【作者】 钱自亮；

【导师】 徐军美；

【作者基本信息】 中南大学 ， 麻醉学， 2010， 博士

【摘要】 目的：研究缺血再灌注大鼠脑组织PPAR-γ1mRNA和蛋白的表达变化,并结合文献分析其和脑缺血再灌注损伤的关系。方法：建立SD大鼠右侧MCAO缺血再灌注模型。实验分为对照组、假手术组和缺血再灌注组。缺血再灌注组取缺血后12h、24h和48h三个时间点进行观察。用RT-PCR方法及Western blotting方法分别检测PPAR-γ1mRNA和蛋白的表达。结果：脑缺血再灌注组PPAR-γ1mRNA和蛋白表达较正常对照组和假手术组明显降低。通过对缺血后12h、24h和48h三个时间点的动态变化观察发现,缺血后12h表达最低,并且随缺血后时间的推移,其表达逐渐增加,存在显著性差异。但缺血后48h的表达值仍低于正常对照组和假手术组。结论：实验SD大鼠缺血再灌注脑组织PPAR-γ1表达下调,提示针对PPAR-γ1作为一靶点进行干预对于缺血性脑血管病的治疗是一个新的研究方向。目的：1.研究通过脑室内途径腺病毒载体转染PPAR-γ1到脑的可行性,如果可行则进一步观察其对缺血再灌注脑是否具有保护作用。2.过表达PPAR-γ1基因和PPAR-γ激活剂吡格列酮合用,观察其对脑I/R保护作用是否有增强。3.研究PPAR-γ1对IL-1β、ICAM-1、Bcl-2、Bax、MMP-9和AQP-4的影响及抗脑I/R损伤的机制。方法：(1)构建复制缺陷性重组腺病毒：采用分子克隆手段获得携带增强型绿色荧光蛋白(EGFP)基因的重组质粒、携带PPAR-γ1基因的重组质粒,以细胞内同源重组法构建复制缺陷型重组腺病毒Adv-EGFP、Adv-PPAR-γ1,在293细胞内分别扩增,应用氯化铯密度梯度离心法纯化病毒。(2)选择95只SD大鼠随机分成6组。按照包新民等报道的定位方法,在立体定位仪下给大鼠脑室内注射相应试剂0.1ml。第1、2组注入不含PPAR-γ1基因的生理盐水0.1ml,第3组注入Adv-PPARγ1 0.1ml,第4注入Adv-EGFP 0.1ml,第5组吡格列酮5mg/kg灌胃Qd×3天,第6组脑室内注入Adv-PPARγ1 0.1ml+吡格列酮5mg/kg灌胃Qd×3天。(3)3天后建立大鼠右侧大脑中动脉缺血再灌注模型。第1组为假手术组,不阻断大鼠大脑中动脉。第2-6组阻断大脑中动脉90分钟再灌注24小时。(4)采集脑组织标本,采用TTC染色法测量脑梗死体积；伊文氏蓝法测定血脑屏障通透性；干-湿重法测定脑含水量；光镜、电镜下进行病理形态学观察,免疫荧光显微镜下观察腺病毒表达情况；脑组织MPO活性测定；用Western Blot方法检测缺血区脑组织IL-1β、ICAM-1、Bcl-2、Bax、AQP-4、MMP-9蛋白的表达。结果：脑室内途径腺病毒载体转染Adv-EGFP,免疫荧光反应阳性。脑缺血再灌注后,脑梗死体积增大、血脑屏障通透性增加、脑组织含水量增加、MPO活性明显增高,IL-1β、ICAM-1、Bax、AQP-4和MMP-9蛋白的表达增加,Bcl-2蛋白表达减少。脑室内注射Adv-PPARγ1或吡格列酮灌胃均能抑制以上脑损伤指标以及IL-1β、ICAM-1、Bax、AQP-4和MMP-9的蛋白表达上调,而使Bcl-2蛋白表达增加。它们联合应用时能增强保护作用。结论：1.通过脑室内注射腺病毒载体转染PPAR-γ1到脑的方法可行,且对缺血再灌注脑具有保护作用。2.脑室内转染PPAR-γ1基因和PPAR-γ激活剂吡格列酮合用,能增强对I/R脑的保护作用。3.PPAR-γ1可以减少缺血再灌注脑组织中性粒细胞的浸润,同时抑制IL-1β、ICAM-1、Bax、MMP-9、AQP-4上调,促进Bcl-2上调。提示PPAR-γ1是通过调控炎性损伤路径发挥抗缺血再灌注损伤的,并且提示抑制凋亡也可能是其机制之一。更多还原

