重组腺相关病毒载体介导的人类缺氧诱导因子-1α抑制Aβ神经毒性作用的实验研究
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摘要
基因治疗是二十世纪八十年代以后发展起来的一种疾病治疗模式,其定义是指将人的正常基因或有治疗作用的基因通过一定方式导入人体靶细胞以纠正基因的缺陷或者发挥治疗作用,从而达到治疗疾病目的的生物医学高新技术。近年来,随着基因治疗技术日趋成熟,基因治疗范围已从遗传病扩展到非遗传病领域,例如肿瘤,传染病,免疫缺陷病,神经系统疾病等,尤其阿尔茨海默病(Alzheimer’s disease, AD)的基因治疗目前倍受学者关注,并且已获得初步成效。其中选择适当的病毒载体将具有治疗价值的基因导入神经细胞并使其有效表达是AD基因治疗研究的关键所在。
腺相关病毒(adeno-associated virus, AAV),也称腺伴随病毒,属于微小病毒科依赖病毒属,是目前发现的一类结构最简单的单链DNA缺陷型病毒。重组腺相关病毒(recombinant adeno-associated virus, rAAV)载体源于非致病的野生型腺相关病毒,安全性好,对人体无致病性,宿主范围广泛,免疫原性小,可通过将外源基因整合到宿主细胞染色体和/或转变成稳定的染色体外附加体形式介导外源基因的长期表达,是基因治疗研究中应用最为广泛的载体系统之一。
缺氧诱导因子-1α(hypoxia inducible factor-1α, HIF-1α)是一种在缺氧应答过程中起重要作用的核转录因子,广泛参与缺氧诱导的细胞适应性反应,已成为缺氧应答时基因表达的调节中心。缺氧时HIF-1α生成增加,通过调控糖酵解酶、促红细胞生成素、血管内皮生长因子和一些涉及细胞生存蛋白质等的表达,调节能量代谢,增加毛细血管网密度,改善组织血流供应,使细胞产生适应性反应,从而恢复机体内环境稳态。最近研究证实HIF-1α能够减弱淀粉样蛋白(βamyloid peptide, Aβ)对神经细胞毒性损伤作用;防止线粒体不可逆抑制剂3-硝基丙酸诱导C6神经胶质瘤细胞损伤以及氧化应激引起的皮层神经细胞凋亡,提示HIF-1α有可能成为治疗AD等神经系统疾病的一个新靶点。
基于以上分析,本研究选用AAV-2为基因载体,人类HIF-1αcDNA为治疗基因,构建重组人类缺氧诱导因子-1α腺相关病毒载体(rAAV-HIF- 1α),深入探讨rAAV-HIF-1α对Aβ25-35诱导原代培养海马神经细胞凋亡及AD动物模型影响的内在机制,为今后应用rAAV-HIF-1α进行AD基因治疗奠定基础。
1 pSNAV-HIF-1α的构建及其转染对Aβ25-35诱导原代培养海马神经细胞凋亡的影响
1.1 pSNAV-HIF-1α的构建及表达
目的:构建携带人类HIF-1α基因腺相关病毒载体穿梭质粒pSNAV- HIF-1α,转染原代培养的海马神经细胞检测HIF-1α蛋白表达。
方法:采用限制性内切酶KpnI、BamHI双酶切pBSKhHIF1αT7质粒获取人类HIF-1αcDNA全长,插入至pSNAV2.0质粒KpnI、BglⅡ位点,获得携带人类HIF-1α基因的腺相关病毒载体穿梭质粒pSNAV-HIF-1α。实验分为3组:normal组:原代培养的海马神经细胞未作任何特殊处理;HIF-1α组:原代培养的海马神经细胞转染pSNAV-HIF-1α(磷酸钙共沉淀法);vector组:原代培养的海马神经细胞转染pSNAV 2.0(磷酸钙共沉淀法)。转染3d后提取细胞核蛋白,Western blot检测HIF-1α蛋白表达。
结果:①酶切、PCR技术证实成功构建了pSNAV-HIF-1α;②Western blot检测结果表明,转染3d后HIF-1α组(2176.04±110.56)细胞HIF-1α蛋白的表达量较normal组(812.96±54.27)、vector组(859.43±72.33)显著升高(P<0.05),而normal组、vector组细胞相比HIF-1α蛋白表达无显著性差异。
结论:成功构建了携带人类HIF-1α基因腺相关病毒载体穿梭质粒pSNAV-HIF-1α,转染原代培养海马神经细胞能够高效表达HIF-1α蛋白。1.2 pSNAV-HIF-1α转染对Aβ25-35诱导原代培养海马神经细胞凋亡的影响
目的:观察pSNAV-HIF-1α转染对Aβ25-35诱导海马神经细胞凋亡的影响
方法:实验分为5组:normal组:原代培养的海马神经细胞未作任何特殊处理;normal+Aβ组:以终浓度为10μmol/L Aβ25-35处理原代培养海马神经细胞24h;vector组:原代培养的海马神经细胞转染pSNAV 2.0;vector+Aβ组:pSNAV 2.0转染3d后以终浓度为10μmol/L Aβ25-35处理24h;HIF-1α+Aβ组:pSNAV-HIF-1α转染3d后以终浓度为10μmol/L Aβ25-35处理24h。采用流式细胞术检测海马神经细胞凋亡百分率。
结果:流式细胞术检测结果显示,normal+Aβ组(31.25±5.14%)、vector+Aβ组(30.48±4.39%)细胞凋亡百分率较normal组(5.27±1.06%)、vector组(6.35±1.34%)显著升高(P<0.05),而HIF-1α+Aβ组(12.39±2.56%)细胞凋亡百分率较normal+Aβ组、vector+Aβ组显著降低(P<0.05),normal+Aβ组与vector+Aβ组细胞凋亡百分率相比无显著性差异,这表明pSNAV-HIF-1α转染能够抑制Aβ25-35诱导海马神经细胞凋亡。
结论:pSNAV-HIF-1α转染能够抑制Aβ25-35诱导海马神经细胞凋亡。
2重组人类缺氧诱导因子-1α腺相关病毒载体的构建及表达
目的:构建重组人类缺氧诱导因子-1α腺相关病毒载体(rAAV-HIF-1α),转染原代培养海马神经细胞检测HIF-1α蛋白表达。
方法:采用Lipofectamine 2000将pSNAV-HIF-1α转染BHK-21细胞,再经G418筛选、挑选G418抗性细胞克隆,得到的混合细胞株命名为BHK/pSNAV-HIF-1α,即AAV载体细胞株。用具有AAV复制和包装功能的重组1型单纯疱疹病毒HSV1-rc/?UL2感染此细胞株,经氯仿处理-PEG/NaCl沉淀-氯仿抽提技术粗纯化AAV病毒,透析袋浓缩法进一步浓缩AAV病毒,即得到用于细胞实验的rAAV-HIF-1α。采用PCR技术鉴定病毒。采用地高辛标记的CMV探针点杂交方法检测病毒液中rAAV-HIF-1α的物理滴度。采用凝胶扫描图象分析系统,分析rAAV-HIF-1α样品经10%的SDS-PAGE电泳后所得蛋白条带测定病毒纯度。实验分为2组:normal组:原代培养的海马神经细胞未作任何特殊处理;rAAV-HIF-1α组:原代培养的海马神经细胞转染rAAV-HIF-1α;于转染3d后提取细胞总蛋白,Western blot检测HIF-1α蛋白表达。
