| Objective:To explore the alteration of CD4+CD25+regulatory T cells (Treg) and Myeloid Derived Suppressor Cells cells (MDSCs) and the effect of anti-tumor treated with low-dose cyclophosphamide (CTX) and gemcitabine (GEM) in tumor-bearing mice.Methods:Melanoma-bearing mice model was established by inoculating s.c. mice with melanoma B16cells, then randomly divided into NS group, CTX group, GEM group, CTX+GEM group.And setting up the normal group, a total of five groups.The model mice was injected i.p. with CTX (20mg/kg) or GEM (50mg/kg) at3,10,17d after injection of tumor cells. At the4th day after last drug administration,The proportion of Treg/CD4+T and MDSCs in spleen were detected by flow cytometry; Proliferation of T and B cells in spleen were detected by CCK-8method; ELISA method to detect the level of IL-10, TGF β1, IFN-γ in serum and spleen cell culture supernatant,and biochemical method to detect the level of NOS in serum and spleen cell culture supernatant;keep a record of the tumour formation time, tumor size, and draw the tumor growth curve.Result:1The percentage of Treg/CD4+T cells in the spleen of each treatment group was significantly decreased compared with NS control group (P<0.05), but higher than normal control group (P<0.05). Although the ratio in GEM+CTX group was closer to GEM group(P>0.05), but lower than GEM group (P<0.05).2The proportion of MDSC in spleens of each treatment group was significantly decreased compared with NS control group (P<0.05), but higher than the normal control group (P<0.05). The ratio of GEM+CTX group was closer to GEM group(P>0.05), but lower than CTX group (P<0.05).3The stimulation index (SI) of T cells and B cells in spleen of each treatment group were significantly higher than NS group (P<0.05); there were no statistically difference among GEM+CTX group,CTX group and normal control group in T cells SI (P>0.05), but slightly higher than GEM group (P<0.05); there were no statistically difference among GEM+CTX group,GEM group and normal control group in B cells SI (P>0.05), but higher than CTX group (P<0.05). 4The killing activity of NK cells in spleen of each treatment group were significantly higher than NS group (P<0.05), but lower than normal control group (P<0.05).The killing activity of GEM+CTX group was close to GEM group(P>0.05),but higher than CTX group(P<0.05).5The level of IL-10, TGF-β1, NOS in serum and spleen cell culture supernatant of each treatment group were lower than NS control group (P<0.05), the level of IFN-y were higher than NS control group (P<0.05). IL-10and TGF-β1in serum and supernatant of GEM+CTX group was lower than other treatment groups (P<0.05). The level of IFN-y of GEM+CTX group was higher than other treatment groups (P<0.05).The level of NOS in serum and supernatant of CTX+GEM group was close to GEM group(P>0.05), but lower than CTX group (P<0.05).6The tumor formation time of each treatment group was delayed compared with NS control group, and the volume of tumor was significantly smaller at the same time poin t(P<0.05), tumor growth in CTX+GEM group was most slowest.Conclusion:1The proportion of MDSC and Treg in spleen of tumor-bearing mice are significantly increased compared with normal mice.2Low dose of CTX and GEM used alone can down-regulate MDSC and Treg in spleen of tumor-bearing mice, and improve the anti-tumor function and delay the growth of melanoma by reducing the secretion of IL-10, TGF-β1and NOS, improving T and B cell proliferation activity and killing activity of NK cells, enhancing the production of IFN-y. But the down regulation of GEM to MDSC,CTX to Treg is more obvious.3Low dose of CTX and GEM used combined show stronger down-regulate MDSC and Treg, reduce the secretion of IL-10, TGF-β1and NOS, improve T and B cell proliferation activity and killing activity of NK cells, enhance the production of IFN-γ,and show stronger antitumor effect in vivo. |
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