摘要
目的:构建携带缺氧诱导因子-1α(HIF-1α)基因的重组腺病毒载体,探索HIF-1α在成年大鼠局灶性脑梗死中的治疗作用及可能机制.方法:应用腺病毒表达系统AdEasy System构建携带缺氧诱导因子-1α和绿色荧光蛋白(GFP)的重组腺病毒(Ad-HIF-1α)并行PCR鉴定.建立大鼠线栓法大脑中动脉缺血再灌注模型,分为Ad-HIF-1α、腺病毒空载体(Ad)、生理盐水(NS)三组;将Ad-HIF-1α、Ad和NS分别注射到模型鼠梗死侧侧脑室.通过大体脑解剖和Nissl染色检测模型是否成功,原位杂交检测脑组织HIF-1αmRNA、VEGFm-RNA、SDF-1αmRNA的表达差异,三组大鼠神经功能缺失评分和2,3,5-三苯基氯化四氮唑(TTC)染色评价Ad-HIF-1α对脑缺血的治疗效果.结果:大体脑解剖标本和Nissl染色均证明大脑中动脉缺血再灌注模型成功建立.经氯化铯梯度离心法纯化后Ad-HIF-1α的滴度为8.2×108pfu/ml,PCR鉴定表明Ad-HIF-1α构建成功.三组大鼠脑梗死后HIF-1αmRNA、VEGFmRNA和SDF-1αmRNA的表达均有明显增加,且均于72h达最高峰.Ad-HIF-1α组HIF-1αmRNA、VEGFmRNA和SDF-1αmRNA在72h的表达与Ad和NS组比较差异有统计学意义(P<0.05),而Ad和NS组比较则差异无统计学意义(P>0.05).Ad-HIF-1α治疗组72 hAd-HIF-1α治疗组神经功能缺失评分分别为1.6±0.7和0.9±0.6,与Ad组(分别为2.9±0.6和3.2±0.6)和NS组(分别为3.0±0.7和3.2±0.8)比较差异均有统计学意义(P<0.05).72 h时脑组织TTC染色显示Ad-HIF-1α治疗组梗死体积为81.2 mm3±1.4 mm3,与Ad组(173.9 mm3±1.3 mm3)和NS组(171.7 mm3±6.2 mm3)比较差异有统计学意义(P<0.05).结论:HIF-1α基因在成年大鼠局灶性脑梗死中具有积极的治疗作用,其作用机制不仅是通过上调抗缺氧基因或脑保护基因如VEGF的表达使缺氧的组织细胞保持氧稳定及耐受低氧状态,而且还通过上调修复基因如SDF-1α的表达达到修复受损的神经组织和重建神经系统的作用.
Objective： To construct a recombinant adenoviral vector carrying HIF - 1α gene and explore the role and mechanism of HIF - 1 α on focal cerebral ischemia in adult rats. Methods ： The AdEasy System was used to construct the recombinant adenoviral vector carrying HIT - 1 α gene and green fluorescent protein, and PCR was used, to identify the HIF -1α gene. Middle cerebral artery occlusion（MCAo） and reperfusion models were established and divided into Ad - HIF - 1α group, Ad group and NS group. After Ad - HIF - 1α,Ad and NS were injected into the ischemic ventricle, in situ hybridization were used to examine the expression of HIF - 1αmRNA, VEGFmRNA and SDF - 1αmRNA, and therapeutic effect of HIF -1α was evaluated by neurological severity scores and 2,3,5 - triphenyltetrasolium chloride（TTC） staining. Results ： The titre of Ad - HIF - 1αwas 8.2 ×10^8 pfu/ml by cesium chloride gradient centrifugation and the recombinant adenoviral vector carrying HIF - 1α gene was constructed successfully by PCR identification. The expression of HIF-1αmRNA, VEGFmRNA and SDF-1αmRNA reached peak at 72h and then fall off. There was no statistical significance between the expression of HIF - 1αmRNA, VEGFmRNA and SDF - 1αmRNA in Ad - HIT - 1α group, Ad group and NS group （P 〉 0.05 ）. The neurological severity scores was 0.9 ± 0.6 at 72 h in Ad- HIF- 1α group, and there was statistical significance compared with Ad group（3.2 ±0.6）and NS group（3.2 ± 0. 8） （P 〈0.05 ）. The infarct volume was 81.2 mm^3 ± 1.4 mm^3 at 72 h in Ad - HIF - 1α group and there was statistical significance compared with Ad group（173.9 mm^3 ± 1.3 mm^3） and NS group（171.7 mm^3 ±6.2 mm^3） （P〈0.05）. Conclusion ： HIF -1α gene had definite therapeutic effect on focal cerebral ischemia in adult rats not only through up - regulating the expression of anti - hypoxia gene or brain protection gene such as VEGF but also through up - regulating the expression of reparation gene such as SDF - 1α.
出处
《贵州科学》
2007年第B05期377-384,共8页
Guizhou Science