结果:①PCR结果证实rAAV-HIF-1α内含有人类HIF-1α基因序列;②点杂交方法检测rAAV-HIF-1α的物理滴度为1×1012v.g./ml;③纯化后的rAAV-HIF-1α经10%SDS-PAGE电泳分离后,AAV病毒颗粒VP1、VP2、VP3三条特征性条带清晰,无其他杂带,大约符合1:1:10的比例,经计算机扫描分析后显示rAAV-HIF-1α纯度大于98%;④Western blot检测结果显示,转染3d后rAAV-HIF-1α组(727.57±36.08)细胞HIF-1α蛋白表达较normal组(328.69±23.71)显著升高(P<0.05)。
结论:成功构建了高滴度、高纯度的重组人类缺氧诱导因子-1α腺相关病毒载体rAAV-HIF-1α,转染原代培养海马神经细胞能够高效表达HIF-1α蛋白,为进一步研究rAAV-HIF-1α神经保护机制奠定基础。
3 rAAV-HIF-1α转染对Aβ25-35诱导原代培养海马神经细胞凋亡的影响及机制初探
目的:应用rAAV-HIF-1α体外转染原代培养海马神经细胞,观察其对Aβ25-35诱导原代培养海马神经细胞凋亡的影响。
方法:实验分为以下3组:normal组:未作任何特殊处理的原代培养海马神经细胞;normal+Aβ组:以终浓度为10μmol/L Aβ25-35处理原代培养海马神经细胞24h;rAAV-HIF-1α+Aβ组:rAAV-HIF-1α转染3d后以终浓度为10μmol/L Aβ25-35处理24h。采用透射电镜技术观察海马神经细胞凋亡形态学改变及流式细胞术检测海马神经细胞凋亡百分率。采用激光共聚焦扫描技术检测海马神经细胞钙离子浓度。
结果:①透射电镜结果显示,normal组海马神经细胞膜边缘清晰、光滑,细胞质结构清晰,细胞染色质均匀;normal+Aβ组海马神经细胞出现皱缩变形;染色质浓缩成块、边集,电子密度升高,厚薄不均,沿核膜新月状排列,部分核周隙扩张等凋亡的特征性形态学改变。而rAAV-HIF-1α+Aβ组海马神经细胞仅出现粗面内质网脱颗粒;线粒体嵴减少、排列紊乱;部分核膜向外锐角突起等现象,这表明Aβ25-35能够引起海马神经细胞凋亡,表现为细胞超微结构发生改变,rAAV-HIF-1α预转染能够减轻这种改变;②流式细胞术结果显示,normal+Aβ组(24.26±3.99%)海马神经细胞凋亡百分率与normal组(4.99±0.83%)相比显著升高(P<0.05),而rAAV-HIF-1α+Aβ组(14.29±1.88%)海马神经细胞凋亡百分率较normal+Aβ组显著降低(P<0.05),这表明Aβ25-35能够引起海马神经细胞凋亡,预转染rAAV-HIF-1α海马神经细胞能够减弱Aβ25-35诱导凋亡的作用;③以Fluo-3/AM荧光染色,在激光共聚焦显微镜下检测显示,normal+Aβ组(197.98±27.60)海马神经细胞钙离子浓度与normal组(61.48±12.61)相比显著升高(P<0.05),而rAAV-HIF-1α+Aβ组(145.66±15.31)海马神经细胞钙离子浓度较normal+Aβ组显著降低(P<0.05),这表明Aβ25-35能够引起海马神经细胞内钙离子浓度升高,预转染rAAV-HIF-1α能够抑制Aβ25-35诱导细胞内钙离子浓度升高。
结论:rAAV-HIF-1α转染能够抑制Aβ25-35诱导海马神经细胞凋亡,其机制可能与HIF-1α降低细胞内游离钙浓度,从而抑制凋亡级联反应有关。
4侧脑室注射rAAV-HIF-1α基因治疗AD模型鼠的实验研究
目的:在体观察侧脑室注射rAAV-HIF-1α对AD模型大鼠海马神经细胞凋亡的影响
方法:健康雄性SD大鼠32只,体重250-300g,随机分为4组:normal组(n=8):未作任何特殊处理;AD组(n=8):右侧脑室(AP:-0.8mm, ML:1.5 mm, DV:4.0mm)注射2μl Aβ25-35(10mg/ml);sham组(n=8):右侧脑室注射2μl生理盐水;AD+rAAV-HIF-1α组(n=8):右侧脑室注射2μl Aβ25-35后1周右侧脑室注射10μl rAAV-HIF-1α(1×1012v.g./ml)。以上各组动物于注射Aβ25-35或生理盐水后5周取材检测海马神经细胞HIF-1α蛋白表达及凋亡百分率。
结果:①Western blot检测结果显示,AD+rAAV-HIF-1α组(451.59±34.39)海马神经细胞HIF-1α蛋白表达较normal组(229.05±41.28)、sham组(216.29±37.08)明显增强(P<0.05),而normal组与sham组相比无显著性差异,这表明侧脑室注射rAAV-HIF-1α能够转染AD模型大鼠海马神经细胞并持续稳定表达HIF-1α蛋白;②流式细胞术结果显示,AD组(19.49±2.59%)海马神经细胞凋亡百分率较normal组(5.41±0.75%)、sham组(5.28±0.66%)显著升高(P<0.05),而normal组与sham组相比无显著性差异;而AD+rAAV-HIF-1α组(12.07±2.06%)细胞凋亡百分率较AD组显著降低(P<0.05),这表明侧脑室注射rAAV-HIF-1α能够抑制AD模型大鼠海马神经细胞发生凋亡。
结论:侧脑室注射rAAV-HIF-1α能够抑制AD模型大鼠海马神经细胞凋亡。
5结语
本研究成功构建了重组人类缺氧诱导因子-1α腺相关病毒载体rAAV- HIF-1α并证实其能够减弱Aβ神经细胞毒性作用,为今后应用rAAV-HIF- 1α进行AD基因治疗奠定了基础。
Gene therapy, a kind of treatment mode for diseases developed since 1980s, is to introduce human normal genes or therapeutic genes into human target cells to remedy the defective genes or play the role in therapy. Thereby, we can achieve the goal of making use of the high technique to treat diseases. The field of gene therapy has extended from hereditary diseases to tumor, contagious diseases, immunologic deficiency diseases, nervous system diseases and so on along with the improvement of gene therapy technique in the past few years. Recently much attention has been focused on the gene therapy for Alzheimer’s disease(AD). The critical step for a successful gene therapy for AD is to select an appropriate viral vector and deliver efficacious therapeutic genes into neurons to make them expressed sufficiently.
