【作者】 李志刚；

【导师】 张智博；

【作者基本信息】 南华大学 ， 内科学， 2011， 硕士

【摘要】 目的本研究通过R0cK抑制剂盐酸法舒地尔(R0cK选择性的抑制剂)对缺血／再灌注模型的干预以及检测和分析cathepsin B与p—JNK之间的蛋白表达的变化、定位的关系，探讨溶酶体组织蛋白酶的促凋亡的分子机制和法舒地尔的药理抗凋亡的分子机制，可能为缺血性脑卒中的防治提供了新的理论依据和新的治疗方向，有着重要的临床意义。方法清洁级健康雄性10—12 N]龄Spr‘ague—I)awley(sD)大鼠72只，体重约270士20g，随机分为假手术组(sham，n=6)、模型组011(M一0h，n=6)、模型组2h(M一2h，n=6)、模型组6h(M一6h，n=6)、模型组24h(M一24h，n=6)、模型组48h(M一48h，n=12)、干预组2h(I一2h，n=6)、干预组6h(I一6h，n=6)、干预组24h(I一24h，n=6)和干预组48h(I一48h，n=12)。线栓法制备McA局灶性缺血再灌注模型。干预组给予盐酸法舒地尔注射液5mg／kg，每12h一次，从缺血再灌注011起给予腹腔注射，直至断头取脑；模型组在对应时间点给予等量的生理盐水。利用Longa的5级标准评分法评价神经功能缺损变化；运用TTc染色法、TuNEL法和免疫组织化学检测方法，分别检测脑梗死体积变化、视前区细胞凋亡变化和视前区cathepsin B与p—JNK蛋白表达变化；通过使用双变量相关分析统计方法和免疫荧光双标激光共聚焦检测法来分析catllepsin B和p—JNK蛋白表达的相关性和这两种蛋白的定位关系。结果1．模型成功率为86．84％；模型组48h相对梗死体积的变异系数为3．89％。2．假手术组未见梗死灶，模型组及干预组缺血侧皮层及皮层下出现白色的梗死灶；模型组48h相对梗死体积为28．52士1．11％，但干预组的相对脑梗死体积为13．43士0．86％，较模型组明显减少(P<0．05)，其神经功能评分也得到明显改善(P<0．05)。3．在大鼠缺血侧视前区，假手术组基本看不到凋亡细胞，模型组2h可观察到细胞凋亡，6h增多，24h达高峰，48h有所下降(P<0.001)；盐酸法舒地尔明显减少缺血再灌注后的凋亡细胞(P<0.001)。4．在大鼠缺血侧视前区，假手术组未见明显catllepsin B免疫活性表达；再灌注开始就可见catllepsin B蛋白表达，2h明显增加，6h达到高峰，24h降到最低，48h又有所增加(P<0．001)；盐酸法舒地尔干预组各个时间点的catllepsin B蛋白表达较模型组无明显变化(P>0．05)。5．在大鼠缺血侧视前区，假手术组未见任何p—JNK阳性表达；模型组再灌注开始未见p—JNK的蛋白表达，再灌注2h可观察到p—JNK阳性细胞表达，6h达到顶峰，再灌注24h降到最低，48h也有所增加(P<0．001)；盐酸法舒地尔可以明显抑制p—JNK免疫活性表达(P<0．05)；干预组的p—JNK蛋白表达再灌注6h比2h还是有增高的掐势．24h降低(P<0 0s)．再灌沣4Rh与24h相比变化不女(P>0 0s)。6．免疫荧光双标显示：在缺血侧视前区缺血再灌注后camepsin B和p—JNK表达共定位于再灌注2h、6h、24h和48h，而再灌注开始时只观察到少量camepsin B阳性细胞，并未见到p—JNK蛋白表达；再灌注6h两种蛋白共染的细胞数量明显增多并达到顶峰，随后下降至再灌注24h，再灌注48h又有所增加(P<0．001)。7．双变量相关分析结果显示：在缺血侧视交叉前区缺血再灌注过程中p—JNK蛋白表达动态变化与camepsin B的免疫活性表达呈直线正相关关系(r。=0．934，P<0．001)。结论1．大鼠脑缺血再灌注后catllepsin B可能通过R0cK—JNK信号通路介导细胞凋亡。2．法舒地尔可能通过抑制JNK通路的凋亡信号转导，减少细胞凋亡，减少脑梗死体积。更多还原

