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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 investigate whether intramuscular injection of Notexin affects the activity and proliferation of exogenous CD8 + T cells (OT-I cells) in skeletal muscle draining lymph nodes of C57BL / 6 mice. Methods Acute myoblasts of the anterior tibial muscle (TA) of B6 mice were prepared by injection of Notexin or mechanical extrusion, and adoptive transfer (iv) OVA-specific OT-I cells (CFSE labeling) Note soluble OVA protein. Tumor-draining lymph nodes (腘, inguinal lymph nodes) were collected and the proliferation of OT-I cells was analyzed by flow cytometry. OVA was used to immunize B6 mice intravenously. Activating spleen dendritic cells (cDCs) were collected and cultured with CFSE-labeled OT-I cells in vitro. Notexin was added at different dilutions to detect the activity of OT-I cells proliferation. Results Fluorescence reduction results of CFSE confirmed that OT-I cells in TA draining lymph nodes rapidly proliferated in mechanical crush injury, but OT-I cells in draining lymph nodes induced by Notexin did not proliferate at 4 days. There was no significant difference between the proliferation rate and non-injection group. OT-I cells co-cultured with activated cDCs cells showed good proliferation and proliferation, but even adding highly diluted (1: 1000) Notexin could seriously interfere with OT-I cell activity and proliferation. Conclusion Intramuscular injection of Notexin will interfere with the activity of CD8 + T cells, indicating that the model of skeletal muscle injury induced by snake venom serum is not suitable for the study of the changes of T cell function induced by muscle injury.