Adeno-associated virus(AAV), which belongs to the dependovirus of parvoviridae, is a species of nonpathogenic defective single strand DNA virus. At present recombinant adeno-associated virus(rAAV), reconstructed from nonpathogenic wild type AAV, is one of the most widely applicable carrier systems with the features of safety, wide host range, weak immunogenicity. And it can persistently express exogenous gene by means of integration into host cells chromosome or/and changing into stable extrachromosome episome.
Hypoxia inducible factor-1α(HIF-1α) is a kind of important nuclear transcription factor during the process of hypoxia, which participate widely in the hypoxia-induced cellular adaptation reaction to recover cells homeostasis. HIF-1αcould regulate energy metabolism, increase the density of capillary network, and improve the blood circulation of living tissue by means of regulating the expression of glycolytic enzyme, erythropoietin, vascular endothelial growth factor and so on, which make cells be capable of resisting the hypoxia-induced injury. Recent studies confirmed that HIF-1αcould attenuate the neurotoxicity ofβamyloid peptide(Aβ), inhibit the insult of C6 glioma cells induced by 3-nitropropionic acid and the apoptosis of cortical neurons induced by oxidative stress, which indicated that HIF-1αmight be used to treat nervous system disease such as AD.
Based on the above analysis, we constructed the recombinant AAV vector containing human HIF-1αgene(rAAV-HIF-1α), and observed the effect of rAAV-HIF-1αon the apoptosis of primary cultural hippocampal neurons and AD animal model, which made it possible to apply rAAV-HIF-1αinto gene therapy for AD in the future.
1 The construction of pSNAV-HIF-1αand the effect of its transfection on the apoptosis of primary cultural hippocampal neurons induced by Aβ25-35
1.1 The construction and expression of pSNAV-HIF-1α
Objective: To construct the AAV vector shuttle plasmid containing human HIF-1αgene(pSNAV-HIF-1α), and to detect the expression of HIF-1αprotein in pSNAV-HIF-1αtransfected primary cultural hippocampal neurons.
Methods: The HIF-1αgene, acquired from pBSKhHIF1αT7 digested by restricted endonuclease enzyme KpnI and BamHI, was inserted into pSNAV2.0 digested by restricted endonuclease enzyme KpnI、BglⅡto obtain the AAV vector shuttle plasmid containing human HIF-1αgene(pSNAV-HIF -1α). The constructed vector was transfected into primary cultural hippocampal neurons using calcium phosphate precipitation. There were three groups in this experiment: normal group: primary cultural hippocampal neurons without any special treatment; HIF-1αgroup: pSNAV-HIF-1αtransfected primary cultural hippocampal neurons; vector group: pSNAV2.0 transfected primary cultural hippocampal neurons. Nuclear protein was extracted from hippocampal neurons three days posttransfection to detect the expression of HIF-1αby western blot.
Results:①Restricted endonuclease enzyme digestion and PCR confirmed that pSNAV-HIF-1αwas successfully constructed;②Western blot showed that the expression of HIF-1αin HIF-1αgroup(2176.04±110.56) was significantly higher than that in normal group(812.96±54.27) and vector group(859.43±72.33)(P<0.05), and the expression of HIF-1αbetween normal group and vector group was not significantly different.
Conclusions: pSNAV-HIF-1αwas successfully constructed and it could express HIF-1αprotein efficiently in primary cultural hippocampal neurons.
1.2 The effect of transfection of pSNAV-HIF-1αon the apoptosis of primary cultural hippocampal neurons induced by Aβ25-35
Objective: To observe the effect of transfection of pSNAV-HIF-1αon the apoptosis of primary cultural hippocampal neurons induced by Aβ25-35.
Methods: There were five groups in this experiment: normal group: primary cultural hippocampal neurons without any special treatment; normal+Aβgroup: primary cultural hippocampal neurons were interfered with 10μmol/L Aβ25-35 for 24h; vector group: pSNAV2.0 transfected primary cultural hippocampal neurons; vector+Aβgroup: three days posttransfection, the pSNAV2.0 transfected primary cultural hippocampal neurons were interfered with 10μmol/L Aβ25-35 for 24h; HIF-1α+Aβgroup: three days posttransfection, the pSNAV-HIF-1αtransfected primary cultural hippocampal neurons were interfered with 10μmol/L Aβ25-35 for 24h. Apoptosis was detected by flow cytometry analysis.
Results: Flow cytometry analysis showed that the apoptosis ratio of hippocampal neurons in normal+Aβgroup(31.25±5.14%) and vector+Aβgroup(30.48±4.39%) were significantly higher than that in normal group (5.27±1.06%) and vector group(6.35±1.34 %)(P<0.05), however the apoptosis ratio of hippocampal neurons in HIF-1α+Aβgroup(12.39±2.56%) was significantly lower than that of normal+Aβgroup and vector+Aβgroup(P<0.05), and the apoptosis ratio of hippocampal neurons between normal+Aβgroup and vector+Aβgroup was not significantly different, which indicated that the transfection of pSNAV-HIF-1could inhibit the apoptosis of hippocampal neurons induced by Aβ25-35.
Conclusions: The transfection of pSNAV-HIF-1αcould inhibit the apoptosis of hippocampal neurons induced by Aβ25-35.
2 Construction and expression of recombinant adeno-associated virus vector containing human hypoxia inducible factor-1αgene
Objective: To Construct recombinant adeno-associated virus vector containing human hypoxia inducible factor-1αgene(rAAV-HIF-1α), and to detect the expression of HIF-1αprotein in rAAV-HIF-1αtransfected primary cultural hippocampal neurons.
