Experimental Study On The Inhibitory Effect Of Anti-VEGF Monocoly Antibody Bevacizumab And DDP To Nude Mice Ascites Which Is Induced By High Level VEGF Expression Of Ovarian Cancer Cell Lines

Background and ObjectiveOvarian cancer is characterized by rapid growth of solid intraperitoneal tumorsand production of large volumes of ascites. The study has shown that Vascularpermeability factor(VPF),aslo known as vascular endothelial growth factor(VEGF)plays an important role in ascites formation associated with Ovarian cancer. This hasbeen a new strategy which inhibits neovascularization and permeability to reduce theascites production by means of targeting the VPF/VEGF. This study plans to establishthe model of the athymic mice ascites tumor with the ovarian cancer cell line which ishighly expressed VPF/VEGF, to observe the inhibitory effect of Bevacizumab(anti-VPF/VEGF Mab) on ovarian cancer ascites,as well as,to investigate themechanisn of action. Investigation the conjoined effect by combination Bevacizumabwith chemotherapeutics DDP on athymic mice ascites formation, to improve the molecule target of ascites management,to search the more effective clinical methodwith bio-chemotherapy to manage ascites.Methods1.To subculture OVCAR3 and HEY-AS cell lines of human ovarian cancer anddetermine their expression of VEGF and the expression of VEGFR of OVCAR3 byRT-PCR.2.To observe the ascites formation after the athymic mice were intraperitoneallyinoculated with two cell lines, Bevacizumab and chemotherapeutics DDP wereadministrated into the peritoneum of the athymic mice.3.Determination the level of VPF/VEGF in ascites by ELISA,after each athymicmouse was injected with 200ul of 5% Evan blue dye from vena caudalis, opticaldensity of Evan blue was measured with 540nm, to reflect indirectly the concentrationof Evan blue dye through the optical density, according it to analyze the membranepermeability.4.To investigate the effect of Bevacizumab and DDP on vascularization ofsolid tumor in ovarian cancer in which VEGF is highly expressed by detectionmicrovessel density with CD34 antibody.5. Determination the level of VEGF by means of ELISA from the clinical ascitessamples, and the important role of VEGF/VPF in ascites formation is further verifiedaccording to the level of VEGF/VPF in patients.6.Statistical treatment: SPSS 10.0 software.Results1. Cultivation and judgement of OVCAR3 and HEY-A8. After resuscitation andsubculturing,the two cell lines grew in a good condition. Cell grew in groups and thesingle cell was big.OVCAR3 cell was round and lucency;HEY-A8 cell was lobulated and transmissivity was very good. The detection of qualitation of VEGF was carried by the method of RT-PCR,and the resultdemonstrated that VEGF was expressed highly in OVCAR3 cell,while it was notexpressed in HEY-AS cell.2.The establishment of the ascites model of athymic mouse with the two ovariancancer cell lines.The two cell lines were intraperitoneally inoculated into differentathymic nude mouse. Athymic nude mice (5 to 7 weeks of age) were inoculatedintraperitoneally with OVCAR3 cells(n=48; 5×10~6 cells per mouse in 200ul of cellsuspension). In addition, 12 Athymic nude mice (5 to 7 weeks of age) were inoculatedintraperitoneally with HEY-AS cells(2×10~6 cells per mouse in 200ul of cellsuspension).Abdominal bulge and body weight were observed twice weekly afterinoculation. After the OVCAR3 cells were inoculated for two weeks, Athymic nudemice began to appeare abdominal bulge,hinting ascites formation.At postmortemexamination,ascites could be seen in abdominal cavity and tumors were found on thesurface of the peritoneum,intestines,mesentery and uterus. But abdominal bulge wasnot appeared in athymic nude mice which inoculated intraperitoneally with HEY-A8cells.3.The effect of Bevacizumab,DDP and Bevacizumab combinatin with DDP onthe ascites of athymic nude mice. Measuring the ascites volume,the content of VEGFin ascites,the permeability of peritoneal(OD value), the number of red cells and tumorcells counted in ascites and microvascular density in tumor tissues.The experimentalresult indicated that three treatment groups had inhibitory action on ascites,the ascitesvolumes of Bevacizumab group,DDP group and therapeutic alliance group are:2.36±0.34, 3.62±0.31, 1.82±0.40; the significant inhibitory action on ascites