Materials and Experimental Methods1. Primary cervical cancer-associated fibroblasts and NFs were isolated from cervical cancer tissue and normal cervical tissue by collagenase digestion. Cell morphology was observed by microscope, cell growth curve was detected by CCK-8. Vimentin, ?-SMA, fibroblast activation protein ?(FAP?) were detected by immunofluorescence and western blot.2. The concentration of TGF-?1 in cell supernatant of CAFs and Hela co-culture, CAFs, Hela was detected by ELISA assay.3. Recombinant Human TGF-?1 treat NFs, seting control group. After 72 h treatment,the cervical fibroblasts were analyzed by Immunofluorescence and Western blotting for myofibroblasts markers FAP? and ?-SMA.4. Statistical analysis: The SPSS statistical package program 17.0 was applied to analyze the results of experiment. Groups were compared using non-parametric test.Significant level is=0.05, P< 0.05 for the difference was statistically significant. Results1. CAFs and NFs are successfully isolated. The morphology of CAFs was variable, as the contact inhibition had been lost. On the contrary, the NFs had the similar size and preserved contact inhibition. The result of CCK-8 assay showed that CAFs grew faster than NFs. The experiment certify that Vimentin had similar expression in CAFs and NFs, while CAFs had higher expression of ?-SMA and FAP? than NFs.2. The concentration of TGF-?1 in cell supernatant of CAFs and Hela co-culture, CAFs, Hela were 1087.26.54±71.42 ng/L,449.93±19.99 ng/L,703.29±27.34 ng/L respectively.3.The group treated with TGF-?1 strongly express FAP? and ?-SMA. The control group do not express FAP? or ?-SMA. Conclusions1. CAFs has both the characteristics of smooth muscle cells and fibroblasts,and it grew faster than NFs.2. TGF-?1 can modulate the myofibroblasts differentiation from normal cervical fibroblast in vitro. |
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