Methods: pSNAV-HIF-1αwas transfected into BHK-21 cells using Lipofectamine 2000 and selected by G418. The G418 resistant BHK-21 cells, BHK/pSNAV-HIF-1α, were obtained. The BHK/pSNAV-HIF-1αcells were infected with HSV1-rc/?UL2, which could express rep and cap genes of wild-type AAV. After chloroform treatment, PEG/NaCl precipitation, chloroform extraction and bag filter concentration, the rAAV-HIF-1αwith high titer and purity was achieved. The authenticity of the rAAV-HIF-1αwas confirmed by polymerase chain reaction. The titer of rAAV-HIF-1αwas detected by dot-blot with digoxin labelled cytomegalovirus probe.The purity of rAAV-HIF-1αwas detected by 10%SDS-PAGE. There were two groups in this experiment: normal group: primary cultural hippocampal neurons without any special treatment; rAAV-HIF-1αgroup: rAAV-HIF-1αtransfected primary cultural hippocampal neurons; Total protein was extracted from hippocampal neurons three days posttransfection to detect the expression of HIF-1αby western blot.
Results:①PCR demonstrated that human HIF-1αgene was included in rAAV-HIF-1α;②The titer of rAAV-HIF-1αdetected by dot-blot with digoxin labelled cytomegalovirus probe was 1×1012v.g./ml;③The purity of rAAV-HIF-1αdetected by 10%SDS-PAGE exceeded 98%;④Western blot showed that the expression of HIF-1αin rAAV-HIF-1αgroup(727.57±36.08) was significantly higher than that in normal group(328.69±23.71)(P<0.05).
Conclusions: rAAV-HIF-1αwith high titer and purity was successfully constructed and it could express HIF-1αprotein efficiently in primary cultural hippocampal neurons.
3 The effect of the transfection of rAAV-HIF-1αon the apoptosis of primary cultural hippocampal neurons induced by Aβ25-35
Objective: To observe the effect of the transfection of rAAV-HIF-1αon the apoptosis of primary cultural hippocampal neurons induced by Aβ25-35.
Methods: There were three groups in this experiment: normal group: primary cultural hippocampal neurons without any special treatment; normal+Aβgroup: primary cultural hippocampal neurons were interfered with 10μmol/L Aβ25-35 for 24h; rAAV-HIF-1α+Aβgroup: primary cultural hippocampal neurons transfected with rAAV-HIF-1αfor three days were interfered with 10μmol/L Aβ25-35 for 24h. Apoptosis was detected by transmission electron microscope and flow cytometry analysis. The intracellular calcium concentration of hippocampal neurons was determined by laser scanning confocal microscopy with Fluo-3/AM as the fluorescent dye.
Results:①Transmission electron microscope showed that normal group hippocampal neuronal membrane was smooth and glossy, cytoplasmic structure was clear, chromatin was well-distribute; normal+Aβgroup hippocampal neurons shrinkaged due to the breakdown of the proteinaceous cytoskeleton by caspases, chromatin underwent condensation into compact patches against the nuclear envelope in a process known as pyknosis; however rAAV-HIF-1α+Aβgroup hippocampal neurons only showed light morphology changes of apoptosis, such as nuclear envelope sprouted irregular buds known as blebs and so on, which indicated that the morphology change of hippocampal neuronal apoptosis induced by Aβ25-35 could be lessened by the transfection of rAAV-HIF-1αin advance;②Flow cytometry analysis showed that the apoptosis ratio of hippocampal neurons in normal+Aβgroup(24.26±3.99%) was significantly higher than that in normal group(4.99±0.83%)(P<0.05), however the apoptosis ratio of hippocampal neurons in rAAV-HIF-1α+Aβgroup(14.29±1.88%) was significantly lower than that in normal + Aβgroup(P<0.05), which indicated that rAAV-HIF-1αtransfected hippocampal neurons could inhibit the apoptosis induced by Aβ25-35;③Laser scanning confocal microscopy showed that the calcium concentration of hippocampal neurons in normal+Aβgroup(197.98±27.60) was significantly higher than that in normal group(61.48±12.61)(P<0.05), however the calcium concentration of hippocampal neurons in rAAV-HIF-1α+Aβgroup(145.66±15.31) was significantly lower than that in normal+Aβgroup(P<0.05), which indicated that the up-regulation of calcium concentration of hippocampal neurons induced by Aβ25-35 could be suppressed by the transfection of rAAV-HIF-1αin advance.
Conclusions: rAAV-HIF-1αtransfection could inhibit Aβ25-35 induced apoptosis of primary cultural hippocampal neurons by suppressing the up-regulation of intracellular calcium concentration.
4 The effect of intracerebroventricular(i.c.v) injection of rAAV-HIF-1αon the hippocampal neuronal apoptosis of AD animal model Objective: To observe the effect of i.c.v injection of rAAV-HIF-1αon hippocampal neuronal apoptosis of AD animal model.
Methods: Thirty-two male SD rats(250-300g) were divided randomly into four groups: normal group(n=8): healthy male animal without any special treatment; AD group(n=8): right i.c.v injection of 2μl Aβ25-35(10mg/ml); sham group(n=8): right i.c.v injection of 2μl NS; AD+rAAV-HIF-1αgroup(n=8): right i.c.v injection of 10μl rAAV-HIF-1α(1×10~(12)v.g./ml) one week after i.c.v injection of 2μl Aβ25-35(10mg/ml). The rats were sacrificed to detect the expression of HIF-1αand apoptosis of hippocampal neurons five weeks after i.c.v injection of Aβ25-35 or NS.
Results:①Western blot showed that the expression of HIF-1αin AD+rAAV-HIF-1αgroup(451.59±34.39) was significantly higher than that in normal group(229.05±41.28) and sham group(216.29±37.08)(P<0.05), and the expression of HIF-1αbetween normal group and sham group was not significantly different.②Flow cytometry analysis showed that the apoptosis ratio of hippocampal neurons in AD group(19.49±2.59%) was significantly higher than that in normal group(5.41±0.75%) and sham group(5.28±0.66%)(P<0.05), and the apoptosis ratio of hippocampal neurons between normal group and sham group was not significantly different; however the apoptosis ratio of hippocampal neurons in AD+rAAV-HIF-1αgroup(12.07±2.06%) was significantly lower than that in AD group(P<0.05), which indicated that the hippocampal neuronal apoptosis of AD animal model could be attenuated by i.c.v injection of rAAV-HIF-1α.
