| BackgroudOvarian cancer is one of three major malignant tumor of department of gynecology, in which more than leather, sex is the most common tumor, accounting for about 70% of ovarian malignant tumors, female malignant tumors of the sixth, the fifth cause of cancer-related death it have the characteristics of onset hidden, easy to transfer, and poor prognosis, since most early in patients with no obvious symptoms, 75% of patients diagnosed has to middle-late, therefore, the mortality rate in female genital malignancies the first.In the past for a long time, with the improvement of modern detection and treatment, a great progress has been made in treatment of ovarian cancer, took surgery in the treatment of ovarian cancer MDT and the latest technological progress of neoadjuvant chemotherapy for ovarian cancer, but the recurrence of ovarian cancer metastasis rate and the mortality is still high, due to its high transfer rate and the recurrence rate, the overall prognosis is still poor, 5 years of survival rate is only 30% to 30%.Therefore, actively looking for a kind of accurately predicting ovarian malignant degree and prognosis of early indicators, so that more early rapid diagnosis and earlyintervention of diseases is a hotspot of current research.Notch1 is a transmembrane protein, Notch1 is one of the Notch signal pathway transmembrane receptors, numerous studies have demonstrated that Notch1 associated with invasion and metastasis of tumor, it is involved in cancer development. In breast cancer, colorectal cancer, and other organizations, Notch1 expression is below tissue adjacent to carcinoma and normal tissues. In gastric cancer tissues, Notch1 associated with distant metastasis, it can promote the nests by epoxidation and enzyme- 2 form [8].In pancreatic cancer, Notch1 signaling pathway activation of nuclear factor lambda B, the release of VEGF and matrix metalloproteinases promote tumor cell invasion and metastasis.PDGF- BB can accelerate cell proliferation, cell chemotaxis, vasoconstriction, such as biological effect, is decided to tumor size, metastasis and recurrence and prognosis of the important factors.PDGF- BB is an important part of the PDGF family, is an important angiogenic factor.Through combine with PDGFR induced its tyrosine kinase phosphorylation, promote the formation of new blood vessels, leading to tumor growth.Many malignant tumors such as breast cancer, prostate cancer and other groups exist in the high expression of PDGF and its receptor, plays an role in the occurrence of tumor development.In tumor occurrence and development process, a variety of environmental and cellular factors can induce the expression of PDGF tumor tissues, such as oxygen immediately release of VEGF and PDGF angiogenesis stimulating factor, thus activate angiogenesis and tumor tissue oxygen supply in the further.In addition, the mutation of oncogene and tumor suppressor genes can induce the expression of PDGF.For Notch1 signaling pathway in a wide variety of tumor research are more, and PDGF- BB for invasion and metastasis of malignant tumor angiogenesis and is directly related to the influence of aspect, whether both for the development of tumor is a progressive relationship and if there is a correlation between remains to be further proved.Gradually, aiming at the above problems, this study reported Notch1 and PDGF BB expression in ovarian cancer organizations, through the study of Notch1 and PDGF-BB in clinical pathology data correlation, reported that Notch1 and PDGF- BB close relationship with the clinical pathological and expressed in ovarian cancer tissue of thelinear relationship.We further confirmed in ovarian cancer cell line Notch1 and biological function of PDGF- BB, preliminary assessment of Notch1 and PDGF- BB relevance in ovarian cancer invasion and metastasis, for future clinical offers a new way for the early diagnosis of ovarian cancer.Objective1. Notch1 and PDGF BB expression in ovarian cancer tissue and correlation analysis;2. Notch1 expression in ovarian cancer cells and the influence of the invasion and metastasis;3. The down-regulation of Notch1 expression for its survival ability and apoptosis in ovarian cancer cells;4. The down-regulation of Notch1 expression, verification PDGF-BB expression in ovarian cancer cells;5. The down-regulation of PDGF-BB expression in ovarian cancer cells and its effect of invasion and metastasis.Methods1. The Notch1 and PDGF-BB expression in ovarian cancer tissue and its clinical pathological analysis of the correlation.a) using the immunohistochemical technique analysis of Notch1 and PDGF-BB in 60 cases of epithelial ovarian cancer tissue specimens and 10 cases of normal ovarian tissue specimens expression of paraffin specimens in the organization;b) spss17.0 statistical software was used to analysis, chi-square test and analysis of Spearman correlation coefficient.2. The application of RT-PCR and Western blot analysis respectively four Notch1 gene expression and protein expression in ovarian cancer cells, Transwell method to detect four ovarian cancer cells invade migration ability.Notch1 si RNA high transfection transfer respectively ovarian cancer cell line HO-8910 PM and low transfer of ovarian cancer cell line HO-8910, observed after transfection Notch1 gene and protein expression in ovarian cancer cell lines, the biological function of ovarian cancer cells after transfection Transwell method and analysis of cell counts.3. Notch1 si RNA high transfection transfer respectively ovarian cancer cell line HO-8910 PM and low transfer of ovarian cancer cell line HO-8910, by using the determined by MTT method and enzyme-linked immune detector at 490 nm wavelength measurement of its light absorption value cell viability, use Annexin V-FITC/PI