The Mechanism Of Recombination Human Endostatin-endostar Inhibiting Growth And Metastasis On The Nude Mice Model Of Human Cervix Cancer

Objective1 To observe the influence of the recombinant human endostatin (Endostar) alone or with cisplatin on the nude mice model of human cervix cancer.2 To evaluate the inhibition of angiogenesis, lymphangiogenesis, lymph node matastasis and the contribution of tumor cells ultrastructure of endostar on the nude mice model of human cervix cancer.3 To detect the expression of VEGF-A, C, D on the nude mice model of human cervix cancer.Methods1 Establish nude mice xenograft modelFemale nu/ mice (5～6 weeks old, 20grams) were raised cages. Hela cells (benefit from longmed incorporation) expressing luciferase were resuspended in MEM (5×106 cells/200ul), and implanted subcutaneously into the hind flank region of the mice.2 Experiment groups Nude mice were randomly grouped until tumor's length reached 7mm. One group of mice (n=8) was treated i.s with Sodium Chloride (NS). A second group of mice (n=8) was treated i.p with cisplatin (DDP) alone (5mg/kg) twice a week. A third group (n=8) was trested i.s with endostar alone (10mg/kg) forth a week. A forth group (n=8) was trested with cisplatin (5mg/kg) twice a week combined with endostar (10mg/kg) forth a week. A fifth group (n=8) was trested with cisplatin (5mg/kg) twice a week combined with endostar (20mg/kg) forth a week. The course of all the treatments would last for 4 weeks.3 Detect the growth of nude mice tumorWhen the tumors'length reached 7mm, the mice were given a 150mg/kg dose of D-luciferin by intraperitoneal injection and were photographed. Before imaging, mice were imaged 15 min after luciferin administration using the IVIS system to determine total photons of emitted light as a measure of the relative number of viable tumor cells in the tumor.4 After 4 weeks'treatment, the nude mice were killed with chloraldurat. The samples of tumor were fixed with 4% paraform for paraffin section and fixed with 4% glutaral for TEM section respectively.5 CD34 was used for the marker of micro vessels and LYVE-1 was used for the marker of micro lymphvessels. Immunohistochemical staining was employed to detect the tumor micro vessels and micro lymphvessels. The expressions of VEGF-A, C, D in implantation tumor tissues of nude mice were determined by immunohistochemistry.6 To observe the transform of ultrastructure on nude mice tumor with transmission electron microscope.Results1 Effects of endostar on the size of transplantation tumor The sizes of subcutaneous transplantation tumor of all the mouse had grown, and they grew fast and obviously in NS group in contrast to that of DDP group, endostar group,endosta(r10mg/kg)with DDP group and endosta(r20mg/kg)with DDP group.So far, there is no better way to examine the activity of tumor and measuring the tumor size has been used to evaluate the growth of the tumor: inhibition or stasis. The aim of this study was to evaluate the activity of tumor cells by"Xenogen IVIS Imaging System"and draw tumor growth curve according to photons in order to recognize the change and growth condition of the tumor. When the study was over, the photons of endostar group,endosta(r10mg/kg)with DDP group and endostar(20mg/kg)with DDP group decreased strikingly compared with that of DDP group and NS group (P<0.05).2 Effects of endostar on the routine lives of nude mice The activity and foodintake of nude mice in DDP group reduced and the weight got lost, which maintained to the end of the study. But the weight of nude mice in NS group, endostar group,endostar(10mg/kg)with DDP group and endosta(r20mg/kg)with DDP group had been on the increase until the end of the study.3 Effects of endostar on lymph node metastasisMetastatic lymph node could be seen near the tumor when dissect nude mice. Metastasis were confirmed by HE staining. The mean rates of lymphatic metastasis on endostar(20mg/kg)with DDP group, endostar(10mg/kg)with DDP group, endostar group,DDP group and NS group are 0%(0/8), 12.5%(1/8), 12.5%(1/8),62.5%(5/8), 75%(6/8) respectively. Statistic analysis showed that the lymphatic metastasis rate of endostar group , endostar(10mg/kg)with DDP group and endostar(20mg/kg)with DDP group reduced remarkably than that of DDP group and NS group.4 Effects of endostar on microvessel density(MVD) CD34 was employed as the maker of vascular endothelial cell and its cytoplasm was dyed yellow-brown.The mean values of MVD on endostar(20mg/kg)with DDP group, endostar(10mg/kg)with DDP group, endostar group,DDP group and NS group are 10.88±1.38,10.25±1.22,10.83±2.29,15.58±2.31,22.08±1.93 respectively. The difference of microvessel density of five groups was significant(P<0.05). The inhibition of tumor vessels was more obvious in endostar group,endostar(10mg/kg)with DDP group and endostar(20mg/kg)with DDP group.5 Effects of endostar on micro lymphvessels densityLYVE-1 was employed as the maker of lymphatic vessel endothelial cell and its cytoplasm was dyed yellow-brown. The mean values of MVD on endostar(20mg/kg)with DDP group, endostar(10mg/kg)with DDP group, endostar group,DDP group and NS group are 5.00±0.63,5.17±0.75,6.00±0.63,14.33±1.63,13.67±1.21 respectively. The difference of microvessel density of five groups was significant(P<0.05). The inhibition of tumor vessels was more obvious in endostar group,endostar(10mg/kg)with DDP group and endostar(20mg/kg)with DDP group6 Effects of endostar on expression of VEGF-A, C, D VEGF-A, VEGF-C and VEGF-D proteins expressed in the cytoplasm of cancerous cells and the immunoreactive protein was yellow-brown deposits. The mean optical density value of positive staining cells was determined by Motic 6.0 digital medical analysis system. As shown by statistic analysis results, endostar group,endostar(10mg/kg)with DDP group and endostar ( 20mg/kg ) with DDP group decreased significantly compared with that of DDP group and NS group.7 The result of TEMApoptosis cells could be found in endostar group,endosta(r10mg/kg)with DDP group and endosta(r20mg/kg)with DDP group. Necrosis cells appeared in NS group and DDP group, but apoptosis cells were not seen.Conclusions1 Both The inhibitory effect of endostar and the effect of endostar with DDP on tumor growth and matastasis are better than that of DDP.2 Endostar can effectively inhibit the lymphangiogenesis and angiogenesis of subcutaneous implantation tumors of nude mice.3 Endostar can inhibits lymphangiogenesis and angiogenesis by a down-regulation of the VEGF-A, C, D expression in tumor cells.4 Endostar can reduce the secondary effect of traditional chemotherapeutics and already hoped become the effective therapeutics of cancer treatment.