Notexin对小鼠体内CD8+ T淋巴细胞活性的干扰作用
马平, 术蓉, 刘幸卉, 史丹丹, 曾慧君, 曹标, 廖华
中国临床解剖学杂志 ›› 2014, Vol. 32 ›› Issue (3) : 296-299.
Notexin对小鼠体内CD8+ T淋巴细胞活性的干扰作用
Notexin effects on the survival and proliferation of CD8+ T cell in the injuried-muscle draining lymph nodes
目的 探讨肌肉注射Notexin是否影响C57BL/6小鼠骨骼肌引流淋巴结内的外源性CD8+ T细胞(OT-I细胞)的活性和增殖。 方法 分别采用Notexin注射或机械挤压法制备B6小鼠的胫骨前肌(TA)急性肌损伤模型,并获得性转移(adoptive transfer, i.v.)OVA特异的OT-I细胞(CFSE标记),随后肌注可溶性OVA蛋白。收集损伤侧TA引流淋巴结 (腘、腹股沟淋巴结),流式分析OT-I细胞的增殖程度。OVA静脉内注射免疫B6鼠,收集激活的脾脏树突状细胞(cDCs),与CFSE标记的OT-I细胞体外联合培养,并添加不同稀释度的Notexin, 流式检测OT-I细胞的活性和增殖。 结果 CFSE的荧光递减结果证实,机械挤压损伤鼠TA引流淋巴结内OT-I细胞迅速活化增殖,但Notexin诱发的肌损伤小鼠引流淋巴结内OT-I细胞在4 d时无增殖反应,7 d的增殖率与非注射组无显著差异。与活化的cDCs细胞共培养的OT-I细胞活性良好,增殖显著,但即使添加高度稀释(1:1000)的Notexin也会严重干扰OT-I细胞的活性和增殖。 结论 肌内注射Notexin注射将干扰CD8+ T细胞的活性,这表明蛇毒血清诱导的骨骼肌损伤模型不适用于肌损伤诱发的T细胞功能改变的相关研究。
Objective To explore the effects of intramuscular injection of Notexin on transferred OT-I cells in muscle draining lymph nodes of C57BL/6 mice. Methods Tibialis anterior(TA)injury were performed by intramuscular injection of Notexin, or crush skill on B6 mice, separately. CFSE-labeled OT-I cells were adoptively transferred to myoinjury mice, and followed by OVA i.m. injection. TA draining lymph nodes were received, and lymphocytes were collected and analyzed the proliferation of OT-I cells by CFSE fluorescence intensity evaluation using flow cytometry. Activated spleen cDC cells were collected and purified from one B6 mouse received OVA i.v. injection. Purified cDC cells were co-cultured with CFSE-labeled OT-I cells. Notexin with different concentration were added into co-culturing system. After 72h, the survival and proliferation of OT-I cells were detected. Results According to the detection results of CFSE fluorescence intensity, OT-I cells in crush induced-damaged muscle draining lymph nodes were activated and proliferated rapidly under the stimulation of OVA antigen. For Notexin injection-induced muscle damage, no cell proliferation of OT-I cells were detected in TA draining lymph nodes at the day 4, but the proliferation of OT-I cells recovered at the day 7. Co-culturing with activated cDCs cells induced a obvious proliferation of OT-I cells in vitro, however, OT-I cells lost the proliferation ability if the co-culturing system were added with Noteixn, even if the concentration of Notexin was very lower(1:1000). Conclusions The activity and proliferation of CD8+ T cells will be interfered by intramuscular injection of Notxin. Therefore, we suggest that Notexin injection is unsuitable for investigating T cell response associated with myoinjury.
[1] Harris JB, Johnson MA, Karlsson E. Proceedings: Histological and histochemical aspects of the effect of notexin on rat skeletal muscle
[J]. Br J Pharmacol, 1974, 52(1):152P
[2] Gutiérrez JM, Cerdas L. Mechanism of action of myotoxins isolated from snake venoms
[J]. Rev Biol Trop, 1984, 32(2):213-222.
[3] Mendler L, Zádor E, Dux L, et al. mRNA levels of myogenic regulatory factors in rat slow and fast muscles regenerating from notexin-induced necrosis
[J].Neuromuscul Disord, 1998, 8(8):533-541.
[4] Miyagawa F, Gutermuth J, Zhang H, et al. The use of mouse models to better understand mechanisms of autoimmunity and tolerance
[J]. J Autoimmun, 2010, 35(3):192-198.
[5] Brigitte M, Schilte C, Plonquet A, et al. Muscle resident macrophages control the immune cell reaction in a mouse model of notexin-induced myoinjury
[J].Arthritis Rheum, 2010, 62(1):268-279.
[6] McGeachie JK, Grounds MD. Initiation and duration of muscle precursor replication after mild and severe injury to skeletal muscle of mice: an autoradiographic study
[J]. Cell Tissue Res,1987, 248(1):125-130.
[7] Calbo S, Delagreverie H, Arnoult C, et al. Functional tolerance of CD8+ T cells induced by muscle-specific antigen expression
[J]. J Immunol, 2008, 181(1):408-417.
[8]Clarke SR, Barnden M, Kurts C, et al. Characterization of the ovalbumin-specific TCR transgenic line OT-: MHC elements for positive andnegative selection
[J]. Immunol Cell Biol, 2000, 78(2):110-117.
[9] Liao H, Franck E, Fréret M, et al. Myoinjury transiently activates muscle antigen-specific CD8+ T cells in lymph nodes in a mouse model
[J]. Arthritis Rheum, 2012, 64(10):3441-3451.
国家自然科学基金(81171724,81371924);广东省科技攻关项目(2012B031800146)
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