| Objective: Epithelial ovarian cancer (EOC) is the most common female malignancy and is the leading cause of death from gynecological malignancies. The hyper-mortality from the ovarian cancer dues to the majority of patients are diagnosed with advanced stage when tumor is already spreaded to abdominal cavity and retroperitoneal lymph node. Despite of aggressive treatment, 5-year survival rate remains about 30%. Unlike other cancers, major route of metastasis of ovarian cancer is not by blood vessel. The growth and metastasis in peritoneal cavity can produce ascites and intestinal obstruction, so peritoneal metastasis of EOC is major cause of mortality. Understanding the mechanisms of ovarian cancer cell adhesion, migration and invasion is very important. Metastasis of ovarian cancer in peritoneum is related with chemotactic factor, growth factor and adhesion factor in ascites beside anatominal and mechanical agent. In 2001, Muller studied chemotactic factor and receptor in breast cancer and melanoma, which made people realize chemotactic metastasis is regulated by CXCL12-CXCR4 biological axis. This study will investigate that CXCL12-CXCR4 biological axis and human peritoneal mesothelial cells'influence is on peritoneal cavity metastasis of EOC. We firstly constructed the epithelial ovarian cancer cell strain which expresses CXCR4 protein stably and explored that CXCR4's influence was to proliferation, migration, invasion of ovarian cancer cells from the study in vitro, then isolated human peritoneal mesothelial cells and verified the expression of some cell factors in human peritoneal mesothelial cells, and studied the effect of peritoneal mesothelial cells to ovarian cancer cell SKOV3 in invasion, adhesion and migration. At last, we established the animal model that were injected tumor cells through intraperitoneal pathway in nude mice, when they died naturally, we dissected abdominal cavity and observed neoplasm quantity and weight, the amount of ascites, the mean survival time, the effect of AMD3100. This is useful to better understand biologic behavior of ovarian cancer, and may provide basis in resisting metastasis in ovarian cancer treatment.Method: This project was divided into three parts:1 Establishment and identification of the EOC cell strain which expresses CXCR4 protein stably and its effect to cell growth and metastasis. We firstly established the eukaryotic expression recombinant plasmid pReceiver-M02- CXCR4, and identified by enzyme cutting and sequencing. The eukaryotic expression recombinant plasimid pReceiver-M02-CXCR4 and vector pReceiver-M02 were transfected into SKOV3 by lipofectamine-mediated gene transfection method, and gained positive cell clone by G418 screening and single cell isolation techniques. Cells that were transfected were examined by RT-PCR, Western Blot, and immmunocytochemistry methods. SKOV3 transfected with plasmid, SKOV3 transfected with vector, SKOV3 were cultured in vitro. MTT was used to analyze effect of different concentration of CXCL12 on three cell lines proliferation in serum-free growing conditions, and to determine inhibition of neutralizing CXCR4 antibody or antagonist AMD3100. Transwell and Matrigel were used to evaluate effect of various concentrationof CXCL12 on three ovarian cell lines'migration and invasion, as well as inhibition of neutralizing CXCR4 antibody or antagonist AMD3100.2 Primary culture of human peritoneal mesothelial cells and study of the impact of peritoneal mesothelial cells to human ovarian carcinoma cell line SKOV3 in invasion adhesion and migration. Human greater omenta was digested with 0.25%trypsin-0.02% ethylene diaminetetraacetic acid (EDTA), and cultured after removing red blood cells. Morphologic change was detected by inverted microscope during cellculture. Morphologic types were observed by HE staining and the purity was calculated. The ultrastructure was observed by scanning electron microscopy (SEM). Isolated cells were characterized by immunocytochemical analysis. To verify the expression of some cell factors in human peritoneal mesothelial cells by immunocytochemical analysis. Invasion, adhesion and migration of SKOV3 to human peritoneal mesothelial were detected by adhesion assay, Transwell Chamber invision assay and migration assay.3 Establishment and research of animal model that were injected tumor cells through intraperitoneal pathway in nude mice SKOV3 transfected with plasmid, SKOV3 transfected with vector and SKOV3 were cultured. Nude mice were divided into three groups at random, every group had twelve mice. 4×106 cells were injected into abdnominal cavity of nude mice respectively. After two days, every group was divided into control group and treatment group at random. Control group was injected PBS, and treatment group was injected AMD3100. When they died naturally, we dissected abdominal cavity and observed neoplasm, quantity and weight, the amount of ascites and the mean survival time.Result:1 Establishment and identification of the EOC cell strain which expresses CXCR4 protein stably and its effect to cell growth and metastasis:①After costruction and sequence of eukaryotic expression recombinant plasmid pReceiver-M02-CXCR4, the result confirms that the order of connection of gene and vector is correct and the sequence of gene is coincidence with CXCR4 of GeneBank.②Screening concentration of G418 is 600μg/ml.③After fourteen days, most of ovarian cancer cells tansfected with plasmid and vector died and formed cell sites. We gained several single cells by limiting dilution assay. The shape and growth velocity of the cells that were transfected had no marked change, but their stretch speed became slow.