Conclusions: The i.c.v injection of rAAV-HIF-1αcould inhibit the hippocampal neuronal apoptosis of AD animal model.
5 Conclusions
Recombinant AAV vector containing human HIF-1αgene(rAAV-HIF-1α) was successfully constructed and it could inhibit the neurotoxicity of Aβ25-35, which laid a foundation for further application of rAAV-HIF-1αinto gene therapy for AD in the future.
腺相关病毒(adeno-associated virus, AAV),也称腺伴随病毒,属于微小病毒科依赖病毒属,是目前发现的一类结构最简单的单链DNA缺陷型病毒。重组腺相关病毒(recombinant adeno-associated virus, rAAV)载体源于非致病的野生型腺相关病毒,安全性好,对人体无致病性,宿主范围广泛,免疫原性小,可通过将外源基因整合到宿主细胞染色体和/或转变成稳定的染色体外附加体形式介导外源基因的长期表达,是基因治疗研究中应用最为广泛的载体系统之一。
缺氧诱导因子-1α(hypoxia inducible factor-1α, HIF-1α)是一种在缺氧应答过程中起重要作用的核转录因子,广泛参与缺氧诱导的细胞适应性反应,已成为缺氧应答时基因表达的调节中心。缺氧时HIF-1α生成增加,通过调控糖酵解酶、促红细胞生成素、血管内皮生长因子和一些涉及细胞生存蛋白质等的表达,调节能量代谢,增加毛细血管网密度,改善组织血流供应,使细胞产生适应性反应,从而恢复机体内环境稳态。最近研究证实HIF-1α能够减弱淀粉样蛋白(βamyloid peptide, Aβ)对神经细胞毒性损伤作用;防止线粒体不可逆抑制剂3-硝基丙酸诱导C6神经胶质瘤细胞损伤以及氧化应激引起的皮层神经细胞凋亡,提示HIF-1α有可能成为治疗AD等神经系统疾病的一个新靶点。
基于以上分析,本研究选用AAV-2为基因载体,人类HIF-1αcDNA为治疗基因,构建重组人类缺氧诱导因子-1α腺相关病毒载体(rAAV-HIF- 1α),深入探讨rAAV-HIF-1α对Aβ25-35诱导原代培养海马神经细胞凋亡及AD动物模型影响的内在机制,为今后应用rAAV-HIF-1α进行AD基因治疗奠定基础。
1 pSNAV-HIF-1α的构建及其转染对Aβ25-35诱导原代培养海马神经细胞凋亡的影响
1.1 pSNAV-HIF-1α的构建及表达
目的:构建携带人类HIF-1α基因腺相关病毒载体穿梭质粒pSNAV- HIF-1α,转染原代培养的海马神经细胞检测HIF-1α蛋白表达。
方法:采用限制性内切酶KpnI、BamHI双酶切pBSKhHIF1αT7质粒获取人类HIF-1αcDNA全长,插入至pSNAV2.0质粒KpnI、BglⅡ位点,获得携带人类HIF-1α基因的腺相关病毒载体穿梭质粒pSNAV-HIF-1α。实验分为3组:normal组:原代培养的海马神经细胞未作任何特殊处理;HIF-1α组:原代培养的海马神经细胞转染pSNAV-HIF-1α(磷酸钙共沉淀法);vector组:原代培养的海马神经细胞转染pSNAV 2.0(磷酸钙共沉淀法)。转染3d后提取细胞核蛋白,Western blot检测HIF-1α蛋白表达。
结果:①酶切、PCR技术证实成功构建了pSNAV-HIF-1α;②Western blot检测结果表明,转染3d后HIF-1α组(2176.04±110.56)细胞HIF-1α蛋白的表达量较normal组(812.96±54.27)、vector组(859.43±72.33)显著升高(P<0.05),而normal组、vector组细胞相比HIF-1α蛋白表达无显著性差异。
结论:成功构建了携带人类HIF-1α基因腺相关病毒载体穿梭质粒pSNAV-HIF-1α,转染原代培养海马神经细胞能够高效表达HIF-1α蛋白。1.2 pSNAV-HIF-1α转染对Aβ25-35诱导原代培养海马神经细胞凋亡的影响
目的:观察pSNAV-HIF-1α转染对Aβ25-35诱导海马神经细胞凋亡的影响
方法:实验分为5组:normal组:原代培养的海马神经细胞未作任何特殊处理;normal+Aβ组:以终浓度为10μmol/L Aβ25-35处理原代培养海马神经细胞24h;vector组:原代培养的海马神经细胞转染pSNAV 2.0;vector+Aβ组:pSNAV 2.0转染3d后以终浓度为10μmol/L Aβ25-35处理24h;HIF-1α+Aβ组:pSNAV-HIF-1α转染3d后以终浓度为10μmol/L Aβ25-35处理24h。采用流式细胞术检测海马神经细胞凋亡百分率。
结果:流式细胞术检测结果显示,normal+Aβ组(31.25±5.14%)、vector+Aβ组(30.48±4.39%)细胞凋亡百分率较normal组(5.27±1.06%)、vector组(6.35±1.34%)显著升高(P<0.05),而HIF-1α+Aβ组(12.39±2.56%)细胞凋亡百分率较normal+Aβ组、vector+Aβ组显著降低(P<0.05),normal+Aβ组与vector+Aβ组细胞凋亡百分率相比无显著性差异,这表明pSNAV-HIF-1α转染能够抑制Aβ25-35诱导海马神经细胞凋亡。
结论:pSNAV-HIF-1α转染能够抑制Aβ25-35诱导海马神经细胞凋亡。
2重组人类缺氧诱导因子-1α腺相关病毒载体的构建及表达
目的:构建重组人类缺氧诱导因子-1α腺相关病毒载体(rAAV-HIF-1α),转染原代培养海马神经细胞检测HIF-1α蛋白表达。
方法:采用Lipofectamine 2000将pSNAV-HIF-1α转染BHK-21细胞,再经G418筛选、挑选G418抗性细胞克隆,得到的混合细胞株命名为BHK/pSNAV-HIF-1α,即AAV载体细胞株。用具有AAV复制和包装功能的重组1型单纯疱疹病毒HSV1-rc/?UL2感染此细胞株,经氯仿处理-PEG/NaCl沉淀-氯仿抽提技术粗纯化AAV病毒,透析袋浓缩法进一步浓缩AAV病毒,即得到用于细胞实验的rAAV-HIF-1α。采用PCR技术鉴定病毒。采用地高辛标记的CMV探针点杂交方法检测病毒液中rAAV-HIF-1α的物理滴度。采用凝胶扫描图象分析系统,分析rAAV-HIF-1α样品经10%的SDS-PAGE电泳后所得蛋白条带测定病毒纯度。实验分为2组:normal组:原代培养的海马神经细胞未作任何特殊处理;rAAV-HIF-1α组:原代培养的海马神经细胞转染rAAV-HIF-1α;于转染3d后提取细胞总蛋白,Western blot检测HIF-1α蛋白表达。