【Abstract】 OBJECTIVE:To explore the expression pattern of PPAR-γ1 mRNA and protein in the ischemia-reperfusion brain tissues, to analyze the relationship between PPAR-yl and cerebral ischemia based on the previous studies.METHODS:Adult male SD rats were subjected to middle cerebral artery occlusion 90min and followed reperfusion by monofilament method. The experimental groups included control group, sham group and ischemia-reperfusion group. The ischemia-reperfusion rats were sacrificed at 12h,24h and 48h after ischemia. The expression of PPAR-γ1, mRNA and protein were measured by RT-PCR and Western blotting respectively.RESULTS:The ischemia-reperfusion group displayed obvious downregulation of PPAR-γ1 mRNA and protein compared with the control group and the sham group. The level of PPAR-γ1 mRNA and protein might reach the lowest point in the 12h after ischemia onset, and increased gradually following the post-ischemic time.The expression of PPAR-γ1 mRNA and protein were still lower on 48h after ischemia onset.CONCLUSIONS:The ischemia-reperfusion injury downregulated PPAR-γ1 mRNA and protein expression. The PPAR-γ1 may be a valuable target and the experiment that make an intervention on it may be very important to improve the brain ischemia injury. OBJECTIVE:1.To explore the feasibility of PPAR-γ1 gene transfection via cerebral ventricle and the protective effect against cerebral ischemia reperfusion injury.2.To observe if the effection against cerebral ischemia reperfusion injury is enhanced when transfecting of PPAR-γ1 gene via cerebral ventricle association with the Pioglitazone which is PPAR-y’s agonist.3.To explore the the expression pattern of IL-1β、ICAM-1、Bcl-2、Bax、MMP-9 and AQP-4 protein in the ischemia-reperfusion brain tissues and to investigate the potential mechanisms.METHODS:1.Construction of the replication-deficient recombinant adenovirus:Recombinant plasmids PDC-EGFP (containing enhanced green fluorescence protein EGFP gene), PDC-PPAR-γ1 (containing PPAR-γ1 gene). Recombinant adenovirus Adv-EGFP, Adv-PPAR-γ1 were produced by homologous recombinant in 293 cells, amplified also in 293 cells on a large scale, and purified by ultracentrifugation in CsCl step gradient solutions. The concentration of recombinant adenovirus Adv-EGFP purified by ultracentrifugation in CsCl step gradient solutions is 2.0×109Pfu/ml; that of Adv-PPAR-γ1 is 1.0×1011Pfu/ml.2.95 S-D rats were randomly divided into 6 groups.Each group was treated with corresponding agent via cerebral ventricle.In group I andⅡwere injected by normal saline.In groupⅢ,rat was injected with 0.1ml Adv-PPAR-γ1 and the groupⅣwas Adv-EGFP. The groupⅤwas intragastric administered with Pioglitazone. The groupⅥwas administered with PPAR-γ1 via cerebral ventricle and intragastric administered with Pioglitazone at the same time.3. After 3 days middle cerebral artery occlusion model was established.The groupⅠwas sham-operation group.In groupⅡ-Ⅵmiddle cerebral artery was obstructed for 90min and followed reperfusion for 24h.4.24 hours after operation sample was collected and cerebral infarction volume was measured by TTC staining, blood brain barrier permeability by Evan’s blue dye Perfusion,brain moisture capacity by W-D weight method. To observe the pathomorphology by light microscope and electron microscope. Measuring the activity of MPO. To observe the expression of IL-1β、ICAM-1、Bcl-2、Bax、AQP-4 and MMP-9 protein by Western Blotting.RESULTS:After transfecting Adv-EGFP plasmid,the fluorescence for EGFP was positive in brain tissue suggesting the successful transfection of virus gene.In response to I/R injury, cerebral infarction was occurred、Blood brain barrier permeability、brain moisture capacity、the activity of MPO were dramatically incresed and the expression of IL-1β、ICAM-1、Bax、AQP-4、MMP-9 protein were upregulated while Bcl-2 was downregulated.After injected Adv-PPAR-γ1 via cerebral ventricle or intragastric administered with Pioglitazone could inhibited the above parameters of injured brain as well as upregulated the Bcl-2.The improvement by adv-PPAR-γ1 gene and Pioglitazone was enhanced by administration together.CONCLUSIONS:1.Transfection of PPAR-γ1 gene via cerebral ventricle was feasible and demostrated the protectiv effect against cerebral ischemia reperfusion injury.2. Combined transefction of PPAR-γ1 gene and intragastric administration with Pioglitazone could enhance the protectiv effect against cerebral ischemia reperfusion injury.3. The infiltrations of neutrophilic leukocytes could be reduced by PPAR-γ1 and the expreeion of IL-1β、ICAM-1、AQP-4、MMP-9 protein were downregulated, Bcl-2 was upregulated. These results suggests that the protective effect of PPAR-γ1 on ischemia-reperfusion injury is through regulating the inflammatory reaction and apoptosis.更多还原