| BackgroundAs a new antineoplastic therapy, the anti-angiogenesis combined conventional drugs can improve the cure effect.Endostar is a new type soluble rhEndostatin,and it can restrain selectly the microvascular endothelial’s proliferation, metaptosis, conglutination and survival.It has reached certain curative effect combined chemotherapeutics in NSCLC.Some studies have indicate that the anti angiogenic drugs can make capillaries normalization transiently.This test is to observe the morphological changes of tumor micro-vascular of ovarian cancer tumor-burdended mice used endostar in different time through the dynamic viewing,to find the tumor micro vascular of normalized time window.On the basis of the time window,we design the different combination scheme with cisplatin,then to search the best dosage schedule. This study had two parts:1.To find the time window of normalization of the tumor vasculature.2. To observe the efficacy of endostar and cisplatin different combination therapyPART 1 To find the time window of normalization of the tumor vasculature. methodsObjective By observing the tumor microvessel structure and tumor tissue hypoxia dynamic change of tumor-bearing mice after Endostar treatment to establish the time window of normalization of tumor microvascularMethods 1.The establish of transplant tumor model2. Experimental groups and drug interventionDivided fifty subcutaneous transplanted tumor mice into ten groups randomly, there were five mices in each group, five groups used endostar 20mg/kg subcutaneous injection. Everyone was injected 0.2ml.Put the mice to death one group on second、forth、sixth、tenth day respectively. The other five groups were as control groups,and used saline 0.2 ml, then made a group to death at the same time.Tumor tissue were taken,paraffin embedding,immunohistochemical study going to do.3. Markers and methods of detection3.1 The test ofα-SMA and Collagen IV:Immunohistochemical staining, count the microvessel of positive expression3.2 The detection of tumor hypoxia status:Immunohistochemical staining, Observe the expression and distribution of hypoxic cells, using semi-quantitative integral method to determine the expression intensity.Results1.Detection of a-SMA and Collagen IV:microvessels expressed a-SMA and Collagen IV mainly located in the peri-stroma of tumor tissue.α-SMA expression of the 4th day and the 6th day treated by Endostar is 15.3±5.2/mm2 VS 4.3±2.1/mm2 (t=4.3860,P=0.0023) 16.4±4.6/mm2 VS 6.6±2.4/mm2 (t=4.2235,P=0.0029) respectively,which is significantly higher than control group.α-SMA expression deacreased in the 8th day and the 10th day,which showed no difference when compared to control group.The expression of Collagen IV in microvessel is generally conformity to a-SMA. The expression of Collagen IV in 4th day and 6th day treated by Endostar can reach to the peak. significant difference is existed when compared to the control group,they are 14.7±4.3/mm2 VS 6.7±5.1/mm2 (t=2.6816,P=0.0279); 18.4±5.5/mm2 VS 7.1±1.7/mm2 (t=4.3892,P=0.0023) respectively.After that, Collagen IV expression deacreased quickly, showed no difference to control group2.Detection of tumor hypoxia:tumor hypoxia cell accumulate around the periphery of tumor necrosis tissue. HIF-1αis expressed in tumor cell,which is highly expressed in tumor hypoxia tissue. Tumor hypoxia cell is highly expressed HIF-1 at the 2th day treated by Endostar.the 4th day and the 6th day hypoxia is improved, HIF-1αis lowly expressed. And the 8th day and the 10th day,expression of HIF-1 is gradually reaching higher,shows middle-intensity expression.ConclusionThere is a definite time window when treated with Endostar. The microvessels tend to mature and t he hypoxic state of tumor is partly improved in the 4th to 6thPART2. To observe the efficacy of endostar and cisplatin different combination therapyObjective According to the time window of normalization of the tumor microvascular well established in the experimental one we designed different therapy regimen of Endostar and cisplatin combination, and observed treatment effect in order to find the best combination regimenMethods1.The establish of transplant tumor model2. Experimental groups and administrationDivided the thirty nude mice formed the transplant tumor subcutaneously into six groups randomly, five mice each group.(1)Endostar group:subcutaneous injection of endostar daily,dose of 20 mg/kg, treatment for ten days.(2)DDP group: abdominal cavity injection of DDP, dose of 3mg/kg, once daily, totally three times.(3)Endostar and DDP (d1-3):subcutaneous injection of endostar daily, dose as before, also for ten days,then abdominal cavity injection of DDP on the day from first to third day, dose as before.(4)Endostar and DDP(d4-6) group: subcutaneous injection of endostar daily,dose as before,and for ten days, then used DDP injected in abdominal cavity on the forth to sixth day, dose as before.(5)Endostar and DDP (d7-9) group:the usage of endostar was as before, DDP was used on the seventh to ninth day, the dose as before too.(6)Control group:injected some volume of normal saline with the same method, putted the mice to death on the fifteenth day and took the tumor tissue. 3.Detected marker and methods3.1 Rate of tumor suppression:Drown tumor growth curve and calculated the rate of tumor suppression.3.2 Micro vascular density(MVD)test use immunohistochemical method3.3 Apoptosis markers PCNA detection of tumor cell:immunohistochemical method(SP), used the half quantitative integral methods to judge the expression strength.Results1. Tumor growth curve:Two weeks after nude mouse inoculated tumor cells subcutaneously.,we can observe about 100 mm3 in volume subcutaneous tumor nodules. tumor formation rate is 100%. After grouping therapy,tumor is progressively enlarged in each group.Control group、Cisplatin(DDP) utilized group and Endostar therapied group、Endostar+ DDP (dl-3) group, tumor grew quickly.When the research is finished,the volume of tumor have no significant difference between these groups. At the Endostar+ DDP (d4-6) group and Endostar+ DDP (d7-9) group,tumor grouth is postponed, tumor inhibition rate of this two groups is significantly higher than the other groups When the research is finished(P<0.05).2. Detection of MVD after therapy in each group:MVD is the lowest in the Endostar+ DDP (d4-6) group after therapy, significant difference is existed when compared to the other groups (P<0.05)3. Detection of tumor cell proliferation marker PCNA:PCNA is lower expressed in the Endostar+ DDP (d4-6) group, middle-intensity expressed in the Endostar+ DDP (d7-9) group,and highly expressed in the rest groups.Conclusion:Different therapy regimen of Endostar and cisplatin combination may influence the therapeutics effects, When the maturity of tumor microvessels is most obvious induced by Endostar, we add DDP to the therapy,and then optimization tumor inhibition effect can be obtained. |
PDF Full Text Request