结果:①PCR结果证实rAAV-HIF-1α内含有人类HIF-1α基因序列;②点杂交方法检测rAAV-HIF-1α的物理滴度为1×1012v.g./ml;③纯化后的rAAV-HIF-1α经10%SDS-PAGE电泳分离后,AAV病毒颗粒VP1、VP2、VP3三条特征性条带清晰,无其他杂带,大约符合1:1:10的比例,经计算机扫描分析后显示rAAV-HIF-1α纯度大于98%;④Western blot检测结果显示,转染3d后rAAV-HIF-1α组(727.57±36.08)细胞HIF-1α蛋白表达较normal组(328.69±23.71)显著升高(P<0.05)。
结论:成功构建了高滴度、高纯度的重组人类缺氧诱导因子-1α腺相关病毒载体rAAV-HIF-1α,转染原代培养海马神经细胞能够高效表达HIF-1α蛋白,为进一步研究rAAV-HIF-1α神经保护机制奠定基础。
3 rAAV-HIF-1α转染对Aβ25-35诱导原代培养海马神经细胞凋亡的影响及机制初探
目的:应用rAAV-HIF-1α体外转染原代培养海马神经细胞,观察其对Aβ25-35诱导原代培养海马神经细胞凋亡的影响。
方法:实验分为以下3组:normal组:未作任何特殊处理的原代培养海马神经细胞;normal+Aβ组:以终浓度为10μmol/L Aβ25-35处理原代培养海马神经细胞24h;rAAV-HIF-1α+Aβ组:rAAV-HIF-1α转染3d后以终浓度为10μmol/L Aβ25-35处理24h。采用透射电镜技术观察海马神经细胞凋亡形态学改变及流式细胞术检测海马神经细胞凋亡百分率。采用激光共聚焦扫描技术检测海马神经细胞钙离子浓度。
结果:①透射电镜结果显示,normal组海马神经细胞膜边缘清晰、光滑,细胞质结构清晰,细胞染色质均匀;normal+Aβ组海马神经细胞出现皱缩变形;染色质浓缩成块、边集,电子密度升高,厚薄不均,沿核膜新月状排列,部分核周隙扩张等凋亡的特征性形态学改变。而rAAV-HIF-1α+Aβ组海马神经细胞仅出现粗面内质网脱颗粒;线粒体嵴减少、排列紊乱;部分核膜向外锐角突起等现象,这表明Aβ25-35能够引起海马神经细胞凋亡,表现为细胞超微结构发生改变,rAAV-HIF-1α预转染能够减轻这种改变;②流式细胞术结果显示,normal+Aβ组(24.26±3.99%)海马神经细胞凋亡百分率与normal组(4.99±0.83%)相比显著升高(P<0.05),而rAAV-HIF-1α+Aβ组(14.29±1.88%)海马神经细胞凋亡百分率较normal+Aβ组显著降低(P<0.05),这表明Aβ25-35能够引起海马神经细胞凋亡,预转染rAAV-HIF-1α海马神经细胞能够减弱Aβ25-35诱导凋亡的作用;③以Fluo-3/AM荧光染色,在激光共聚焦显微镜下检测显示,normal+Aβ组(197.98±27.60)海马神经细胞钙离子浓度与normal组(61.48±12.61)相比显著升高(P<0.05),而rAAV-HIF-1α+Aβ组(145.66±15.31)海马神经细胞钙离子浓度较normal+Aβ组显著降低(P<0.05),这表明Aβ25-35能够引起海马神经细胞内钙离子浓度升高,预转染rAAV-HIF-1α能够抑制Aβ25-35诱导细胞内钙离子浓度升高。
结论:rAAV-HIF-1α转染能够抑制Aβ25-35诱导海马神经细胞凋亡,其机制可能与HIF-1α降低细胞内游离钙浓度,从而抑制凋亡级联反应有关。
4侧脑室注射rAAV-HIF-1α基因治疗AD模型鼠的实验研究
目的:在体观察侧脑室注射rAAV-HIF-1α对AD模型大鼠海马神经细胞凋亡的影响
方法:健康雄性SD大鼠32只,体重250-300g,随机分为4组:normal组(n=8):未作任何特殊处理;AD组(n=8):右侧脑室(AP:-0.8mm, ML:1.5 mm, DV:4.0mm)注射2μl Aβ25-35(10mg/ml);sham组(n=8):右侧脑室注射2μl生理盐水;AD+rAAV-HIF-1α组(n=8):右侧脑室注射2μl Aβ25-35后1周右侧脑室注射10μl rAAV-HIF-1α(1×1012v.g./ml)。以上各组动物于注射Aβ25-35或生理盐水后5周取材检测海马神经细胞HIF-1α蛋白表达及凋亡百分率。
结果:①Western blot检测结果显示,AD+rAAV-HIF-1α组(451.59±34.39)海马神经细胞HIF-1α蛋白表达较normal组(229.05±41.28)、sham组(216.29±37.08)明显增强(P<0.05),而normal组与sham组相比无显著性差异,这表明侧脑室注射rAAV-HIF-1α能够转染AD模型大鼠海马神经细胞并持续稳定表达HIF-1α蛋白;②流式细胞术结果显示,AD组(19.49±2.59%)海马神经细胞凋亡百分率较normal组(5.41±0.75%)、sham组(5.28±0.66%)显著升高(P<0.05),而normal组与sham组相比无显著性差异;而AD+rAAV-HIF-1α组(12.07±2.06%)细胞凋亡百分率较AD组显著降低(P<0.05),这表明侧脑室注射rAAV-HIF-1α能够抑制AD模型大鼠海马神经细胞发生凋亡。
结论:侧脑室注射rAAV-HIF-1α能够抑制AD模型大鼠海马神经细胞凋亡。
5结语
本研究成功构建了重组人类缺氧诱导因子-1α腺相关病毒载体rAAV- HIF-1α并证实其能够减弱Aβ神经细胞毒性作用,为今后应用rAAV-HIF- 1α进行AD基因治疗奠定了基础。
Gene therapy, a kind of treatment mode for diseases developed since 1980s, is to introduce human normal genes or therapeutic genes into human target cells to remedy the defective genes or play the role in therapy. Thereby, we can achieve the goal of making use of the high technique to treat diseases. The field of gene therapy has extended from hereditary diseases to tumor, contagious diseases, immunologic deficiency diseases, nervous system diseases and so on along with the improvement of gene therapy technique in the past few years. Recently much attention has been focused on the gene therapy for Alzheimer’s disease(AD). The critical step for a successful gene therapy for AD is to select an appropriate viral vector and deliver efficacious therapeutic genes into neurons to make them expressed sufficiently.
