The Role Of Heparanase In The Angiogenesis Of Ovarian Cancer And The Inhibiting Effect Of Curcumine On The Cell Growth Of Blood Vessel Endothelium Cell Of Ovarian Cancer

Background and AimsThe incidence of ovarian cancer is the second in the malignant tumor of department of gynecology,just next to uterine cervix cancer.It has some characteriscs such as earlier occurrence and metastasis.The development mechanisms and therapeutic study is the topic of biomedicine investigation.Heparanase is a kind of glucosidase,which can degrade heparin sulfate in extracellular matrix and basement membrane.Heparanase exist in malignant tumor widespreadly.The expression of heparanase in ovarian cancer and the relationship with clinical characteriscs of patients need more study.The growth of tumor depended on the persistence and extensive angiogenesis of tumor. New vessels not only provide tumor for nutrient substance and oxygen,but also is the important pathway of tumor metastasis.Vascular endothelial cell growth factor(VEGF) bind with receptorVEGFR-1,VEGFR-2 and VEGFR-3 on the cell membrane.The binding cause a serial signal conduction,which promote the proliferation and migration of vascular endothelial cell and neovascularization.VEGF play an important role in the occurrence, development and metastasis of tumor.RNAi technology is a new biomedical research method.Double strand RNA(dsRNA) consists of sense RNA and anti-senseRNA corresponding to messenger RNA(mRNA).The mRNA was degraded after double strand RNA imported into cells.The result is the gene silenced.This technology is called RNA interference.RNAi technology will be used in functional gene study,gene therapy and new drug research widespreadly.Curcumine has significant economic value and extensive pharmacological action,such as antioxygen,anti-inflammatory,anti-artherosclerosis and degradation of blood fat. Curcumine is an anticancer drug.More attention was paid to its anti-tumor effect.The aim of this research is to observe the role of heparanase in the angiogenesis of ovarian cancer and the effect of curcumine on the growth of blood vessel endothelium cell from ovarian cancer.The study was divided into four parts.In the 1^st part,65 cases of ovarian cancer were included into the experiment. Immunohistochemistry staining was used to detect the expression of heparanase,VEGF, VEGFR1 and Factor 8 of ovarian cancer.The relationship of heparanase with ovarian cancer clinical pathological characteristics and with the tumor angiogenesis was investigated.In the 2^nd part,the blood vessel endothelium cell from ovarian cancer was primary cultured.The cell bionomics was observed.Immunofluorescence method and RT-PCR method were used to detect the heparanase expression in blood vessel endothelium cell from ovarian cancer.In the 3^rd part,the RNAi experiment of heparanase expression in the blood vessel endothelium cell from ovarian cancer was performed.The cell activity,heparanase mRNA level and protein expression,VEGF expression,cell proliferation and apoptosis were detected.In the 4^th part,curcumine of different dose was added to the the blood vessel endothelium cell from ovarian cancer.The cell survival rate,cell proliferation were observed.Immunofluorescence and RT-PCR were used to detect heparanase expression.The main results and conclusions were summarized as follows:The 1^st part:expression characteristic of heparanase in ovarian cancer and the relationship with angiogenesis1.In the 12 cases of normal ovary,heparanase expression was not observed.The poaitive rate was 0%.In the 65 cases of ovarian cancer,heparanase was positive in 51 cases.The positive rate was 78.5%.The heparanase expression difference was significant.2.In the group of 27 cases whose ages were younger than 50,heparanase expression of 21 cases were positive.The positive rate was 77.8%.In the group of 38 cases whose ages were older than 50,heparanase expression of 30 cases were positive.The positive rate was 78.9%.In the 30 cases of serous cystadenocarcinom,25 cases were heparanase expression positive.The positive rate was 83.3%.In the 21 cases of serous cystadenoma, 16 cases were heparanase expression positive.The positive rate was 76.2%.In the 14 cases of other type groupof ovarian cancer,10 cases were positive.The positive rate was 71.4%. There was not significant difference with them.It shows that there is not dependablity between heparanase expression and ovarian cancer tissue type and patients age.3.In the earlier period group of 12 cases of ovarian cancer,heparanase was positive in 3 cases.In the advanced stage group of 53 cases of ovarian cancer,heparanase was positive in 48 cases.The difference was significant.In the well-differentiated group of 5 cases, heparanase expression was positive in 1 case.In the moderately differentiated group of 15 cases,heparanase expression was positive in 6 cases.In the poorly differentiated group of 45 cases,heparanase expression was positive in 44 cases.The difference was significant.It shows that there was dependablity between heparanase expression and ovarian cancer tissue pathological grade and clinical