减压性损伤大鼠脑脊液对离体小胶质细胞肿瘤坏死因子-α的影响

Effects of cerebrospinal fluid from rats suffered by ultra-fast decompression on microglia TNF-αin vitro

目的观察气压性中枢神经损伤动物脑脊液(cerebrospinal fluid,CSF)对离体小胶质细胞的刺激作用,探讨小胶质细胞激活参与气压性中枢神经损伤的机制。方法极快速减压制备气压性中枢神经损伤大鼠模型,收集致伤后或高压氧(hyperbaric oxygen,HBO)处理后的动物CSF,分别以正常动物CSF、致伤动物CSF、HBO动物CSF和/或基质金属蛋白酶(matrix metalloproteinase,MMPs)阻断剂GM6001为条件介质刺激新生的SD大鼠大脑皮层原代培养小胶质细胞。用流式细胞分析术检测肿瘤坏死因子-α(mTNF-α)、肿瘤坏死因子-α转换酶(TNF-αconverting enzyme,TACE)含量的变化,L929细胞毒性实验测定上清内可溶性TNF-α(sTNF-α)的生物活性。结果致伤组和HBO组sTNF-α均较对照组显著增加(P<0.01,P<0.05),HBO组比致伤组显著减少(P<0.01),增加GM6001处理后的结果与对照组间的差异均无统计学意义(P>0.05);致伤组小胶质细胞表面的mTNF-α蛋白比对照组增加了约2.35倍,HBO组与致伤组无明显差异,GM6001显著增加致伤组的mTNF-α。但对HBO组的作用不明显;致伤组小胶质细胞表面的TACE蛋白总量比对照组增加2.98倍。HBO组比致伤组降低1.97倍。结论极快速减压损伤动物CSF中存在大量刺激小胶质细胞反应的活性物质,刺激小胶质细胞TNF-α和TACE的合成,增加sTNF-α的分泌,可起到继发损伤作用。HBO治疗可以减少极快速减压致伤动物CSF中小胶质细胞刺激物的含量,起到神经元保护作用。TACE在小胶质细胞分泌TNF-α中起重要作用。GM6001可以通过减少小胶质细胞TNF-α的分泌,在改善小胶质细胞激活诱导的继发性损伤中与HBO起协同作用。

Objective To observe the irritant actions of cerebrospinal fluid （CSF） from rats suffered by ultra-fast decompression on microglia in vitro to make a scientific approach to the mechanism of microglial cell activating the central nervous system （CNS） injury induced by ultra-fast decompression. Methods Models of CNS injured rats were established by ultra-fast decompression. Collecting CSF by vulnerate or hyperbaric oxygen （HBO）, primary-cultured microglial cells in cerebral cortex of neonatal SD rats were stimulated under conditions： CSF from normal animal, CSF from injured animal and CSF from HBO and/or blocking agent of matrix metalloproteinase （MMPs） GM6001 treatments. Flow cytometry （FCM） were used to determine changes of content in tumor necrosis factor-alpha （mTNF-ct） and TNF-α convertase （TACE）. Biological activities of solubility TNF-α （ sTNF-α ） were detected by L929 cell cytotoxicity bioassay. Results Compared with the control group, biologic activities of sTNF-α injured group and HBO group were significantly higher （ P 〈 0. 01, P 〈 0.05） and there were no significant differences after GM6001 disposal. At the same time,biologic activities of sTNF-α were lower than those of HBO group （ P 〈 0.01 ）. Contents of mTNF-α in injured group were increased 2.35 times than those in control group,while there were no differences between HBO group and injured group. GM6001 increased mTNF-α of microglia disposed by injured CSF,but not by HBO CSF. Contents of TACE on microglia membrane in injured group were increased 2.98 times than those in control group, and 1.97 times than those in HBO group. Conclusions Ultra-fast decompression may irritate amount of microglia-activated materials in CSF,stimulate synthesis of TNF-α and TACE,increase secretion of sTNF-α and induce secondary damage to CNS tissues. HBO treatment can decrease the content of microglia-activated materials in CSF induced by ultra-fast decompressing,which may be contributed to its protecting effects. TACE play a key role in TNF-α secretion of microglia. GM6001 may cooperate with HBO in alleviating secondary damage induced by activatedmicroglia in a way of decreasing microglia＇s secretion of TNF-α.