Adeno-associated virus(AAV), which belongs to the dependovirus of parvoviridae, is a species of nonpathogenic defective single strand DNA virus. At present recombinant adeno-associated virus(rAAV), reconstructed from nonpathogenic wild type AAV, is one of the most widely applicable carrier systems with the features of safety, wide host range, weak immunogenicity. And it can persistently express exogenous gene by means of integration into host cells chromosome or/and changing into stable extrachromosome episome.
Hypoxia inducible factor-1α(HIF-1α) is a kind of important nuclear transcription factor during the process of hypoxia, which participate widely in the hypoxia-induced cellular adaptation reaction to recover cells homeostasis. HIF-1αcould regulate energy metabolism, increase the density of capillary network, and improve the blood circulation of living tissue by means of regulating the expression of glycolytic enzyme, erythropoietin, vascular endothelial growth factor and so on, which make cells be capable of resisting the hypoxia-induced injury. Recent studies confirmed that HIF-1αcould attenuate the neurotoxicity ofβamyloid peptide(Aβ), inhibit the insult of C6 glioma cells induced by 3-nitropropionic acid and the apoptosis of cortical neurons induced by oxidative stress, which indicated that HIF-1αmight be used to treat nervous system disease such as AD.
Based on the above analysis, we constructed the recombinant AAV vector containing human HIF-1αgene(rAAV-HIF-1α), and observed the effect of rAAV-HIF-1αon the apoptosis of primary cultural hippocampal neurons and AD animal model, which made it possible to apply rAAV-HIF-1αinto gene therapy for AD in the future.
1 The construction of pSNAV-HIF-1αand the effect of its transfection on the apoptosis of primary cultural hippocampal neurons induced by Aβ25-35
1.1 The construction and expression of pSNAV-HIF-1α
Objective: To construct the AAV vector shuttle plasmid containing human HIF-1αgene(pSNAV-HIF-1α), and to detect the expression of HIF-1αprotein in pSNAV-HIF-1αtransfected primary cultural hippocampal neurons.
Methods: The HIF-1αgene, acquired from pBSKhHIF1αT7 digested by restricted endonuclease enzyme KpnI and BamHI, was inserted into pSNAV2.0 digested by restricted endonuclease enzyme KpnI、BglⅡto obtain the AAV vector shuttle plasmid containing human HIF-1αgene(pSNAV-HIF -1α). The constructed vector was transfected into primary cultural hippocampal neurons using calcium phosphate precipitation. There were three groups in this experiment: normal group: primary cultural hippocampal neurons without any special treatment; HIF-1αgroup: pSNAV-HIF-1αtransfected primary cultural hippocampal neurons; vector group: pSNAV2.0 transfected primary cultural hippocampal neurons. Nuclear protein was extracted from hippocampal neurons three days posttransfection to detect the expression of HIF-1αby western blot.
Results:①Restricted endonuclease enzyme digestion and PCR confirmed that pSNAV-HIF-1αwas successfully constructed;②Western blot showed that the expression of HIF-1αin HIF-1αgroup(2176.04±110.56) was significantly higher than that in normal group(812.96±54.27) and vector group(859.43±72.33)(P<0.05), and the expression of HIF-1αbetween normal group and vector group was not significantly different.
Conclusions: pSNAV-HIF-1αwas successfully constructed and it could express HIF-1αprotein efficiently in primary cultural hippocampal neurons.
1.2 The effect of transfection of pSNAV-HIF-1αon the apoptosis of primary cultural hippocampal neurons induced by Aβ25-35
Objective: To observe the effect of transfection of pSNAV-HIF-1αon the apoptosis of primary cultural hippocampal neurons induced by Aβ25-35.
Methods: There were five groups in this experiment: normal group: primary cultural hippocampal neurons without any special treatment; normal+Aβgroup: primary cultural hippocampal neurons were interfered with 10μmol/L Aβ25-35 for 24h; vector group: pSNAV2.0 transfected primary cultural hippocampal neurons; vector+Aβgroup: three days posttransfection, the pSNAV2.0 transfected primary cultural hippocampal neurons were interfered with 10μmol/L Aβ25-35 for 24h; HIF-1α+Aβgroup: three days posttransfection, the pSNAV-HIF-1αtransfected primary cultural hippocampal neurons were interfered with 10μmol/L Aβ25-35 for 24h. Apoptosis was detected by flow cytometry analysis.
Results: Flow cytometry analysis showed that the apoptosis ratio of hippocampal neurons in normal+Aβgroup(31.25±5.14%) and vector+Aβgroup(30.48±4.39%) were significantly higher than that in normal group (5.27±1.06%) and vector group(6.35±1.34 %)(P<0.05), however the apoptosis ratio of hippocampal neurons in HIF-1α+Aβgroup(12.39±2.56%) was significantly lower than that of normal+Aβgroup and vector+Aβgroup(P<0.05), and the apoptosis ratio of hippocampal neurons between normal+Aβgroup and vector+Aβgroup was not significantly different, which indicated that the transfection of pSNAV-HIF-1could inhibit the apoptosis of hippocampal neurons induced by Aβ25-35.
Conclusions: The transfection of pSNAV-HIF-1αcould inhibit the apoptosis of hippocampal neurons induced by Aβ25-35.
2 Construction and expression of recombinant adeno-associated virus vector containing human hypoxia inducible factor-1αgene
Objective: To Construct recombinant adeno-associated virus vector containing human hypoxia inducible factor-1αgene(rAAV-HIF-1α), and to detect the expression of HIF-1αprotein in rAAV-HIF-1αtransfected primary cultural hippocampal neurons.
