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Effects of CXCR4 on Epithelial Ovarian Cancer Growth Characteristics as Well as Invasion, Metastasis

Author: JiXiangLi
Tutor: WuXiaoHua
School: Hebei Medical University
Course: Obstetrics and Gynaecology
Keywords: Transfection CXCL12 CXCR4 SKOV3 Epithelial ovarian cancer Proliferation Shift
CLC: R737.31
Type: Master's thesis
Year: 2009
Downloads: 88
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Abstract


Purpose: Chemokines are small single-stranded molecule protein superfamily, is a class that cells chemotaxis of cytokines, chemokines exercise its functions by the chemokine receptor mediated. CXCL12 chemokine family belongs to (the last called SDF-1stromal cell-derived factor-1), secreted by the stromal cells. CXCR4 is a chemokine receptor, in studies of ovarian cancer, 14 observed only CXCR4 chemokine receptor expression in ovarian cancer cells. CXCR4 is the only physiological receptor of CXCL12, CXCL12 is the only physiological CXCR4 ligand between the two has a very high affinity, CXCL12 chemokine receptor CXCR4 and its specific posed by biological CXCL12-CXCR4 axis the spread of a variety of tumors and organ-specific metastasis plays an important role. Our previous study found no expression in normal ovarian epithelial CXCL12 and CXCR4; ovarian cancer tissues with high expression CXCR4; ovarian cancer ascites and peritoneal mesothelial cells highly expressed CXCL12, therefore, presumably CXCL12-CXCR4 biological axis in peritoneal metastasis of ovarian cancer play an extremely important role. This study was to investigate the in vitro expression of CXCR4 level changes on peritoneal metastasis of ovarian cancer. Methods: a eukaryotic expression recombinant plasmid pReceiver-M02-CXCR4 build CXCR4 gene by PCR using the PCR product was obtained. The empty vector pReceiver-M02 and PCR products were annealed, ligated and other reactions, eukaryotic expression recombinant plasmid pReceiver-M02-CXCR4, and sequenced. Use Sequence3.0 sequencing and analysis software to analyze data in GeneBank. Two ovarian cancer cell lines stably expressing CXCR4 Establishment and identification of the use of liposome-mediated transfection method, the eukaryotic expression recombinant plasmid pReceiver-M02-CXCR4 and empty vector pReceiver-M02 were transfected ovarian cancer SKOV3, with G418 screened a number of single cells and expanding culture, cells were obtained by RT-PCR, Western blot and immunocytochemistry were identified. 3 CXCL12-CXCR4 interactions in vitro experiments on ovarian cancer cell growth and the level of invasion and metastasis of transfected eukaryotic expression recombinant plasmid pReceiver-M02-CXCR4, transfected with empty vector pReceiver-M02 and the parental SKOV3 cells were cultured in vitro . Using methyl thiazolyl tetrazolium (MTT) assay, observation of three different concentrations of CXCL12 and CXCR4 cell growth neutralizing antibodies and CXCR4 antagonist AMD3100 inhibition; order of Transwell chamber as a model, the application of Matrigel explore various concentrations of CXCL12 gradient of the three cell migration, invasion impacts and CXCR4 neutralizing antibody and CXCR4 antagonist AMD3100 inhibition. Results: 1 target gene CXCR4 CXCR4 gene PCR product was amplified by PCR using a single size of 1059 bp specific band, consistent with CXCR4 fragment size. 2 eukaryotic expression recombinant plasmid extraction and sequencing of eukaryotic expression recombinant plasmid pReceiver-M02-CXCR4 build successful, DNA sequencing, cloning and confirmed CXCR4 empty vector pReceiver-M02 connection in the correct order, the sequence of the human CXCR4 GeneBank gene (NM003467) standard sequence exactly. 3 ovarian cancer cell line stably expressing CXCR4 Screening Screening G418 concentration 600μg/ml. Cells transfected with 14 d after limiting dilution method to obtain a number of single cells, the single cells were expanding culture. Transfected cell morphology and growth rate did not change significantly, stretching speed slower than the non-transfected cells, transfected eukaryotic expression recombinant plasmid pReceiver-M02-CXCR4 and empty vector pReceiver-M02 cells and were named SKOV3/CXCR4 SKOV3/neg cells. 4 ovarian cancer cell line stably expressing CXCR4 The transfected cells identified by RT-PCR, Western blot, immunocytochemistry identified three methods. Confirmed by the above three methods average transfection efficiency was 73%. 5 CXCL12-CXCR4 interactions on ovarian cancer cell growth MTT assay 100ng/mlCXCL12 SKOV3/CXCR4 alone can promote cell proliferation, and can be 10μg/ml CXCR4 neutralizing antibodies with 1μg/ml CXCR4 antagonist AMD3100 inhibited ( F = 256.89, P <0.05). For SKOV3/neg and SKOV3 cells, joining various concentration gradient CXC12 and CXCR4 neutralizing antibody or CXCR4 antagonist AMD3100, cell growth did not change significantly between the groups was not statistically significant (F = 0.31, P > 0.05) (F = 0.92, P> 0.05). 6 CXCL12-CXCR4 interaction effect on ovarian cancer cell migration is for SKOV3/CXCR4 cells, 100ng/ml CXCL12 make migrating cells was significantly increased, and can be 10μg/ml CXCR4 neutralizing antibodies and 1μg/mlCXCR4 antagonist AMD3100 inhibited (F = 393.21, P <0.05), for SKOV3/neg and SKOV3 cells, joining various concentration gradient CXCL12 and CXCR4 neutralizing antibody and CXCR4 antagonist AMD3100, cell migration numbers did not change significantly in all groups no significant difference between (F = 0.41, P> 0.05) (F = 0.62, P> 0.05). 7 CXCL12-CXCR4 interactions invasion of ovarian cancer cells to the current room to serum-free culture fluid, SKOV3/CXCR4 cells very few cells through Matrigel, CXCL12 effect, the number of cells through the Matrigel increased compared with the control group ; when the room Join 10μg/ml CXCR4 neutralizing antibodies with 1μg/mlCXCR4 antagonist AMD3100, can inhibit chemotaxis invasion (F = 16.65, P <0.05); For SKOV3/neg and SKOV3 cells, joining various concentration gradient CXCL12 and CXCR4 neutralizing antibodies and CXCR4 antagonist AMD3100, the number of cells in each group of the invasion was no significant difference compared to (F = 0.04, P> 0.05) (F = 0.05, P> 0.05). Conclusion: a eukaryotic expression recombinant plasmid pReceiver-M02-CXCR4 successfully constructed confirmed by sequencing; ovarian cancer cell line stably expressing CXCR4 SKOV3/CXCR4 successfully constructed by RT-PCR, Western blot, immunocytochemistry identified three methods for Subsequent research tools and platforms. 2 CXCL12-CXCR4 interactions promote ovarian cancer cell proliferation, migration, invasion; CXCR4 neutralizing antibodies or CXCR4 antagonist AMD3100 inhibit ovarian cancer growth and metastasis, suggesting that CXCL12-CXCR4 biological axis in ovarian cancer growth, invasion and metastasis plays an important role.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Female genital tumors > Ovarian tumors
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