stage.4.VEGF expression had not significant difference within patients of ovarian cancer of different age.There was not dependablity between VEGF expression and ovarian cancer tissue type.The VEGF expression of ovarian cancer in advanced stage was higher than that of earlier period.The VEGF expression of well-differentiated and moderately differentiated group was lower than that of poorly differentiated group.The VEGF expression of ovarian cancer had the same tendency as heparanase expression.They were positive correlation. The coefficient correlation was 0.647.5.VEGFR1 expression had not significant difference within patients of ovarian cancer of different age.There was not dependablity between VEGFR1 expression and ovarian cancer tissue type.The VEGFR1 expression of ovarian cancer in advanced stage was higher than that of earlier period.The VEGFR1 expression of well-differentiated and moderately differentiated group was lower than that of poorly differentiated group.The VEGFR1 expression of ovarian cancer had the same tendency as heparanase expression.They were positive correlation.The coefficient correlation was 0.612.6.MVD had not significant difference within patients of ovarian cancer of different age.There was not dependablity between MVD and ovarian cancer tissue type.The MVD of ovarian cancer in advanced stage was higher than that of earlier period.The MVD of well-differentiated and moderately differentiated group was lower than that of poorly differentiated group.The MVD ovarian cancer had the same tendency as VEGF,VEGFR1 and heparanase expression.They were positive correlation.The coefficient correlation was 0.754,0.713 and 0.669.It shows that heparanase and VEGF have congenerous effect in the angiogenesis of ovarian cancer.The 2^nd part:blood vessel endothelium cell isolated culture and heparanase expression observation of ovarian cancer1.The blood vessel endothelium cell of ovarian cancer was primary cultured.The cellactivity was above 95%.The 1Fâ…§immunofluorescence detection was positive.The blood vessel endothelium cell of ovarian cancer can be used in the study of tumor angiogenesis.2.Heparanase was positive in the cytoplasm of blood vessel endothelium cell in the detection of immunofluorescence.The heparanase expression in the OdMEC group was higher than that in the HUVEC group.In the RT-PCR detection,the heparanase level in the OdMEC group was higher than that in the HUVEC group too.It shows that heparanase maybe the biological marker in the OdMEC.The 3^rd part:The effect of RNA interference on the heparanase expression of blood vessel endothelium cell of ovarian cancer1.The transfection rate of heparanase siRNA in blood vessel endothelium cell of ovarian cancer was above 95%.2.In the experiment group,the blood vessel endothelium cell of ovarian cancer was transfected by heparanase siRNA;the heparanase expression was detected at 2,4,6 and 8 day.The results showed that heparanase mRNA level and protein significantly decreased.It shows that heparanase siRNA transfection can significantly inhibit the heparanase of blood vessel endothelium cell of ovarian cancer.3.In the experiment group of the blood vessel endothelium cell of ovarian cancer transfected by heparanase siRNA,VEGF expression significantly decreased.It shows that heparanase siRNA transfection can significantly inhibit the VEGF expression of blood vessel endothelium cell of ovarian cancer.4.In the MTT detection of the experiment group,cell activity had not significant changes.but in the normal group and the control group,cell activity increased.In the experiment group,the proliferation index significantly decreased and apoptosis index significantly increased.It shows that RNAi interference can inhibit heparanase expression efficiently,and inhibit VEGF expression and cell proliferation too.The cell apoptosis was induced.RNAi maybe is one way for the treatment of tumor. The 4^th part:The effect of curcumine on the bionomics and heparanase expression of blood vessel endothelium cell of ovarian cancer1.In the cfurcumine group,cell proliferation step down and cell growth density decreased.With the dose increase of the curcumine,cells shed,floated or broke into pieces. It shows that curcumine(2-250ug/ml) can significant inhibit the activity of blood vessel endothelium cell of ovarian cancer dose dependently and time dependently.2.In the curcumine group,curcumine can inhibit the cell proliferation significantly.It shows that curcumine(2-250ug/ml) can significant inhibit the proliferation of blood vessel endothelium cell of ovarian cancer dose dependently and time dependently.3.In the curcumine group,the cell proliferation index decreased significantly.The subdiploid was observed.4.According to the immunofluorescence detection,the heparanase expression in the curcumine group decreased significantly.The effect was dose dependent and time dependent.It shows that curcumine(2-250ug/ml) can significant inhibit the heparanase expression of blood vessel endothelium cell of ovarian cancer.5.In the curcumine group,the MDA content and LDH activity of cell culture supernatant significant increased.