| Background:Ovarian cancer is a kind of common cancer in female reproductive system which havea high incidence rate, as follows are cervical cancer and endometrial cancer. The mortalityrate is higher than any other female reproductive system cancer because its morbidity covertcharacteristic, easily to transfer and more likely to relapse. In addition, lacking ofprevention and treatment and the low5-year survival rate at this stage, Ovarian cancergradually become a serious threat to women life which bring huge burden to society. Therecent research indicate that the active angiogenesis has been involved to the developmentof the ovarian cancer, which affects tumor invasion, metastasis and disease outcome. Recentstudies have demonstrated that endothelial progenitor cells (EPCs) is a key factor of theangiogenesis, which may be derived from bone marrow incorporation and differentiation.Thus, we hypothesize that EPCs coexist with ovarian carcinoma cells in the tumormicroenvironment, tumor cells secrete a variety of cytokines or activate the relativeenzymes, then have a interaction with EPCs to promote tumor angiogenesis. In this study,we intend to inhibit the expression of ovarian cancer cells cyclooxygenase2(cox-2) toobserve the influence of the migration chemotactic ability of EPCs by the way ofnon-contact co-culture.Objectives:To suppress ovarian cancer SKOV-3cells expressing COX-2by the cyclooxygenaseinhibitor sulindac, and then co-cultured with EPCs to observe the migration chemotacticability, then explore its possible mechanism.Methods:1. Using the gradient centrifugation to isolate mononuclear cells in cord blood,combined use growth factors and screening method to purify cells, then using doublefluorescent staining and immunofluorescence technique to identify umbilical cord blood-derived EPCs.2. Using the stepwise concentration of sulindac to act on different times in ovariancancer SKOV-3cells, then using RT-PCR and Western Blot technique to detect theexpression of COX-2to identify the most obvious combination of inhibition by theMultivariate Analysis Of Variance.3. Co-cultured with the inhibition of the expression of COX-2ovarian cancer cells andEPCs. Comparing the change of the EPCs migration chemotactic ability by contrastiveobservational method, and check the amount of HPA expression in cells and VEGF contentin culture medium, correlation analysis the relationship with cox-2, HPA, VEGF and EPCsmigration quantities.Results:1. In the early stage, most of the EPCs are round, after three days, the cultured cellsgrew against the wall of flask, after seven days formed a monolayer after fusion andfortnight later showed a mosaic arrangement and the typical "cobblestone" morphology.FITC-UEA-1and DiI-Ac-LDL intake tests on EPCs showed double positive responses andimmunofluorescence surface markers CD34and CD133were both positive.2. Using cyclooxygenase inhibitor sulindac treatment of ovarian cancer SKOV-3cells,RT-PCR showed that dose and time-dependent relationship exist with drug and inhibitionefficiency. Then we selected a combination of three strong inhibitory effects, used WesternBlot technique to detect COX-2protein expression, the results showed that: after Sulindac(2mmol/L) treatment for48h, the COX-2expression was the most significant decrease.3. Ovarian cancer inhibition of cox-2cells could make the EPCs migration chemotacticability significantly decreased (P <0.01), cox-2, HPA, VEGF were related to the number ofEPCs migration (P <0.01or P <0.05), and the expression of cox-2was related to theamount of HPA, VEGF expression (P <0.01or P <0.05).Conclusions:1. After separation, culture and identification of mononuclear cells from umbilical cordblood, we got EPCs successfully.2. After sulindac treatment in ovarian cancer SKOV-3cells, the cox-2expression wassuppressed. We successfully built a model of the cox-2expression inhibition ovarian cancercells. 3. The cox-2expression inhibition ovarian cancer cells significantly reduced the EPCsmigration chemotactic ability, which was associated with lower levels of HAP, VEGF inreaction system. |
