缺血缺氧致新生鼠脑白质损伤模型中骨形态生成蛋白4表达及作用的研究

目的 研究在缺血缺氧致新生鼠脑白质发生、发展中,脑白质损伤超微结构变化,以及骨形态生成蛋白(bone morphogenetic protein,BMP)4基因及蛋白表达动态变化和对髓鞘化的影响.方法 3日龄Sprague-Dawley 新生大鼠共152只,按随机数字表法随机分为脑白质损伤组(n=76)和对照组(n=76).采用结扎右侧颈总动脉伴低氧(8%O2 92%N2)方法制备脑白质损伤模型,分别于手术后3d、7d、14d及21d采集标本.光镜下观察脑组织形态学改变,透射电镜下观察髓鞘形成情况,免疫组织化学观察BMP4和髓鞘化标志物髓磷脂碱性蛋白(myelin basic protein,MBP)的表达和定位,Western blot方法测定BMP4和MBP的蛋白表达水平,real-time PCR方法分析BMP4 mRNA的表达情况.结果 对照组大鼠的脑白质中细胞结构清晰,纤维走行紧密有序,脑白质损伤组大鼠脑白质细胞稀疏,白质疏松呈筛网状,纤维走向紊乱.电镜下可见对照组髓鞘形态规则,密度均匀,厚度一致,而脑白质损伤组髓鞘疏松、变薄,板层分离,边界不清.免疫组织化学、Western blot及real-time PCR结果均提示,对照组内BMP4蛋白及mRNA表达水平随日龄增大无明显变化,脑白质损伤组内随缺血时间延长BMP4表达迅速增加.两组间比较,从手术后3d起,脑白质损伤组较对照组表达增多(P<0.05),随缺血时间延长两组间差异更加明显.免疫组织化学及Western blot方法分析MBP蛋白表达,发现两组内MBP蛋白表达均随日龄增长而逐渐增加,但两组间比较,14d和21d时脑白质损伤组MBP蛋白表达明显较对照组减少,组间差异有统计学意义(P<0.05).结论 缺血缺氧致新生鼠脑白质损伤发生时存在髓鞘化障碍,而此过程中BMP4基因及蛋白表达均显著增加,提示BMP4可能参与脑白质损伤时髓鞘化障碍的发生.

The expression of bone morphogenetic protein 4 and its effects in ischemia-anoxemia induced white matter injury model

Objective To investigate the ultrastructural alteration in brain tissues as well as the expression of bone morphogenetic protein(BMP) 4 and its effects on regulating myelination in the process of white matter injury development.Methods A total of 152 Sprague-Dawley newborn rats(3 days old) were randomly divided into white matter injury group(n=76) or control group(n=76).The white matter injury model was established by ligation of the right common carotid artery and hypoxic exposure(8% O2 and 92%N2),and samples were collected at 3d,7d,14d and 21d after operation.Morphological changes of the brain tissues were observed under a light microscope,while myelination was analyzed using a transmission electron microscope.The expression and location of BMP4 and myelin basic protein(MBP),a marker for myelination,was detected by immunohistochemistry staining,expression levels of BMP4 and MBP proteins were analyzed by Western blotting,and BMP4 mRNA expression was measured by real-time PCR.Results Observed under the light microscope,the cellular structure was clear,fibers arranged closely and orderly in the white matter of the control group.Whereas in the white matter injury group,sparse cells,loose mesh shaped white matter,and disorderly oriented fibers were observed.In the control group,myelin sheath had regular morphology,uniform density,and same thickness,observed using the transmission electron microscope.While in the white matter injury group,the myelin sheath was loosened,thinned,lamellar separated,and boundary obscured.Using immunohistochemistry staining,Western blot,and real-time PCR analyses,it was found that the protein and mRNA expression of BMP4 had no significant change with the increase of age in the control group,while it was rapidly increased with the extending of ischemic time in the white matter injury group.Comparing with the control group,the expression of BMP4 was significantly increased since 3d after operation in the white matter injury group(P<0.05),and the difference between two groups became more significant with the extending of ischemic time.The expression of MBP protein was analyzed by immunohistochemistry staining and Western blot,and a gradual increase was found in both groups with the increase of age.However,the expression of MBP protein was significantly decreased on 14d and 21d after operation in the white matter injury group compared with the control group(P<0.05).Conclusion Myelination disorders exists in white matter injury induced by ischemia-anoxemia.Meanwhile,the expression of BMP4 is significantly increased in the white matter injury group,indicating a possibility that BMP4 involves in the regulation of myelination disorders in white matter injury.