Effects Of HPA To Form Tubular Structure、Prolifer And Migrate On Umbilical Cord Blood Derived EPC In Vitro

Backgrounds：The incidence of ovarian cancer accounts for46%of gynecological malignancies.Signs and symptoms of ovarian cancer are frequently absent early on and when they existthey may be subtle, with easy transferred, easy to relapsed and so on. Patients with ovariancancer have poor clinical outcomes, low in5-year survival rate. It has became a seriousthreaten to women health[1,2]. Early diagnosis and effective treatment was the bottleneck ofclinical diagnosis and treatment. With the understanding of mechanisms of tumor invasionand metastasis, the existence of the phenomenon in ovarian cancer is angiogenesis andactive angiogenesis is an important pathological feature, which have Important influence oncancer development, invasion, metastasis and so on. Isolated from the bone marrow,umbilical cord blood or peripheral blood, EPC (endothelial progenitor of the cell), whichraised in the chemokine and growth factor stimulation, home in tumor angiogenesis sites,involved in tumor angiogenesis and promote the microvascular bedsprouting angiogenesis.More and more studies have confirmed that the tumor has a rich supply of blood vessels.EPCs participated in tumor angiogenesis and promote tumor growth.1Endothelialprogenitor cells play an important role in tumor angiogenesis. Some growth factor such asVEGF secreted by the tumor, is not only local effect, but can mobilized the bonemarrow-derived EPC to be settled in the vascular bed. Some studies of insulinoma foundthat bone marrow-derived EPC participate in the generation of tumor angiogenesis bed[14].Glioma study confirmed that tumor stem cells secrete a large number of VEGF whichpromote EPC settled in the tumor bed, differentiation and forming pipeline, and also impacton self-renewal and differentiation of cancer stem cell. In the early stage of liver cancer,bone marrow-derived EPC accounted for6%of the endothelium of tumor angiogenesis,increased to27%in advanced hepatocellular carcinoma. In the process of tumorangiogenesis, there are also have the EPC participation[12]. Heparanase (HPA) as malignant one of the important functional enzyme, is a keyenzyme involved in the degradation of extracellular matrix and basilar membrane topromote tumor cell invasion and metastasis. With the progress of the study at home andabroad and more and more depth understanding of HPA, HPA expression is found inmalignant ovarian tumors significantly higher than the normal ovarian tissue. Theexpression level of HPA tended to increase, when the degree of malignancy and staginggradually increased, which confirmed that HPA play a vital role in the malignant ovariantumor invasion, metastasis and angiogenesis. Moreover, the expression intensity of VEGFand HPA was positively correlated in ovarian cancer, suggesting that the HPA promoteangiogenesis through the release, activation of VEGF. Therefore, we speculate that theremaybe a direct interaction between malignant tumor cells and EPC which constitute thetumor microenvironment. This interaction may be bidirectional: the vascular cells in theniche support cancer stem cell differentiation and update, while the cancer cells maintainthe cells iv microenvironment at the same time[15]. Therefore, we speculate that there is amicroenvironment-niche in the ovarian, where cancer tumor cells release a variety ofcytokines, which interact to endothelial progenitor cells promoting tumor angiogenesis. Inview of our preliminary tests have confirmed that HPA expression in human ovarian cancertissue is related to tumor invasion, metastasis, clinical stage and microvessel density[13]andHPA highly expressed in human ovarian cancer cell line SKOV3. There ismicro-environment between the EPC and ovarian cancer, so the aim of this study is todetermine whether HPA secretion by ovarian cancer cells can promote EPC angiogenesis.And further study to discuss the phenomenon of HPA promoting EPC proliferation andmigration. Based on the previous findings, this study propose the hypothesis of tumor cellstumor the cell-cell niches (T-E niches) of ovarian cancer, which suggested that in the tumorprogression, recurrence and metastasis, the interaction between ovarian cancer cells andEPC