The Changes Of Myeloid Suppressor Cells And Immune Function In Tumor-bearing Mice And The Regulatory Effects Of Gemcitabine On Them

ObjectiveTo study the change of myeloid suppressor cells in blood,spleen,bone marrow and CD4~+,CD8~+ cells in blood with the growth of the tumor,and the effects of Gemcitabine on these cells and the tumor inhibitory effects in H22-bearing Kunming mice models,and to investigate whether MSCs are immune suppressor in tumor.MethodsThe H22 cell line was cultured in ascites of Kunming mice and collected cells were transplanted subcutaneously(s.c.)to the anterior right limb of every mouse by 5×10~6 per mice.After transplanted,the mice were separated into three groups:control,high dose of Gem(80mg/kg)and low dose of Gem(60mg/kg).Seven days later, Gemcitabine were injected abdominally once with the volume of 0.2mL.The control group was given naturism chloride with the same volume to Gemcitabine and tested the tumor volumes in definite time.At the 0,7th,14th,21st after tumor transplantation,3 mice of every group were put to death,then weight the tumor and calculate the tumor inhibitory rate,and we separate the PBMC and mononuclear cells from blood,spleen and bone marrow and test the proportion of CD11b~+/Gr-1~+ MSCs and CD4~+,CD8~+ lymphocytes by flow cytometry(FCM)used CD11b-PE/Gr-1-FITC and CD4 - FITC/ CD8-PE.The morphology of MSCs was observed by microscopy after immunocytolochemistry stained. ResultsAt the 7th,14th,21st day after the s.c transplantation of H22 tumor cells,the tumor volumes of control group were(63.68±17.37)mm~3,(6931.96±1648.16)mm~3 and (11856.80±4053.17)mm~3;that of Gemcitabine 60mg/kg were(64.80±15.20)mm~3, (4739.36±1589.73)mm~3 and(9520.85±2669.32)mm~3;in the group of Gem 80mg/kg, they were(65.56±18.58)mm~3,(4325.72+2267.73)mm~3 and(6323.15±2000.86)mm~3. The tumors in control group grew rapidly.Compared with control group,there were no statistical difference in the group of Gem 60,80mg/kg at the 7th day after s.c. transplantation of H22,but so were they at the 14th day(P values were 0.014 and 0.017).And at the 21st day,tumors in the group of Gemcitabine 80mg/kg were smaller statistically than those in control group(P values was 0.009).In PBMC of control group,the MSCs proportion at the 0,7th,14th,21st day after s.c.transplantation of H22 were(5.17±1.80)%,(9.87±0.71)%,(16.28±4.75)%and (13.05±1.74)%,which showed the larger tumor,the more MSCs in blood;those in groups of Gem 60mg/kg and Gem 80mg/kg were(9.18±1.46)%and(9.40±1.73)%, (8.86±0.78)%and(8.40±2.21)%at the 14th,21st day after s.c.transplantation of H22 respectively.P values were 0.069 and 0.062,0.056 and 0.046,which showed that there were less MSCs in Gem 80mg/kg than that of control group occurred(P＜0.05).In spleen of control group,the MSCs proportion at the 0,7th,14th,21st day after s.c.transplantation of H22 were(5.61±0.37)%,(8.68±0.73)%,(13.93±0.70)%and (14.12±5.09)%,which was the same result with blood;those in groups of Gem 60mg/kg and Gem 80mg/kg were(11.12±1.20)%and(12.55±0.73)%,(8.92±1.53)% and(8.37+0.52)%at the 14th,21st day after s.c.transplantation of H22 respectively.P values were 0.024 and 0.626,0.0068 and 0.123,which showed that there was difference only in the early phase in the MSCs proportion between control group and drug-given groups.In bone marrow,the MSCs proportion at the 0 day were(26.9±4.2)%,and at the 7th day after s.c.transplantation of H22,it was(22.96±1.85)%.The proportion of MSCs in control group were(29.4±3.19)%and(22.31±4.56)%;those in groups of Gem 60 mg/kg and Gem 80 mg/kg were(25.47±0.89)%and(20.49±1.99)%,(21.89±3.62)% and(11.97±2.20)%at the 14th,21th day after s.c.transplantation of H22 respectively. P values were 0.108 and 0.054,0.561 and 0.024,which showed that there was statistical difference in the group of Gem80mg/kg in bone marrow between control group and drug-given groups(P＜0.05).At the 0 day after s.c.transplantation of H22,the proportion of CD4~+,CD8~+ and CD4/CD8 are(19.67±3.26)%,(7.50±1.63)%and(2.64±0.14).At the 7th day after s.c. transplantation of H22,they are(4.36±0.39)%,(1.72±0.28)%and(2.57±0.39).There were no different at the 7th,14th day after Gem given in the ratio of CD4/CD8(P values were more large than 0.05),but at the 7th day after drug given,there were more CD4~+,CD8~+ cells in the group of Gem 80mg/kg than those in control group.At the 7th day after s.c.transplantation of H22,the mean quality of tumor was (0.50±0.23)g.At the 7th days after drug given,the tumor quality of control group was (4.09±1.27)g,that of Gem 60mg/kg group was(3.72±1.18)g and Gem80mg/kg group was(2.88±0.72)g.Whereas at the 14th day after drug given,the control group was (7.99±1.58)g,Gem 60mg/kg group was(5.71±0.77)g,Gem 80mg/kg was(5.00±0.21)g (P=0.031,P＜0.05,there is statistical different.).The inhibitory rate of Gem 80mg/kg was 29.58%at the 7th day after drug given,37.42%at the 14th day after drug given, which were larger than that of Gem 80mg/kg group.Contrast with control group,the mice survival in Gem 60mg/kg group was prolonged 8 days(P=0.0018,P＜0.05), whereas 4 days in Gem 60mg/kg group.Conclusions1.The percentages of MSCs in blood,spleen and bone marrow increased with the growth of the tumor.2.High dose of Gemcitabine can inhibit the growth of H22 in Kunming mice models and decreases the percentages of MSCs,but when we used low dose of Gemcitabine,the decreased MSCs went back to control level,which verified the relationship of MSCs with tumor burden.3.Although there were no significant difference in the ratios of CD4~+/CD8~+ cells between control and tested group,there were more CD4~+,CD8~+ cells in the group of Gem 80mg/kg than those in control group.We concluded that Gem might no effect on CD4~+,CD8~+ cells and the ratios of CD4~+/CD8~+ cells.