针灸及其血清对荷瘤小鼠CD4~+CD25~+Treg细胞体外增殖的影响

目的:观察针灸治疗对H_(22)荷瘤小鼠CD4~+CD25~+Treg细胞(CD4~+CD25~+regulatory T cells)体外增殖的影响和针灸血清刺激下的CD4~+CD25~+Treg细胞的体外增殖变化,探讨针灸及其血清对荷瘤小鼠免疫调节细胞的干预作用。方法:48只小鼠随机分为电针治疗组、艾灸治疗组、肿瘤对照组、正常对照组。采用H_(22)肿瘤细胞移植性实体瘤模型,电针和艾灸"大椎"治疗后,磁珠分离CD4~+CD25~+Treg细胞,氚标记胸腺嘧啶核苷掺入法观察不同组小鼠CD4~+CD25~+Treg细胞体外增殖能力和针灸血清对CD4~+CD25~+ Treg细胞体外增殖的影响。结果:肿瘤对照组CD4~+CD25~+Treg细胞增殖水平比正常对照组明显增高(P<0.05);电针治疗组和艾灸治疗组CD4~+CD25~+Treg细胞增殖水平与肿瘤对照组比较均明显降低(P<0.01)。电针治疗组和艾灸治疗组1:1、1:8稀释度血清刺激正常小鼠CD4~+CD25~+Treg细胞体外增殖,较正常对照、肿瘤对照组血清显著增高(P<0.05),各组1:16、1:32稀释度血清对Treg细胞作用的差别无统计学意义(P>0.05)。结论:针灸治疗能下调荷瘤小鼠CD4~+CD25~+Treg细胞体外增殖能力。不同浓度针灸血清体外刺激CD4~+CD25~+Treg细胞增殖表现出不一致的效应。

Influence of Electroacupuncture and Moxibustion and Their Treated Mouse Serum on the Proliferation of the Cultured Splenetic CD4~+CD25~+ Regulatory T Cells of Tumor-bearing Mice

Objective To observe the effect of electroacupuncture (EA) and moxibustion of "Dazhui" (G,V 14) on the proliferation levels of the splenetic CD4~+ CD25~+ regulatory T cells (Tregs) of H_(22) tumor-bearing mice in vitro. Methods Forty eight Balb/c mice were randomized into control, model, moxibustion and EA groups, with 12 cases in each. H_(22) tumor-bearing model was set up by hypodermic injection of H_(22) tumor cells (0. 2 ml, 1×10~7 cells/ml). EA (2 Hz, 2 mA) was applied to "Dazhui" (GV 14) and left "Huantiao" (GB 30) for 20 min, and moxibustion was applied to "Dazhui" (GV 14) 2 moxa-cones every time. The treatment was given from the 2~(nd) day on after innoculation of tumor cells, once every other day, 6 times altogether. After the treatment, the mice were killed by peeling off the eyeball and blood samples were collected to be separated into serum. Then, Tregs of the spleen tissues of Balb/c mice in different groups were isolated by using megnetic activated cell sorting (MACS) sys-tem to be cultured independently, and co-cultured with EA-treated serum and moxibustion-treated serum separately in culture fluid for 96 h, added with 3H-tritiate thymidine (TdR) in the culture-fluid 12 h before the end of culture, followed by collecting the cells and detecting their proliferation levels (count per minute, cpm) by using a lipid scintillation device. Results The proliferation level of Tregs in model group was elevated significantly compared to normal control group (P<0. 05), while in comparison with model group, those of Tregs of EA and moxibustion groups decreased considerably (P<0. 01). After separate application of the diluted acupuncture-treated serum and moxibustion-treated serum at 1:1 and 1:8 (not 1:16 and 1 :32) to the cultured Tregs, their proliferation levels (cpm) in EA and moxibustion groups were obviously upregulated in comparison to those of normal control group (P<0. 05), and the cpm in EA group was significantly higher than that in model group (P<0. 05), suggesting a different action mechanism between acupuncture-moxibustion treatment and serum stimulation. Conclusion EA of "Dazhui" (GV 14) and "Huantiao" (GB 30) and moxibustion of "Dazhui" (GV 14) can effectively downregulate the proliferation level of the cultured sple-netic Tregs of the tumor bearing mice. EA-treated serum and moxibustion-treated serum diluted at 1 : 1 and 1 : 8 can evidently up-regulate the proliferation level of Tregs in vitro.