④We identified the epithelial ovarian cancer cells which express CXCR4 protein stably by RT-PCR, Western blot, immunocytochemistry methods. All these indicate that CXCR4 is positive in ovarian cancer cells transfected with plasmid. Transfection efficiency is about 73%. At the same time, we named ovarian cancer cells that transfected with eukaryotic expression recombinant plasimid pReceiver-M02-CXCR4 and vector pReceiver-M02 as SKOV3/CXCR4 and SKOV3/neg cells.⑤Under serum-free suboptimal condition, 100ng/ml CXCL12 can promote the proliferation of SKOV3/CXCR4 cells (F=256.89, P<0.05), and this effect can be inhibited by 10μg/ml neutralizing CXCR4 antibody or antagonist AMD3100. 100ng/ml CXCL12 can not promote the proliferation of SKOV3/neg and SKOV3 cells.⑥Under serum-free suboptimal condition, with regard to SKOV3/CXCR4 cells, 10ng/ml CXCL12 can promote cell migration, 100ng/ml CXCL12 can also promote cell migration (F=702.6, P<0.05). This enhancing effect of CXCL12 on cell migration is increased with increasing concentration of CXCL12, and is strongly inhibited by treatment with 10μg/ml neutralizing CXCR4 antibody or antagonist AMD3100. With regard to SKOV3/neg and SKOV3 cell, CXCL12 can not promote cell migration, regardless of the concentration of CXCL12.⑦Matrigel is reconstituted artificial basel membrane, which have the shape and function of internal basel membrame. Under serum-free suboptimal condition, with regard to SKOV3/CXCR4 cells, they displayed minimal invasiveness through Matrigel when CXCL12 was no present in the lower chamber of transwell (25.00±8.42). 10ng/ml CXCL12 promoted cell invasion across Matrigel. The number of invading cells through Matrigel in the prensnce of 100ng/ml CXCL12 was significantly higher than that of 10ng/ml CXCL12 (56.00±9.23) (F=16.65, P<0.05). The enhancing effect of CXCL12 on cell invasion was increased with increasing concentration of CXCL12, and is strongly inhibited by treatment with 10μg/ml neutralizing CXCR4 antibody or antagonist AMD3100. With regard to ovarian cancer cell SKOV3/neo and SKOV3 cell, CXCL12 can not promote cell invasion, regardless of the concentration of CXCL12. Every group had no significant difference (P>0.05) .2 Primary culture of human peritoneal mesothelial cells and study the impact peritoneal mesothelial cells to human ovarian carcinoma cell line SKOV3 in invasion adhesion and migration:①Cultured cells were multipolar and presented a cobblestone-like appearance when they reached confluence, with a purity of 95%.②SEM verified the abundant microvilli on the surface of the cells.③Immunocytochemical studies showed positive staining for cytokeratin and vimentin, but negative staining for white blood cell CD45 antigen and factorⅧassociated antigen. All the characters of the isolated cells were coincided with mesothelial cells.④Hydrocortisone (0.5%) and insulin (20μg/ml) had an exact effect to stimulate the growth of mesothelial cells.⑤Expression of CXCL12, MSLN in human peritoneal mesothelial cells were verified. The study also imformed that CXCR4 was negative in human peritoneal mesothelial cells.⑥Adhesion assay confirmed that human peritoneal mesothelial cells promoted the adhesion of SKOV3. Invision and migration assay confirmed that human peritoneal mesothelial cells promotes the invasion and migration of SKOV3.3 Effects of CXCL12-CXCR4 axis can the growth in skov3 ovarian cancer cells peritoneal Xengraft-bearing mice:①The rate of tumor formation is 100%.②Nude mice have ascites in every group. The quantity of ascites have no significant difference(F=48.09,P >0.05).③All nude mice have tumors in peritoneal cavity, gastrocolic omentum and mesentery. Liver, spleen, uterus and annexa had some metastasis. Heart, kidney, lung had no metastasis.④The mean survival time of the nude mice that were injected SKOV3/CXCR4 cells is 15.00±0.90 days in control group, 17.50±1.64 days in treatment group, two groups had significant dfference (Z=-2.40, P<0.05). The mean survival time of the nude mice that were injected SKOV3/neg cells is 16.83±1.33 days in control group, 17.00±0.89 days in treatment group, two groups had no significant difference (F=-2.29, P>0.05). The mean survival time of the nude mice that were injected SKOV3 cells is 17.20±1.90 days in control group, 17.17±1.94 days in treatment group, two groups had no significant difference (P>0.05). The mean survival time of the nude mice of the three groups in control group had significant difference.⑤The number of the metastatic tumors in nude mice that were injected SKOV3 was 78.16±1.17, which of significantly lower than that of nude mice that were injected SKOV3/CXCR4 cells 87.67±1.86 (P<0.05), and which have no obvious difference with nude mice that were injected SKOV3/neg cells 79.17±2.32 (P>0.05). The weight of the metastatic tumors in nude mice that were injected SKOV3 cells was 2.82±0.12, which was significantly lower than that of nude mice that were injected SKOV3/CXCR4 cells 3.74±0.11 (P<0.05), and which had no obvious difference with nude mice that were injected SKOV3/neg cells 2.82±0.13 (P>0.05).Conclusion:1 Establishment of the epithelial ovarian cancer cell which expresses CXCR4 protein stably is successful, and which is identified by RT-PCR, Western blot, immunocytochemistry. CXCL12-CXCR4 can promote proliferation, migration, invasion of ovarian cancer cells in vitro.2 Trypsin-EDTA enzymatic disaggregation method is a simple, effective and repetitive protocol for isolation of human peritoneal mesothelial cells. CXCL12, MSLN are positive and CXCR4 is negative in human peritoneal mesothelial cells. HPMC can promote proliferation, migration, invasion of ovarian cancer cell.3 Establishment of animal model that are injected tumor cells through intraperitoneal pathway in nude mice is successful. Ovarian cancer cells can promote cell proliferation, migration in vivo.These data indicate that interfering with interaction of CXCL12 and CXCR4 will improve treatment of patients with ovarian cancer.
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