磁共振成像动态观察重组MANF蛋白在大鼠缺血再灌注损伤中的治疗作用

发布时间：2018-12-08 18:11

【摘要】：目的:应用磁共振成像(MRI)技术动态评价重组MANF(中脑星形胶质细胞来源的神经因子)蛋白对大鼠缺血再灌注损伤的保护作用。方法:利用实验室pET28a-MANF质粒转染好的E.coli BL21菌株诱导表达MANF蛋白,经BCA蛋白测定法测出蛋白浓度。利用大鼠大脑中动脉栓塞法建立局灶性脑缺血再灌注损伤模型(MCAO),经磁共振扫描证实有明显的脑缺血损伤症状后,经尾静脉和侧脑室注射两种方式将适宜剂量的重组MANF蛋白注入大鼠体内,同时静脉注射依达拉丰(3 mg/kg)作为阳性对照组。在给药后的第1天、第2天、第3天、第5天及第7天对各实验组大鼠进行利用磁共振扫描,同时结合死亡率统计、体重评估、神经功能评分和梗死体积的变化评价重组MANF蛋白对脑缺血再灌注损伤的治疗效果。用MRIcro 2.0对MRI扫描结果进行梗死体积和梗死侧/正常侧信号强度的统计;用Image J对TTC染色结果进行梗死体积统计;将所得结果应用SPASS 16.0软件分析是否有统计学意义。结果:MRI和神经功能评分筛选出成功的脑缺血模型,在T2WI图像上观察到大鼠缺血侧脑实质皮层及基底节区出现片状高信号区;同时大鼠神经功能评分为3分,不能伸展对侧前肢,并向对侧转圈。在脑缺血再灌注后经MR连续扫描发现,再灌注3h后大鼠梗塞灶的信号强度比值和再灌注0 h相比具有显著性差异,表明脑部出现早期损伤。然后将纯化好的重组MANF蛋白经尾静脉和侧脑室注射两种方式注入大鼠体内,同时注射PBS组作为阴性对照组,注射依达拉丰(3mg/kg)作为阳性对照组。在注射药物后的第1天、第2天、第3天、第5天及第7天,利用MRI来评价各实验组治疗脑缺血再灌注损伤的疗效。结果发现,第三天大鼠侧脑室注射组脑梗死体积(45.31±21.13)小于尾静脉注射组(97.86±8.64),经独立样本t检验P0.05;侧脑室注射10μg及20μg MANF蛋白治疗组与PBS治疗组比较,有效减轻了大鼠脑缺血再灌注损伤,且有统计学意义;同时20μg MANF蛋白治疗组的脑缺血保护作用优于10μg组。除此之外,在缺血再灌注损伤早期经侧脑室注入MANF蛋白相比对照组可以有效降低大鼠死亡率,促进大鼠体重增长,提高神经功能学评分。基于MRI和TTC染色两种方法统计的梗死体积都表明侧脑室注射MANF组在给药后第7天梗死体积相比对照组及明显减少。对神经元的免疫组化染色分析结果表明,侧脑室注射MANF组神经元存活率明显高于对照组。结论:MANF蛋白对大鼠缺血再灌注损伤具有保护作用,可以有效降低大鼠死亡率,促进体重增长,提高神经功能学评分,减少脑梗死体积,改善神经元的存活。MRI结果与TTC染色结果及免疫组化结果相一致,表明该技术是评价重组MANF蛋白治疗脑缺血疗效的有效方法。
[Abstract]:Aim: to dynamically evaluate the protective effect of recombinant MANF protein on ischemia-reperfusion injury in rats by magnetic resonance imaging (MRI) technique. Methods: the expression of MANF protein was induced by E.coli BL21 strain transfected with pET28a-MANF plasmid in laboratory, and the protein concentration was measured by BCA protein assay. The focal cerebral ischemia-reperfusion injury model was established by middle cerebral artery embolization (MCAE) in rats. The model of focal cerebral ischemia-reperfusion injury (MCAO),) was proved to have obvious symptoms of cerebral ischemia injury by magnetic resonance imaging (MRI). The appropriate dose of recombinant MANF protein was injected into the rat body through caudal vein and lateral ventricle injection, and Edarafen (3 mg/kg) was injected intravenously as the positive control group. On the first day, the second day, the third day, the fifth day and the seventh day, the rats in each experimental group were scanned by magnetic resonance imaging (MRI), combined with mortality statistics and weight evaluation. The effect of recombinant MANF protein on cerebral ischemia-reperfusion injury was evaluated by neurological function score and infarct volume. The infarct volume and signal intensity of infarct side / normal side were calculated by MRIcro 2.0 scan results of MRI, the infarct volume of TTC staining results were calculated by Image J, and the results were analyzed by SPASS 16.0 software. Results: the successful cerebral ischemia model was selected by MRI and nerve function score. On the T2WI images, the ischemic cortex and basal ganglia were observed in the ischemic cortex and basal ganglia. At the same time, the neurological function score of the rats was 3, which could not extend the contralateral forelimbs and turn to the opposite side. Continuous MR scanning after cerebral ischemia and reperfusion showed that there was significant difference in signal intensity ratio between 3 h reperfusion and 0 h reperfusion, indicating early brain injury. Then the purified recombinant MANF protein was injected into the rat via caudal vein and lateral ventricle. The PBS group was used as the negative control group and the Edarafone (3mg/kg) group as the positive control group. On day 1, day 2, day 3, day 5 and day 7 after injection, MRI was used to evaluate the efficacy of each experimental group in the treatment of cerebral ischemia-reperfusion injury. The results showed that the volume of cerebral infarction in the intracerebroventricular injection group (45.31 卤21.13) was smaller than that in the caudal vein injection group (97.86 卤8.64) on the third day. Compared with PBS group, ICV injection of 10 渭 g and 20 渭 g MANF protein significantly alleviated cerebral ischemia-reperfusion injury in rats, and 20 渭 g MANF protein treatment group had better cerebral ischemic protection than 10 渭 g group. In addition, injection of MANF protein through the lateral ventricle at the early stage of ischemia-reperfusion injury can effectively reduce the mortality, promote the growth of body weight and improve the neurological score compared with the control group. The infarct volume calculated by MRI and TTC staining showed that the infarct volume in the MANF group was significantly lower than that in the control group on the 7th day after administration. The results of immunohistochemical staining showed that the survival rate of neurons in MANF group was significantly higher than that in control group. Conclusion: MANF protein has protective effect on ischemia-reperfusion injury in rats. It can effectively reduce the mortality, promote body weight, improve neurological function score and reduce the volume of cerebral infarction in rats. The results of MRI were consistent with the results of TTC staining and immunohistochemistry, which indicated that this technique was an effective method to evaluate the therapeutic effect of recombinant MANF protein on cerebral ischemia.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743;R445.2

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