【Abstract】 ObjectiveThe aim of the present study was to investigate molecular mechanisms of lysosomal cathepsins-induced apoptosis and pharmacological anti-apoptosis of fasudil,a selective inhibitor of ROCK,through the intervention of fasudil hydrochloride and the detection and analysis of the expression and colocalization of cathepsin B and p-JNK,which may provide the theoretical basis and new direction of the prevention and cure of cerebral ischemic stroke.MethodsSeventy-two male Sprague-Dawley(270±20g,10-12 weeks old,CLA)were randomly divided into ten groups:sham-operated group(Sham,n=5),model group 0h(M-0h,n=6),model group 2h(M-2h,n=6),model group 6h(M-6h,n=6),model group 24h (M-24h,n=6),model group 48h(M-48h,n=6),intervention group 2h (I-2h,n=6), Intervention group 6h(I-6h,n=6),intervention group 24h (I-24h,n=6) and intervention group 48h (I-48h,n=6).Transient middle cerebral artery occlusion was performed with some modifications,as Longa described.The Intervention group was intraperitoneally injected with fasudil hydrochloride injection (5mg/kg) every 12 hours beginning with the withdrawal of the suture.The rats were injected with the same volume of 0.9%NaCl at the same time points for the other groups.The neurologic deficit scores were evaluated with longa’s five-point scale standard scoring method. TTC Staining, TUNEL,immunohistochemistry were performed for assessment of infarct volumes, apoptosis and expressions of cathepsin B and p-JNK in the preoptic region.The colocalization of cathepsin B and p-JNK and the correlation between the expression of both the proteins were analyzed through double-label immunofluorescence with the laser scanning confocal microscope and bivariate correlation analysis. Results1. Transient middle cerebral artery occlusion were achieved in 66 of 76 rats 86.84%;the coefficient of variation of the relative infarct volume of model group 48h is 3.89%.2. No infarction was observed in sham-operated group,whereas cortical and subcortical infarctions(white color) were detected in model group and intervention group;The relative infarct volume of the intervention group (13.43±0.86%) were significantly decreased compared with the model group(28.52±1.11%)(P<0.001),and the neurologic deficit score of its was significantly reduced (P<0.05).3. In the sham-operated rats,few apoptotic cells were detected in the ipsilateral preoptic region;In the model rats,TUNEL-positive cells appeared in the ipsilateral preoptic region,and were gradually increased at 6 hours of reperfusion,to a peak of 24 hours,and were subsequently decreased at 48 hours (P<0.001).Fasudil hydrochloride significantly reduced cell apoptosis after ischemia-reperfusion (P<0.001).4. In the sham-operated rats,no cathepsin B immunoreactivity was detected in the ipsilateral preoptic region;In the model group,there was a little growth in cathepsin B cytoplasmic staining in ischemic neurons of the preoptic region at the onset of reperfusion,and a significant increase during the 2-hour reperfusion,and a peak at 6 hours of reperfusion,and an obvious decrease at 24 hours,and subsequent increase at 48 hours (P<0.001).The intervention group by fasudil hydrochloride had no significant change in the expression of cathepsin B compared with the model group at the various time points(P>0.05).5. In the sham-operated rats,no p-JNK staining was detected in the ipsilateral preoptic region;In the model rats,no p-JNK immunoreactivity was observed in the ischemic preoptic region at the onset of reperfusion,and p-JNK expession was obvious at 2-hour reperfusion,and peaked at 6-hour reperfusion,and was minimized at 24-hour reperfusion,followed by being moderately increased at 48-hour reperfusion(P<0.001); Fasudil hydrochloride significantly inhibited the immunoreactivity of p-JNK (P<0.05); In the intervention group,p-JNK expression increased moderately at 6-hour reperfusion,compared with its expression at 2-hour reperfusion,and decreased at 24-hour reperfusion (P<0.05),but p-JNK immunoreactivity at 48-hour reperfusion was not significantly enhanced(P >0.05).6. Immunofluorescent double labeling revealed that the colocalization of cathepsin B with phospho-JNK appeared in the ischemic preoptic region 2,6,24,48 hours after ischemia–reperfusion,and that a few of cathepsin B-staining neurons were observed at the onset of reperfusion,when p-JNK expression was not seen at the same region;Co-staining cells of cathepsin B and p-JNK were significantly increased and peaked at 6 hours of reperfusion,and were decreased at 24 hours of reperfusion,and were moderately increased at 48 hours of reperfusion (P<0.001).7. Bivariate correlation analysis showed a strong positive linear correlatio-n between the expression of cathepsin B and p-JNK with Spearman’s rho analysis(rs=0.934,P<0.001).Conclusions1. Cathepsin B may mediate apoptosis through ROCK-JNK signaling pathway after cerebral ischemia-reperfusion in rat.2. Fasudil may inhibit the JNK signal transduction pathway of apoptosis,and reduce cell apoptosis, and decrease cerebral infarct volume.更多还原