亚低温延缓脑创伤后神经元迟发性凋亡进程的研究

Postponed Effect of Mild Hypothermia on Neuron Apoptosis Following Traumatic Brain Injury

目的:观察亚低温对脑创伤后神经元程序性细胞死亡进程的延缓作用,从细胞水平测试亚低温治疗的意义。方法:分离培养乳鼠脑皮质神经元,免疫荧光方法鉴定;部分神经元反复冻融,将细胞裂解液加入正常神经元细胞表面,分别置于33℃和37℃温控培养箱中培养;测定培养上清液中乳酸脱氢酶(LDH)含量,TUNEL法检测原位凋亡细胞数量,以hoechst33258标记法检测亚低温对凋亡速度的影响。结果:免疫荧光检测神经元特异标记蛋白神经元特异性烯醇化酶(NSE)和微管相关蛋白(MAP-2)阳性表达率分别为(74.7±8.4)%和(80.1±6.7)%。培养于33℃的LDH水平和发生原位凋亡的细胞数量均低于37℃培养组(P<0.05),hoechst33258标记法显示亚低温可延缓神经元调亡速度(P<0.05)。结论:亚低温可明显减缓脑创伤后神经元调亡进程,为其他治疗争取救治时间。

Objective： To observe the postponed effect of mild hypothermia on neuron apoptosis process after traumatic brain injury,and study the positive significance at cellular level. Methods： The neurons were isolated from the suckling rat brain cortex, and identified by immunofluorescence assay. Part of neurons was frozen and thawed repeatedly, and the lysate was added onto the normal neurons. Then the neurons were cultured in temperature-controlled incubator at 33 ℃ or 37 ℃ respectively. The lactic acid dehydrogenase （LDH） level was tested at different time point. The apoptosis cell population was detected by TUNEL,and cellular nucleus was labeled by hoechst 33258 to observe neuron apoptosis speed. Results： The results from immunofluorescence indicated that cultured cells were positively expressed neuron specific enolase[NSE（74.7 ± 8.4）%] and microtubule-associated protein-2[MAP-2 （80.1 ± 6.7）%]. At the same time point,the LDH level and apoptosis cell population cultured at 33 ℃ were lower significantly than those of cultured at 37 ℃ ,and the cell apoptosis speed at 33 ℃ was slower than that of 37 ℃ （P 〈 0.05）. Conclusion： Mild hypothermia can markedly postpone the neuron apoptosis process after traumatic brain injury ,which reduces the degree of injury, and prolongs the time of remedy window.