Separation And Cultivation Of Ovarian Carcinoma-associated Fibroblasts And The Effects On Ovarian Cancer Cell

Part I The Differential Gene Expression between Cancer-Associated Fibroblasts and Normal Fibroblasts separated,cultured and identified from Human Ovarian CarcinamasObjective: To separate, cultivate, purify and identify ovarian carcinoma-associated fibroblasts (CAFs) and ovarian normal fibroblasts (NFs) preliminarily. Analyze the distinct molecular expression profiles between CAFs and NFs.Methods:1. The primary ovarian CAFs and NFs of ovarian were obtained by tissue culture. The second passage was obtained after the cell overgrew the bottom of the culture flask and purified by trypsinization and adherence repeatedly methods. 2. The identity of the CAFs and NFs was through the observation of the shape of the cell with inverted microscope and the cellular organ with transmission electron microscope, the protein expression of vimentin, keratin andα-SMA in the cellular membrane with immunocytochemistry and mRNA expression of vimentin andα-SMA with RT-PCR. 3. Analyze the distinct molecular expression profiles between CAFs and NFs with RT-PCR.Results:1. The purified ovarian CAFs and NFs were maintained. 2. The characteristics of shape and growth of the ovarian CAFs changed significantly comparing to the NFs. They are in long-fusiform shape, cytoplasmic reduction, and expansion of rough endoplasmic reticulum. 3. The ovarian CAFs showed negative staining for cytokeratin , and positive staining for vimentin andα-SMA, while NFs showed negative staining for cytokeratin andα-SMA,positive staining for vimentin. 4.α-SMA was found mRNA expression in both CAFs and NFs, but the expression increased significantly in CAFs (P﹤0.05). 5. Control with the NFs, molecular expression profiles increased in CAFs, including growth factors, MMPs, angiogenesis factors, at the same time, MMP1 was found decreased.Conclusions:There are obvious differences of the morphological characteristics and expression of certain mRNA and proteins between the CAFs and NFs. The distinct molecular expression profiles of CAFs in ovarian cancer support the notion that these fibroblasts form a favorable microenvironment for cancer cells. The microecology of the oral tumor-host interface might be one of the most important factors affecting the progression of epithelial ovarian cancer.Part II The effect of Ovarian Carcinoma-associated Fibroblasts on the Proliferation, Apoptosis, Migration and Invasion of Epithelial Ovarian Carcinoma CellObjective: To observe the effects of CAFs on the proliferation, apoptosis, migration and invasion of ovarian caicinoma cell line CAOV3 cells, and to elucidate the role of CAFs in ovarian carcinoma progression.Methods:1. The interaction model between primary ovarian CAFs,NFs and ovarian carcinoma cell line CAOV3 was established by Transwell chamber of 0.4μm diameter. In order to find out the effect of proliferation of CAFs to CAOV3, the RT-PCR assay was used to observe the changing of PCNA mRNA expression. 2. Cell cycle examination and Annexin V-FITC/PI assays to verify the apoptosis of CAOV3 after the co-culture with CAFs or NFs with flow cytometry. 3. The interaction model between primary ovarian CAFs,NFs and ovarian carcinoma cell line CAOV3 was established by Transwell chamber of 8μm pore to observe the effect of migration of CAOV3. 4. Matrigel was used to remodel the basement membrane, and the interaction model between primary ovarian CAFs,NFs and ovarian carcinoma cell line CAOV3 was established by Transwell chamber of 8μm diameter to observe the effect of invasion of CAOV3.Results:1. The expression of PCNA mRNA in CAOV3 was significantly increased after the co-culture with CAFs, and with the number of CAFs increased, the motility of proliferation in CAOV3 was significantly increased. 2. By flow cytometry of the cell cycle, the apoptosis of CAOV3 was found significantly decreased after the co-culture with CAFs, while it was not found in CAOV3 co-cultured with NFs. 3. Meanwhile, the decreasing apoptosis of CAOV3 was observed with flow cytometry in Annexin V-FITC/PI assay, but the apoptosis of CAOV3 did not change significantly after the co-culture with NFs. 4. When contrast with the control group, the migration of CAOV3 was significantly increased after the co-culture with CAFs or NFs for 8hours, moreover, there was an extremely significant increase in CAFs group. 5. In the invasion experiment, when CAOV3 was co-cultured with CAFs or NFs for 24hours, the invasion of CAOV3 was significantly increased, especially in CAFs group.Conclusions:CAFs can promote the proliferation and decrease the apoptosis of ovarian carcinoma CAOV3 cells, both CAFs and NFs can promote the migration and invasion of CAOV3, but it was much more obvious in CAFs. All the findings suggest, during the interaction between CAFs and ovarian carcinoma CAOV3 cells, the signaling can promote the progression of ovarian carcinoma. Part III The cross-talk of the change of genes expression between CAFs and ovarian cancer cell CAOV3 co-culture and the possible mechanismsObjective:To investigate the change of genes expression between CAFs and ovarian cancer cell CAOV3 co-culture and the possible mechanisms,and the effects of ovarian CAFs on ovarian cancer,new targets were to be found.Methods:1. To explore the reciprocal effects of host-tumor interaction, we developed a system to assess the gene expression patterns and the diversity of gene expression of ERK and PI3K pathway in CAOV3 human ovarian cancer cells and primary carcinoma-associated fibroblasts cocultured with each other for 48 hours with RT-PCR. 2. Western blot was used to detect the expression of extracellular signal-regulated kinase1/2 prtotein in ovarian cancer cell CAOV3 after the co-culture with CAFs. 3. We established the model of human ovarian carcinoma with human ovarian carcinoma cell CAOV3 and CAFs 1:1 of nude mice to observe the weight and volume of those carcinomas in nude mice,and hematoxylin and eosine staining of tumor-bearing nude mice in general. 4. Immunohistochemistry was used to determine the expression ofⅧfactors antigen and PCNA protein in those carcinomas in nude mice,compared with the carcinomas of CAOV3 in nude mice.Results:1. After the co-culture with CAFs, the expression of FGF1,tTG,PDGF-A were significantly increased(P﹤0.05).2. The expression of TGF-β2 was significantly decreased(P﹤0.05). In MMPs,the expression of MMP1 was decreased when co-cultured with NFs,while MMP7 was increased. But TIMP3 both were decreased after the co-culture with CAFs and NFs. 2. After the co-culture with CAOV3, the expression of tTG,VEGF-C,MMP1,MMP9 and TIMP3 were significantly decreased. 3. The expression of ERK2,PI3K mRNA and ERK1/2 protein in CAOV3 were increased after the co-culture with CAFs. 4. The weight of treated nude mice was decreased fast by CAFs, and a large number of vessels were generated,the proliferation inceased and the MVD raised.Conclusions:The expression of several growth factors and MMPs changed in CAFs and CAOV3 after the co-culture of them,and CAFs have the activation effect on ERK and PI3K pathway in CAOV3 cells. The cancer angiogenesis and progression were stimulated by CAFs in vivo. Therefore,the study targeting CAFs would afford us a new thincking of therapeutic strategies.