Methods: pSNAV-HIF-1αwas transfected into BHK-21 cells using Lipofectamine 2000 and selected by G418. The G418 resistant BHK-21 cells, BHK/pSNAV-HIF-1α, were obtained. The BHK/pSNAV-HIF-1αcells were infected with HSV1-rc/?UL2, which could express rep and cap genes of wild-type AAV. After chloroform treatment, PEG/NaCl precipitation, chloroform extraction and bag filter concentration, the rAAV-HIF-1αwith high titer and purity was achieved. The authenticity of the rAAV-HIF-1αwas confirmed by polymerase chain reaction. The titer of rAAV-HIF-1αwas detected by dot-blot with digoxin labelled cytomegalovirus probe.The purity of rAAV-HIF-1αwas detected by 10%SDS-PAGE. There were two groups in this experiment: normal group: primary cultural hippocampal neurons without any special treatment; rAAV-HIF-1αgroup: rAAV-HIF-1αtransfected primary cultural hippocampal neurons; Total protein was extracted from hippocampal neurons three days posttransfection to detect the expression of HIF-1αby western blot.
Results:①PCR demonstrated that human HIF-1αgene was included in rAAV-HIF-1α;②The titer of rAAV-HIF-1αdetected by dot-blot with digoxin labelled cytomegalovirus probe was 1×1012v.g./ml;③The purity of rAAV-HIF-1αdetected by 10%SDS-PAGE exceeded 98%;④Western blot showed that the expression of HIF-1αin rAAV-HIF-1αgroup(727.57±36.08) was significantly higher than that in normal group(328.69±23.71)(P<0.05).
Conclusions: rAAV-HIF-1αwith high titer and purity was successfully constructed and it could express HIF-1αprotein efficiently in primary cultural hippocampal neurons.
3 The effect of the transfection of rAAV-HIF-1αon the apoptosis of primary cultural hippocampal neurons induced by Aβ25-35
Objective: To observe the effect of the transfection of rAAV-HIF-1αon the apoptosis of primary cultural hippocampal neurons induced by Aβ25-35.
Methods: There were three groups in this experiment: normal group: primary cultural hippocampal neurons without any special treatment; normal+Aβgroup: primary cultural hippocampal neurons were interfered with 10μmol/L Aβ25-35 for 24h; rAAV-HIF-1α+Aβgroup: primary cultural hippocampal neurons transfected with rAAV-HIF-1αfor three days were interfered with 10μmol/L Aβ25-35 for 24h. Apoptosis was detected by transmission electron microscope and flow cytometry analysis. The intracellular calcium concentration of hippocampal neurons was determined by laser scanning confocal microscopy with Fluo-3/AM as the fluorescent dye.
Results:①Transmission electron microscope showed that normal group hippocampal neuronal membrane was smooth and glossy, cytoplasmic structure was clear, chromatin was well-distribute; normal+Aβgroup hippocampal neurons shrinkaged due to the breakdown of the proteinaceous cytoskeleton by caspases, chromatin underwent condensation into compact patches against the nuclear envelope in a process known as pyknosis; however rAAV-HIF-1α+Aβgroup hippocampal neurons only showed light morphology changes of apoptosis, such as nuclear envelope sprouted irregular buds known as blebs and so on, which indicated that the morphology change of hippocampal neuronal apoptosis induced by Aβ25-35 could be lessened by the transfection of rAAV-HIF-1αin advance;②Flow cytometry analysis showed that the apoptosis ratio of hippocampal neurons in normal+Aβgroup(24.26±3.99%) was significantly higher than that in normal group(4.99±0.83%)(P<0.05), however the apoptosis ratio of hippocampal neurons in rAAV-HIF-1α+Aβgroup(14.29±1.88%) was significantly lower than that in normal + Aβgroup(P<0.05), which indicated that rAAV-HIF-1αtransfected hippocampal neurons could inhibit the apoptosis induced by Aβ25-35;③Laser scanning confocal microscopy showed that the calcium concentration of hippocampal neurons in normal+Aβgroup(197.98±27.60) was significantly higher than that in normal group(61.48±12.61)(P<0.05), however the calcium concentration of hippocampal neurons in rAAV-HIF-1α+Aβgroup(145.66±15.31) was significantly lower than that in normal+Aβgroup(P<0.05), which indicated that the up-regulation of calcium concentration of hippocampal neurons induced by Aβ25-35 could be suppressed by the transfection of rAAV-HIF-1αin advance.
Conclusions: rAAV-HIF-1αtransfection could inhibit Aβ25-35 induced apoptosis of primary cultural hippocampal neurons by suppressing the up-regulation of intracellular calcium concentration.
4 The effect of intracerebroventricular(i.c.v) injection of rAAV-HIF-1αon the hippocampal neuronal apoptosis of AD animal model Objective: To observe the effect of i.c.v injection of rAAV-HIF-1αon hippocampal neuronal apoptosis of AD animal model.
Methods: Thirty-two male SD rats(250-300g) were divided randomly into four groups: normal group(n=8): healthy male animal without any special treatment; AD group(n=8): right i.c.v injection of 2μl Aβ25-35(10mg/ml); sham group(n=8): right i.c.v injection of 2μl NS; AD+rAAV-HIF-1αgroup(n=8): right i.c.v injection of 10μl rAAV-HIF-1α(1×10~(12)v.g./ml) one week after i.c.v injection of 2μl Aβ25-35(10mg/ml). The rats were sacrificed to detect the expression of HIF-1αand apoptosis of hippocampal neurons five weeks after i.c.v injection of Aβ25-35 or NS.
Results:①Western blot showed that the expression of HIF-1αin AD+rAAV-HIF-1αgroup(451.59±34.39) was significantly higher than that in normal group(229.05±41.28) and sham group(216.29±37.08)(P<0.05), and the expression of HIF-1αbetween normal group and sham group was not significantly different.②Flow cytometry analysis showed that the apoptosis ratio of hippocampal neurons in AD group(19.49±2.59%) was significantly higher than that in normal group(5.41±0.75%) and sham group(5.28±0.66%)(P<0.05), and the apoptosis ratio of hippocampal neurons between normal group and sham group was not significantly different; however the apoptosis ratio of hippocampal neurons in AD+rAAV-HIF-1αgroup(12.07±2.06%) was significantly lower than that in AD group(P<0.05), which indicated that the hippocampal neuronal apoptosis of AD animal model could be attenuated by i.c.v injection of rAAV-HIF-1α.
Conclusions: The i.c.v injection of rAAV-HIF-1αcould inhibit the hippocampal neuronal apoptosis of AD animal model.
5 Conclusions
Recombinant AAV vector containing human HIF-1αgene(rAAV-HIF-1α) was successfully constructed and it could inhibit the neurotoxicity of Aβ25-35, which laid a foundation for further application of rAAV-HIF-1αinto gene therapy for AD in the future.
引文
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