Study On The Inhibition Of Aβ Neurotoxicity By Recombinant Adeno-associated Virus With Human Hypoxia Inducible Factor-1α Gene

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 Aβ25-351.1 The construction and expression of pSNAV-HIF-1αObjective: 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,Bglâ…¡to 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:â‘ Restricted endonuclease enzyme digestion and PCR confirmed that pSNAV-HIF-1αwas successfully constructed;â‘¡Western 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 Aβ25-35Objective: 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-1αgeneObjective: 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:â‘ PCR demonstrated that human HIF-1αgene was included in rAAV-HIF-1α;â‘¡The titer of rAAV-HIF-1αdetected by dot-blot with digoxin labelled cytomegalovirus probe was 1×1012v.g./ml;â‘¢The purity of rAAV-HIF-1αdetected by 10%SDS-PAGE exceeded 98%;â‘£Western 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 Aβ25-35Objective: 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:â‘ Transmission 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;â‘¡Flow 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;â‘¢Laser 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(1×10¹²v.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:â‘ Western 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.â‘¡Flow 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 ConclusionsRecombinant 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.