Experimental Study On Anti-tumor Effect Of Neovascular Based On Cyclophosphamide Regulating Treg

Objective:To observe the curative effect of bevacizumab combined with cyclicaladministration of low dose cyclophosphamide （CTX） on hepatocellular carcinoma inmice and investigate the relevant mechanism.Methods:1、After seven days,when the tumor-burdened model in C7BL/6J mice wasestablished, injecting25mg/kg and200mg/kg CTX in the abdominal cavity ofmice, using flow cytometer to detect the proportion of CD4⁺CD25⁺FOXP3⁺/Treg in mouse spleen tissue, filter out the optimal dose of CTX;2、Comparing the influence of Treg in mice spleen tissue after using Single andcycle CTX: when the mice have tumor, after7d,11d,14d,18d,21d,25d,28d,selcet6mice in each time point （n=6）, using cervical dislocation in the mice, thenremove spleen tissue, detecting the proportion of CD4⁺CD25⁺FOXP3⁺/Treg;3、 After intervent angiogenesis and cycle using small doses ofcyclophosphamide, Observing the antineoplastic effect of subcutaneoushepatocellular carcinoma in mice: establish subcutaneous liver cancer model ofC57BL/6J mice, when the tumor grew to the size of100-150mm2spare, divide the32mice into four groups randomly, the first group is the control group, injecting thePBS in the abdominal cavity, the second group is single CTX group, on Day0,injecting CTX （100mg/kg, once a week）; the third group is single Bevacizumab （BV）group, injecting BV （5mg/kg, twice a week） on Day0and CTX （100mg/kg, once aweek）;the forth group is the combined group in the same time, injecting the samedose BV （5mg/kg, twice a week） on Day0and CTX （100mg/kg, once a week）.when the treatment are over, using flow cytometry to detect the CD4⁺CD25⁺andTreg in spleen tissue, analyze the influence of the CD4⁺CD25⁺and Treg afterinterventing angiogenesis and Cycle using small doses of cyclophosphamide. andobserve the growth of mouse subcutaneous tumor、the survival of mice, then drawing tumor growth curve.Results:1. When extend of the tumor-bearing time, CD4⁺CD25⁺FOXP3⁺Tregproportion of mice spleen tissue gradually, the14th day is significantly higher thecontrol group （P <0.05）.2. The dose of CTX was100mg/kg,the CD4⁺CD25⁺FOXP3⁺/Tregproportion of tumor-bearing mouse was reduced significantly （P <0.05）.3. When Single applicatiing CTX,it can suppress Treg during a short time, butcycle using CTX can extend the inhibition of Treg （P <0.05）, in that time, the Tregcan keep lower level （P <0.05）.4. Tumor growth was significantly suppressed in CTX group, CTX+BV groupand CTX⁺BV group, the tumor growth of CTX+BV group was significantlyslower than the above group (P <0⁺improvement,compared with the BV group,the survival period of CTX+BV group was also significantly increased, thisindicated that the combination of BV and beat applications CTX can enhance thetherapeutic effect of BV. CTX+BV joint treatment group has two mouse tumordecreased gradually and subsided, the mice has long-term survival, and no tumorrecurrence.5. Applicating Bevacizumab monotherapy or jointing the CTX group,the Treglevels of mouse spleen were7.17±2.18,5.07±1.47, this is significantly lowerthan the control group, CTX single group（P <0.05）. Concurrent chemotherapy groupis higher than single BV group, the difference was statistically significant （P <0.05）.Conclusion:1. Treg ratio of tumor-bearing mice spleen tissue was gradually increased withthe progress of tumor growth.2. The optimal dose of CTX deleting Treg is100mg/kg for C57BL/6Jtumor-bearing mice,3. When single applicating CTX,it can suppress the Treg in a short time. Butusing optimal dose after interval7days, the beat application CTX can maintain Tregat a relatively low level in a longer period of time, this shows using beat application CTX has more value.4. Bevacizumab monotherapy treatment or combined with metronomiclow-dose CTX treatment can significantly inhibit Treg generating in the tumormicroenvironment, the effectiveness of combined chemotherapy is higher than thesimple BV.5. Metronomic low dose cycle applications CTX can inhibit Treg infiltration inthe spleen and reversal the immune suppression micro-environment. On the basis ofinhibiting Treg, joining neovascularization intervention BV treatment can improvethe treatment of liver cancer in mice.