promote tumor angiogenesis.Purpose：The aim of study is to clarify how HPA secreted by ovarian cancer cells SKOV3haveinfluence of its proliferation and migration to cord blood EPC cells. And to discuss thespecific mechanisms of interaction between the ovarian cancer cells and endothelialprogenitor cells. Methods：1. Mononuclear cells prepared from healthy donor cord blood by centrifugation on aFicoll-Hypaque density, then were cultured in EBM-2medium supplemented with10%or20%FBS for7to14days, in order to differentiate to EPC. Detect the capacities ofphagocytosis to DiI-acLDL and binding of FITC-UEA-1(double-fluorescence staining);EPC was determined expressions of CD34and CD133, VEGFR-2by flow cytometry testsin order to identification features of EPC.2. Build the HPA-shRNA Lentiviral Particles to transfect ovarian cancer cells SKOV3and to inhibit HPA expression; then detect gene and protein expression levels of HPA,which secreted from ovarian cancer cells, using real-time PCR, Western blot analysis.Moreover, detect their blocking effect. Aquiered the best siRNA cells were subcultured.Collected before and after transfection supernatant of ovarian cancer cell, and detected theexpression level by ELISA.3. The influence of HPA to EPC growth (lymphatic of endothelial cells, of LECs)was determined using MTT assay. Using transwell experiments and tube-like structureformation assay to analysis the effect of of HPA concentration from different ovarian cancercell supernatant to EPC cell migration and tube-like structure formation.Result：1. Early primary cultured cell were morphological heterogeneity, and most of themwere spiculiform and polygonal, some almost circular, mixed with red blood cells.After24hours incubation, the cells were small round, floating in the culture medium. After3days,the cells began to extend into the spindle or spindle-shaped. After5days, colony formation,set off the central circular cell populations, surrounding were spindle cells appeared. After7days, cells in the central round were floating, surrounding spindle cells remained adherentstate and vigorous growth.About14days cells integrated into the endothelial cell monolayer.Flow cytometry showed that the5th day to14th day, EPC expressed of VEGFR (95.30%)of CD34(47.22%) and CD133(57.29%), stable and uniform. Then detected EPCphagocytosis of DiI-acLDL and binding capacity of FITC-UEA-1using dual fluorescentstaining.2. Build the HPA-shRNA Lentiviral Particles to transfect ovarian cancer cells SKOV3and observed under fluorescence microscope. That effectively reducing the expression of HPA to obtain efficient transfection. There were different three pairs interfere sequencesdesigned in experiment. The interference effect of HPA-shRNA-1and HPA-shRNA-2sequence are the best. The HPA-shRNA-3sequence does not achieve the interference effect.Acquired stably transfected cells, then detected gene and protein expression levels of HPAusing real-time PCR, Western blot analysis. The results show that the shRNA specificblocking the expression of HPA.3. Analysis how the different HPA concentration promote EPC cell proliferation,migration and tube-like structure formation (P <0.05) in different volume of ovarian cancercell supernatant. When the volume fraction was40%, we got the strongest promotion.Extended over time can promote the fouction of EPCs and in a dose-dependent. Comparedto blocking the secretion of HPA supernatant, there were the significant differences.Conclusion：1. Successfully isolated EPC in vitro, we cultured monocytes in conditioned mediuminduced5-14days and obtain a more stable EPC. About two weeks of integration into theendothelial cell monolayer, losing the characteristics of progenitor cells.2. Build the HPA-shRNA Lentiviral Particles to transfect ovarian cancer cells SKOV3and effectively inhibit HPA expression. And we acquired efficiently transfected cells.3. Add different volume fractions of the ovarian cancer cell supernatant in EPCscondition medium, which contained different concentrations of HPA. HPA directlypromoted EPC cell proliferation, migration and tube-like structure formation. The reasonwas related to ovarian cancer cell surface expression of HPA. Therefore, HPA secretedfrom ovarian cancer cell further promote the angiogenesis of the latter.