骨髓单个核细胞移植对脑梗死大鼠ERK1／2信号通路的影响

Effects of bone marrow mononuclear cells transplantation on ERK1/2 signal pathway after cerebral infarction in rats

目的研究骨髓单个核细胞（BMMNCs）移植对脑梗死损伤保护作用及ERKl／2信号通路是否参与BMMNCs的抗凋亡保护机制。方法选择健康清洁级雄性6～8周龄sD大鼠共384只，体质量250～280g；采用改良线栓法制作大脑中动脉栓塞模型（MCAO）。SD大鼠按随机数字表法分为：假手术组，模型组，BMMNCs组和ERK1／2抑制剂组，每组96只；模型组于造模成功后24h经尾静脉注入200μlPBS溶液，BMMNCs组注入200μl含5×10^6个BMMNCs的PBS溶液，ERK1／2抑制剂组在注入BMMNCs的同时经侧脑室注射5μlPD98059；分别于3d、7d和14d采用改良的神经功能损害评分法（mNSS）进行神经功能评分，3TC染色评价脑梗死体积，Westernblot技术检测pERK1／2、Bax、Bcl-2和caspase-3蛋白水平，免疫荧光法检测BMMNCs对小胶质细胞活化的影响。结果（1）各时间点模型组mNSS和脑梗死体积明显高于假手术组（P〈0．05），BMMNCs组低于模型组，高于假手术组，随治疗时间延长逐渐降低（P〈0．05），ERK1／2抑制剂组与模型组比较差异无统计学意义（P〉0．05）。（2）各时间点模型组pERK1／2、Bcl-2、Bax和caspase-3蛋白水平明显高于假手术组（P〈0．05）；与模型组[pERK1／2：（0．17±0．05），（0．14±0．04），（0．13±0．03），Bel-2：（0．23±0．11），（0．24±0．12），（0．27±0．14），Bax：（0．39±0．13），（0．40±0．14），（0．45±0．23），Caspase-3：（0．52±0．26），（0．56±0．27），（0．58±0．28）]比较，BMMNCs组pERKI／2和Bel-2蛋白水平[pERKl／2：（0．38±0．16），（0．39±0．15），（0．40±0．20），Bcl-2：（0．38±0．14），（0．39±0．15），（0．37±0．13）]明显升高，Bax和easpase-3蛋白水平[Bax：（0．25±0．13），（0．19±0．06），（0．21±0．08），caspase-3：（0．35±0．13），（0．34±0．16），（0．29±0．09）明显降低（P〈0．05），ERKl／2抑制剂组pERKl／2蛋白水平均低于其他各组，ERKl／2与模型组比较，Bcl-2、Bax和easpase-3蛋白水平差异无统计学意义（Jp〉0．05）。（3）BMMNCs组缺血半暗带中小胶质细胞（Ibal阳性）明显多于模型组，且随时间延长而增多（P〈0．05）；ERK1／2抑制剂组与模型组比较差异无统计学意义（P〉0．05）。结论BMMNCs可通过ERK1／2信号通路减轻脑梗死后细胞凋亡，从而改善神经功能，降低梗死范围。

Objective To explore the neuroproteetion and mechanisms of bone marrow mononuclear cells （BMMNCs）, and evaluate whether ERK1/2 signaling pathway was involved in it. Methods 384 healthy male SD rats,which were 6-8 week old,weighting 250-280 g,were selected. The middle cerebral ar- tely occlusion （MCAO） model was established in SD rats using the suture method. The rats were randomly divided into sham operation group ,model group, BMMNCs group and ERK1/2 inhibitor group ,with 96 rats in each group. At the time of 24 h after the successful modeling,200 μl PBS solution was injected into the cau- dal vein of the rats in the model group ,200 μl PBS solution containing 5μl×10^6 BMMNCs was injected into the rats in the BMMNCs group and the ERK1/2 inhibitor group.meanwhile,5 Ixl PD98059 was injected into the lateral ventricle of the brain of rats in the ERK1/2 inhibitor group. At the time points of 3 d,7 d and 14 d,the modified neurological severity scores （mNSS） was used to evaluate the neurological function,the volmne of cerebral infarction was assessed by TTC staining,the pERK1/2,Bax,Bcl-2 and caspase-3 levels were de- tected by Western blot, and the effect of BMMNCs on activation of microglia was detected by immunofluores- cence assay. Results （ 1 ） At each time point, the mNSS arid the volume of cerebral infarction of the model group were significantly higher than those of the sham operation group （P〈0.05）, while the mNSS and the volume of cerebral infarction of the BMMNCs group were lower than those of the model group, higher than those of the sham operation group, and it was gradually decreased with the treatment time extension （P〈 0.05）. There was no difference in comparison between the ERK1/2 inhibitor group and the model group （P〉 0.05）. （2）At each time point, the pERK1/2, Bcl-2, Bax and caspase-3 protein levels of the model group were significantly higher than those of the sham operation group （P〈0.05）. The pERK1/2 and Bcl-2 protein levels of the BMMNCs group（ pERK1/2 ： （ 0.38±0.16）, （ 0.39±0.15 ）, （ 0.40±0.20）, Bcl-2 ： （ 0.38 ±0.14）, （0.39±0.15）, （0.37±0.13）） were higher than those of the model group（pERK1/2： （0.17±0.05）, （0.14± 0.04）, （ 0.13±0.03 ）, Bcl-2 ： （ 0.23±0.11 ）, （ 0.24±0.12）, （ 0.27±0.14）, Bax： （ 0.39±0.13 ）, （ 0.40± 0.14 ）, （ 0.45±0.23 ） , caspase-3 ： （ 0.52±0.26 ） , （ 0.56±0.27）, （ 0.58±0.28 ） ）, while Bmx and caspase-3 protein lev- els（Bax： （0.25±0.13） , （0.19±0.06） , （0.21±0.08） , caspase-3： （0.35±0.13） , （0.34±0.16） , （0.29±0.09） ） were decreased （P〈0.05）. The pERK1/2 protein level of ERK1/2 inhibitor group was lower than other groups,There was no difference in the level of Bcl-2, Bax and caspase-3 between the ERK1/2 group and the model group. （/9〉0.05）. （3） At each time point, microglia （Ibal positive） in isehemic penmnbra of the BMMNCs group was significantly more than those of the model group, and it was increased with the time extension （P〈0.05）. There was no difference in comparison between the ERK1/2 inhibitor group and the modei group （P〉0.05）. Conclusion BMMNCs can reduce the apoptosis through ERK1/2 signaling path- way, thus improving the neurological function and reducing the infarct scope.