發(fā)布時間：2018-12-08 18:11

【摘要】：目的:應(yīng)用磁共振成像(MRI)技術(shù)動態(tài)評價重組MANF(中腦星形膠質(zhì)細胞來源的神經(jīng)因子)蛋白對大鼠缺血再灌注損傷的保護作用。方法:利用實驗室pET28a-MANF質(zhì)粒轉(zhuǎn)染好的E.coli BL21菌株誘導(dǎo)表達MANF蛋白,經(jīng)BCA蛋白測定法測出蛋白濃度。利用大鼠大腦中動脈栓塞法建立局灶性腦缺血再灌注損傷模型(MCAO),經(jīng)磁共振掃描證實有明顯的腦缺血損傷癥狀后,經(jīng)尾靜脈和側(cè)腦室注射兩種方式將適宜劑量的重組MANF蛋白注入大鼠體內(nèi),同時靜脈注射依達拉豐(3 mg/kg)作為陽性對照組。在給藥后的第1天、第2天、第3天、第5天及第7天對各實驗組大鼠進行利用磁共振掃描,同時結(jié)合死亡率統(tǒng)計、體重評估、神經(jīng)功能評分和梗死體積的變化評價重組MANF蛋白對腦缺血再灌注損傷的治療效果。用MRIcro 2.0對MRI掃描結(jié)果進行梗死體積和梗死側(cè)/正常側(cè)信號強度的統(tǒng)計;用Image J對TTC染色結(jié)果進行梗死體積統(tǒng)計;將所得結(jié)果應(yīng)用SPASS 16.0軟件分析是否有統(tǒng)計學意義。結(jié)果:MRI和神經(jīng)功能評分篩選出成功的腦缺血模型,在T2WI圖像上觀察到大鼠缺血側(cè)腦實質(zhì)皮層及基底節(jié)區(qū)出現(xiàn)片狀高信號區(qū);同時大鼠神經(jīng)功能評分為3分,不能伸展對側(cè)前肢,并向?qū)?cè)轉(zhuǎn)圈。在腦缺血再灌注后經(jīng)MR連續(xù)掃描發(fā)現(xiàn),再灌注3h后大鼠梗塞灶的信號強度比值和再灌注0 h相比具有顯著性差異,表明腦部出現(xiàn)早期損傷。然后將純化好的重組MANF蛋白經(jīng)尾靜脈和側(cè)腦室注射兩種方式注入大鼠體內(nèi),同時注射PBS組作為陰性對照組,注射依達拉豐(3mg/kg)作為陽性對照組。在注射藥物后的第1天、第2天、第3天、第5天及第7天,利用MRI來評價各實驗組治療腦缺血再灌注損傷的療效。結(jié)果發(fā)現(xiàn),第三天大鼠側(cè)腦室注射組腦梗死體積(45.31±21.13)小于尾靜脈注射組(97.86±8.64),經(jīng)獨立樣本t檢驗P0.05;側(cè)腦室注射10μg及20μg MANF蛋白治療組與PBS治療組比較,有效減輕了大鼠腦缺血再灌注損傷,且有統(tǒng)計學意義;同時20μg MANF蛋白治療組的腦缺血保護作用優(yōu)于10μg組。除此之外,在缺血再灌注損傷早期經(jīng)側(cè)腦室注入MANF蛋白相比對照組可以有效降低大鼠死亡率,促進大鼠體重增長,提高神經(jīng)功能學評分。基于MRI和TTC染色兩種方法統(tǒng)計的梗死體積都表明側(cè)腦室注射MANF組在給藥后第7天梗死體積相比對照組及明顯減少。對神經(jīng)元的免疫組化染色分析結(jié)果表明,側(cè)腦室注射MANF組神經(jīng)元存活率明顯高于對照組。結(jié)論:MANF蛋白對大鼠缺血再灌注損傷具有保護作用,可以有效降低大鼠死亡率,促進體重增長,提高神經(jīng)功能學評分,減少腦梗死體積,改善神經(jīng)元的存活。MRI結(jié)果與TTC染色結(jié)果及免疫組化結(jié)果相一致,表明該技術(shù)是評價重組MANF蛋白治療腦缺血療效的有效方法。
[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 鹵21.13) was smaller than that in the caudal vein injection group (97.86 鹵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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R743;R445.2

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