double staining flow cytometry to detect cell apoptosis.4. Notch1 siRNA high transfection transfer respectively ovarian cancer cell line HO-8910 PM and low transfer of ovarian cancer cell line HO-8910, observed after transfection PDGF-BB gene expression in ovarian cancer cell lines and Notch1 and PDGF-BB protein expression.ELISA method detected PDGF-BB concentration in cell supernatant.5. PDGF-BB si RNA high transfection transfer respectively ovarian cancer cell line HO-8910 PM and low transfer of ovarian cancer cell line HO-8910, observed after transfection PDGF-BB protein expression in ovarian cancer cell lines.Transwell method to detect biological function of ovarian cancer cells after transfection and cell count analysis.Rusults1. The observation of Notch1 and PDGF-BB expression in ovarian cancer tissue and correlation analysis of clinical pathology.a) Notch1 and PDGF-BB were expressed in ovarian cancer tissue;b) Notch1 positive staining was mainly located in cell membrane, nucleus and cytoplasm were expressed. In ovarian cancer tissues and 63%(38/60) high expression, 37%(22/60) lower expression; In 10 cases of normal ovarian tissue and part of the staining showed high expression of 20%(2/10), the rest were lower expression;c) PDGF-BB positive staining mainly in the cytoplasm.In ovarian cancer tissues and 60%(36/60) high expression;40%(24/60) lower expression.Part in normal ovarian tissue staining showed high expression of 10%(1/10), the rest were low.d) PDGF-BB and Notch1 expression level and ovarian cancer FIGO staging, pathologic stage, lymph node metastasis(P < 0.05), had nothing to do with the ovarian cancer patients’ age, tissue types(P > 0.05).e) Spearman correlation analysis showed that the expression of PDGF-BB expression and Notch1 was positively related(r = 0.696, P < 0.696)2. The down-regulation of Notch1 expression in ovarian cancer cells, biological effect analysis of ovarian cancer cells.a) RT-PCR and Western blot respectively for them of Notch1 gene expression and protein expression in HO-8910 PM,SKVO3,3AO and HO-8910 ovarian cancer cells for testing, HO-8910 PM ovarian cancer cells HO-8910 gene expression and protein expression were the highest, ovarian cancer cells HO-8910 both were low.b) HO-8910 PM, SKVO3, 3AO and HO-8910 ovarian cancer cell invasion and migration statistics showed: invasion and migration group HO-8910 PM cells number was more, its strong ability to affect migration, invasion and migration group HO-8910 cells was less, explain its affect migration ability was weak.c) For high transfer of ovarian cancer cell line HO-8910 PM and low transfer of ovarian cancer cell line HO-8910 transfection Notch1 si RNA, RT-PCR detection Notch1 gene expression level transfection group were lower than the control group and untransfected group and Western blot analysis that Notch1 protein expression transfection group were lower than the control group and untransfected group, Transwell method to detect HO-8910 PM and HO-8910 ovarian cancer cell invasion and migration ability, the cell count showed that Notch1 transfection group were lower than the control group and untransfected group(P < 0.05).3. The down-regulation of Notch1 expression in ovarian cancer cells, the cell viability and apoptosis of impact assessment.a) Determined by MTT method is used to inspect the transfection Notch1 siRNA 490 nm wavelength of ovarian cancer cells, according to the determination of its light absorption value transfection group and untransfected group compared with control group, no significant difference(P > 0.05).b) Using Annexin V- FITC/PI double staining flow cytometry to detect cell apoptosis results: transfection group and no- transfection group compared with control group, no significant difference(P > 0.05).4. The high transfer of ovarian cancer cell line HO-8910 PM and low transfer of ovarian cancer cell line HO- 8910 transfection Notch1 si RNA, By RT-PCR analysis PDGF-BB was lower than the control group and the level of gene expression in untransfected group.Western blot analysis of Notch1 and PDGF-BB protein expression in ovarian cancer cells were lower than the control group and untransfected group, ELISA analysis of PDGF-BB cells in cell supernatant were lower than the control group and untransfected group.(P < 0.05).5.By Western blot analysis PDGF-BB protein expression in transfection group were lower than the control group and untransfected group.Transwell method to detect the HO-8910 PM and HO-8910 ovarian cancer cell invasion and migration ability, the cell count showed that cell count PDGF- BB transfection group were lower than the control group and untransfected group(P < 0.05).Conclusion1. This experiment determined the Notch1 and PDGF-BB expression in ovarian cancer tissues and cells.Notch1 and PDGF-BB in ovarian cancer tissues with linear correlation, both for high expression and a common signaling pathways in ovarian cancer development.2. Found Notch1 and PDGF-BB expression level were associated with the FIGO staging, pathologic stage, lymphnode metastasis of ovarian cancer tissue.PDGF- BB was associated with ovarian cancer tissue pathology classification, we proved that the two played an important role in ovarian malignant potential.3. By down-regulation of Notch1, the invasive migration ability of the ovarian cancer cells could be reduced, and at the same time decreased the expression of PDGF-BB, but not on ovarian cancer cell viability and apoptosis significantly influence, confirmed the Notch1 positive regulate PDGF-BB, there may be a PDGF-BB positive ovarian cancer cells under the effect of Notch 1 pathway, the excessive promote new blood vessels to form, to induce the occurrence of tumor development. |
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