颗粒酶-B在大鼠心肺复苏后脑组织中的表达及其意义

The expression and significance of granzyme-B after cardiac arrest and resuscitation in rats

目的观察大鼠心搏骤停（cardiacarrest，CA）、心肺复苏（cardiopulmonary resuscitation，CPR）后早期，脑中颗粒酶-B（granzymeB，Gra-B）、穿孔素表达及CD8±T细胞含量的变化，探讨其在CPR后脑损伤中的意义。方法雄性Wistar大鼠按随机数字表法分为两组：复苏组和假手术组，复苏组又按取材时间不同分为12h组、1d组、3d组3个亚组。假手术组大鼠仅进行麻醉和气管插管、血管穿刺，不进行窒息及CPR，于术后1d处死取样。以夹闭气管导管，窒息6min的方法成功建立大鼠CPR模型后，用平衡木行走实验及肌力测定实验检测大鼠神经功能的变化。通过检测脑组织中伊文蓝（evansblue，EB）的含量说明血脑屏障（blood-brain barrier，BBB）的破坏程度，应用免疫组化染色，Western blot以及免疫荧光染色技术检测脑组织内CD8±T细胞浸润、Gra-B和穿孔素表达的变化以及CDnT细胞和Gra-B的共表达情况。结果与假手术组比较，复苏组大鼠CPR后的感觉运动功能和肌力明显减退，其评分在第3天达到最低[复苏组（4．0±0．7）分，假手术组（0．9±0．3）分（P〈0．01）]，第4、5天开始恢复。CPR后大鼠BBB通透性增大，脑皮质和纹状体中CD8±T细胞的浸润以及Gra-B的表达增多，在1dGra-B的表达增加最明显[皮质中1d组（O．95±0．08）比假手术组（O．35±0．09）（P〈0．05）；纹状体中1d组（1．08±0．15）比假手术组（0．39±0．09）（P〈0．05）]。CD8±T细胞与Gra-B在脑中共表达。结论CA-CPR后，大鼠脑中BBB通透性增加，CD8±T细胞浸润并释放Gra-B及穿孔素，参与脑神经损伤。

Objective To observe the expression of granzyme B （Gra-B）, perforin and CD8＋ T cells in rat brain and to investigate how they take part in the neuronal death after cardiac arrest （CA） and cardiopulmonary resuscitation （CPR）. Methods Male Wistar rats were randomly divided into sham-operation group and resuscitation group. Cardiac arrest was induced by clamping the tracheal tube for 6 min. The Beam-Walking Test and the hanging wire were used to detect CA-CPR induced neurologic and motor deficits. Blood-brain barrier （BBB） permeability was detected by evans blue （EB） content in brain tissue. The expressions of CD8＋T cells, Gra -B and perforin were determined with immunohistochemistry, Western blot and immunofluorescent technique. Results Compared with the sham-operated group, the sensorimotor function and muscle strength of CPR rat was significantly decreased, and the difference reached to the top level at 3 d （4.0±0.7） vs （0.9±0.3）（P〈0.01）, and then began to recover at 4 d and 5 d. The BBB permeability was significantly increased, CDSCF ceils infiltrate the cortex and corpora striatum, as well as a significant increase in Gra-B and perforin levels within 12 h of resuscitation（P〈0.01 ）. These changes reached to the peak value at 1 d[ （compared with the sham-operated group, Gra-B（0.95±0.08） vs （0.35±0.09） and perforin（1.08±0.15） vs （0.39±0.09） in cortex. Gra-B was located in CD8＋ T cells in rat brain after CPR. Conclusions The increased BBB permeability, CD8＋T cells infiltration and the release of Gra- B and perforin may be involved in mediating neuronal death after CA-CPR.