【作者】 孔滨；

【导师】 陈礼刚；

【作者基本信息】 泸州医学院 ， 外科学， 2010， 硕士

【摘要】 目的：观察腺病毒介导的血红素加氧酶-1(heme oxygenase-1,HO-1)基因转染在大鼠脑缺血再灌注损伤中的作用；探讨调节血红素加氧酶基因表达对临床脑卒中的治疗前景。方法：建立携带血红素加氧酶-1基因的腺病毒载体：取大鼠脾脏细胞提取总RNA,参考GerieBank公布的大鼠HO-1序列(J02722)设计上下游引物,采用反转录-聚合酶链反应扩增HO-1基因；采用AdEasy System方法构建包含HO-1基因的重组腺病毒载体(Ad-HO-1)。测得Ad-HO-1和空载体腺病毒(Ad-GFP)滴度分别为2.5×1011PFU/ml、2.0×1011PFU/ml。健康雄性SD大鼠72只随机分为4组：假手术对照组(sham group,SH)、生理盐水组(vehicle group,V)、空载体组(adenovirus group,Ad)和Ad-HO-1转染组(HO group),各组18只。后3组在缺血前3d于大鼠右侧脑室分别注射20μl生理盐水、20μl生理盐水含空载体腺病毒(1.0×1010 Plaque-forming unit/ml,PFU/ml)和重组HO-1腺病毒(1.0×1010PFU/ml)1μl,注射方法部位参照Paxinos和Watson的大鼠脑立体定位图谱。连续注射3d后,参照zea—Longa线栓法建立大鼠大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)缺血再灌注模型,大鼠予以2%戊巴比妥钠腹腔注射麻醉,术中麻醉机控制持续吸入2%异氟烷,用线栓阻断右侧MCA的血液供应,阻断大脑中动脉2h后退出线栓,给予再灌注。术中以手术恒温设备将直肠温度控制在36.5-37.5℃,以探针监测大鼠颞温,多普勒探针检测脑缺血局部血流。每组大鼠在缺血再灌注后24小时测定Garcia神经功能评分,然后处死大鼠并取全脑标本,行脑切片采用TTC染色法测定大脑梗死体积,并计算梗死体积百分比。于脑缺血再灌注2h后取脑组织匀浆检测细胞凋亡指标Caspase-3活性；于再灌注2h后取脑切片行TUNEL染色检测细胞凋亡率。在荧光显微镜下观察连续转染后24h时脑组织荧光蛋白的表达情况,并计算转染率。Western blot检测转染后24h脑组织HO-1的表达。结果：转染后HO-1表达的变化：Western blot检测示：HO组大鼠脑内HO-1表达显著高于Ad组和V组,Ad组和V组比较HO-1表达无明显差异。绿色荧光蛋白示转染率：显微镜下观察Ad组和HO组的大鼠脑切片,均可见有绿色荧光蛋白表达且两组比较绿色荧光蛋白表达量无明显差异,并测定转染率为(34.5±3.4)%。神经功能评分：在脑缺血再灌注后24h, SH、V、Ad、HO四组大鼠的神经功能评分分别为18、9.571±0.254、9.857±0.149、12.75±0.211,HO组大鼠神经功能评分均显著优于Ad组和V组,差异有统计学意义(P<0：001)；Ad组与V组比较神经功能评分无明显差异。梗死体积及细胞凋亡的检测：a.脑梗死体积：在脑缺血再灌注后24h, SH、V、Ad、HO四组大鼠的脑梗死体积百分比分别为0%、60.36±6.750%、61.80±7.544%、27.31±2.913%,与SH组比较,V组、Ad组脑梗死体积百分比显著增高；与V组及Ad组比较,HO组脑梗死体积百分比显著降低(P<0.01)；Ad组与V组比较脑梗死体积百分比无显著差异。b.神经细胞凋亡：在脑缺血再灌注后2h, SH、V、Ad、HO各组中纹状体、海马(CA1区)、皮层(缺血半暗带)的凋亡细胞阳性率及Caspase-3活性分别为SH组：<0.1%/0.800±0.170、<0.1%/0.441±0.193、<0.10%/0.674±0.186。V组：(53.39±1.742)%/8.889±0.352、(34.81±1.206)%/7.750±0.446、(57.67±2.591)%/7.144±0.424。Ad组：(52.27±1.051)%/8.273±0.359、(36.31±2.210)%/8.284±0.264、(59.33±2.951)%/7.744±0.398。HO组：(43.83±1.15)%/6.217±0.313、(27.31±1.382)%／6.428±0.342、(48.94±2.12)%/6.472±0.347。与SH组比较,V组、Ad组神经细胞凋亡率及Caspase-3活性均显著增高；与V组及Ad组比较,HO组神经细胞凋亡率及Caspase-3活性均显著降低(P<0.01)；Ad组与V组比较神经细胞凋亡率及Caspase-3活性均无明显差异。结论：通过AdEasy Sytem成功构建含有HO-1基因的腺病毒载体；通过zea—Longa线栓法建立稳定的MCAO模型；腺病毒携带的HO-1基因通过立体定向法脑室内转染能有效的转染脑组织,并在脑内稳定表达；Ad-HO-1对局灶性脑缺血再灌注大鼠脑组织有保护作用,其机制是外源性HO-1基因促使HO-1表达上调,高度表达的HO-1早期(2h)发挥抗凋亡作用并在晚期(24h)转化为具有临床意义的减少梗死的作用。更多还原