wasBevacizumab group and therapeutic alliance group,especially the therapeutic alliancegroup (p=0.000; p＜0.05). The number of red cells and tumor cells counted inmalignant ascites were beyond 10~7/ml and 10~6/ml, the red cells in ascites of control group was 78.83±7.63; the number of red cells of Bevacizumab group,DDP groupand therapeutic alliance group was: 49.33±6.15, 63.67±9.83, 34.00±14.27,compared to the control group,the red cells in ascites of every treatment group wassignificantly less than the control group (p=0.000; p＜0.05); the tumor cells inascites of control group was 77.00±6.60; the number of tumor cells of Bevacizumabgroup,DDP group and therapeutic alliance group are: 56.67±4.68, 67.83±6.88,40.33±9.59, there was significant difference between treatment groups and the controlgroup, especially, the most significance was the therapeutic alliance group (p=0.000;p＜0.05); The concentration of VEGF in ascites of the Avastin group,DDP group,therapeutic alliance group and control group was: 193.3±10.09, 402.0±11.59,138.8±10.28 and 411.2±10.80, the concentration of VEGF in ascites of theBevacizumab group was obviously less than that of DDP group, and control group;while there was not significant difference between DDP group and controlgroup(p=0.154, p＞0.05); these all suggested the therapeutic alliance combinationwith Bevacizumab and DDP could strengthen the inhibitory action on the VEGF inascites (p=0.000; p＜0.05). In the membrane permeability,the result suggested therewas no obvious difference between DDP group and control group,as well asBevacizumab group and therapeutic alliance (p=0.078; p＞0.05),from this,we couldconclude that Bevacizumab could significantly improve the membranepermeability,while DDP had no significant impact on membrane permeability,.4. The influence of Bevacizumab group,DDP group and therapeutic alliancegroup with Bevacizumab and DDP on microvessel density in tumor tissue.Theexperiment result had shown: the mean value of microvessel density in control groupwas 33.00±3.74; the mean value of microvessel density in DDP group was31.50±3.08; the mean value of microvessel density in Bevacizumab group was21.67±2.73; the mean value of microvessel density in therapeutic alliance group was 16.83±2.48. There was no obvious difference between DDP group and control group(P=0.404, P＞0.05); there was significant difference between Bevacizumab groupand therapeutic alliance group (p=0.000; p＜0.05); compared to DDP group andcontrol group, Aavastin group and the therapeutic alliance of Bevacizumab with DDPcould significantly reduce the microvessel density (p=0.000; p＜0.05).5. The level of VPF /VEGF in ascites of clinical patients demonstrated thatVEGF/VPF played an important role in ascites formation. in the ascites of clinicalpatients, the level of VPF/VEGF in malignant ascites was (1294.9±761.9) pg/ml,while it in the non-malignant ascites was (224.6±198.6) pg/ml, there hadsignificant difference between them(p=0.000; p＜0.05).Conclusions1. There are some differences in the ability of development of ascites betweenOVCAR3 and HEY-A8, that VEGF is high expressed in OVCAR3,while HEY-A8 isnot expressed VEGF. Maybe this difference is concerned with the diversity ofexpression of VEGF in the two cell lines.2. In the ascites model of the OVCAR3 athymic mice,the experiment resultwhich administrated with Bevacizumab,DDP and combination Bevacizumab withDDP demonstrated that Bevacizumab could significantly inhibit Ascites production ofthe athymic mice; reduce the content of VPF/VEGF in ascites; degrade themembrane permeability and decrease the number of tumor cells and red cells in theascites, these results suggested that the therapeutic alliance of Bevacizumab and DDPcan efficiently control the ascites.3. Bevacizumab and DDP can obviously reduce the level of microvessel densityin tumor tisse of ovarian cancer, which suggests the combination of them can inhibitthe new vessels formation in ovarian cancer, thus,restrain the growth and development of ovarian cancer, and delay the ascites formation.4. The level of VPF/VEGF of ascites in clinical patients also can demonstratethat VPF/VEGFis the key factor in ascites production, clinically, the clinical workercan determine the character of ascites and guide the treatment, VPF/VEGF is beingexpected to be a valuable tumor marker in ascites.