【Abstract】 Abstract:bjective:To observe the effects on cerebral ischemia-reperfusion(CIR) injury after transfection of heme oxygenase-1 gene in rat for discussing the therapy prospect of regulating the heme oxygenase gene to stroke in practice. Methods:Adenovirus Vector Production:HO-1 gene was cloned by using the reverse-transcription polymerase chain reaction (RT-PCR) method from the total RNA extracted from rat spleen cells.According to published GeneBank sequence of rat HO-1(J02722) the primer sequence was designed. Adenoviral vectors carrying a rat HO-1 gene were constructed by Adenovirus-bacterial recombination system (AdEasy).The viral titers of Ad-Ho-1 and Ad-GFP were 2.5×1011 pfu/ml and 2.0×1011 pfu/ml.Male Sprague Dawley (SD) rats (n=78) were randomly divided into four groups: sham group (SH),vehicle group (V), empty adenovirus vector group (Ad) and Ad-HO-1 transfection group(18 each group).Rats of V, Ad and Ad-HO-1 groups were respectively injected with 20μl saline,20μl saline containing 1μl of empty vector adenovirus or 1μl of recombinant HO-1 adenovirus through the right ventricle for three days before ischemia. Injection site was reference to Paxinos and Watson rat brain stereotactic atlas.Then,cerebral ischemia-reperfusion was established by middle cerebral artery occlusion (MCAO) according to a method Zea—Longa described.Under halothane anesthesia (induction 2%, maintenance:2% in an oxygen and nitrous oxide 30:70 mixture), a nylon monofilament was introduced into internal carotid artery to occlude the middle cerebral artery. Two hours later, the filament was withdrawn and the wound was sutured.Rectal temperature was maintained at physiological levels (36.5-37.5℃) using a thermal blanket. Regional cerebral blood flow (rCBF) was measured using a laser Doppler flow meter probe.Neurological deficits were evaluated at 24 hours after ischemia using the scoring system reported by Garcia. Then the brain tissue was removed and was sectioned into slices, infarct volume was evaluated with 2,3,5-triphenyltet-razolium hydrochloride (TTC) staining.Infarct volumes were expressed as percentage of the contralateral hemisphere.Two hours after ischemia, brain tissue was homogenized.The homogenates were using to detect neuronal caspase-3 activity.24h after ischemia, brain tissue sections were using to detect apoptotic cells by TUNEL stain, GFP expression was observed by fluorescence microscope, and calculated the transfection rate.The expressions of HO-1 in brain tissue were detected by Western blot. Results:Western blot show the change of heme oxygenase-1 level after transfection:In HO gene treated rats, heme oxygenase-1 expression was significantly increased compared with that in vehicle and Ad groups.The HO-1 expression in Ad group was no difference than those in and V group.Expression of green fluorescent protein show transfection rate:Fluorescent protein expression was seen in brain tissue in both Ad group and HO group with no significant difference,and the transfection rate was 34.5%±3.4%. The neurological behavior score:The neurological behavior score among 4 groups:18、9.571±0.254、9.857±0.149、12.75±0.211.The neurological behavior score of HO group were significantly higher than those in Ad group and V group (P<0.001).It is no significant difference between Ad group and V group.Infarction volume and neuronal apoptosis:a. Percentage of infarction volume among 4 groups:0%、60.36±6.750%、61.80±7.544%、27.31±2.913%.Compared with the SH group, volume of cerebral infarction in V group and Ad group increased. Compared with V group or Ad group, these indexes in HO group significant reduced (P< 0.01).Compared with V group or Ad group, these indexes in HO group significant reduced (P<0.01).Those indexes were no significant difference between Ad group and V group.b.Neuronal apoptosis:Rate of apoptosis positive staining cell and Caspase-3 activity in caudate putamen,CAl of hippocampal and cortex among 4 groups:SH group:<0.1%/0.800±0.170, <0.1%/0.441±0.193,<0.1%/0.674±0.186.V group:(53.39±1.742)%/8.889±0.352,(34.81±1.206)%/7.750±0.446,(57.67±2.591)%/7.144±0.424.Ad group: (52.27±1.051)%/8.273±0.359,(36.31±2.210)%/8.284±0.264,(59.33±2.951)%/ 7.744±0.398。HO group:(46.55±1.001)%/6.217±0.313,(27.31±1.382)%/ 6.428±0.342, (48.94±2.120)%/6.472±0.347.Compared with the SH group, the. neuronal apoptosis in V group and Ad group increased.Compared with V group or Ad group,these indexes in HO group significant reduced (P<0.01). Compared with V group or Ad group, these indexes in HO group significant reduced (P<0.01).Those indexes were no significant difference between Ad group and V group.Conclusion:Adenoviral vectors carrying a rat HO-1 gene were constructed by AdEasy Vector System; the stabled animal models were established by transient occlusion of MCA according to a method Zea—Longa described. Brain stereotactic intraventricular injection of HO-1 gene carried by adenovirus can be effectively transfected into the brain tissue and express stably in vivo.Ad-HO-lhas neuroprotective effect against cerebral ischemic reperfusion in rats.The neuroprotective role of them to CIR may contribute to the increased expression of HO-1 protein. High expression of HO-1 can play an early (2h after reperfusion)anti-apoptosis effect may translate into clinically meaningful infarct reduction at a later time point (24h after reperfusion).更多还原