基因疗法是将基因导入靶细胞内,以纠正缺陷和异常基因引起的疾病的治疗方法。目前,多个基因治疗药物已经进入临床研究阶段或已上市,为癌症、罕见病、神经性疾病等多个领域的疾病治疗带来了希望。基因治疗中最关键的步骤就是如何将外源...基因疗法是将基因导入靶细胞内,以纠正缺陷和异常基因引起的疾病的治疗方法。目前,多个基因治疗药物已经进入临床研究阶段或已上市,为癌症、罕见病、神经性疾病等多个领域的疾病治疗带来了希望。基因治疗中最关键的步骤就是如何将外源基因准确安全地导入宿主细胞。腺相关病毒(adeno-associated virus, AAV)载体是当前最受欢迎的人类基因治疗载体之一,它是唯一能定点整合到人类基因组特定位点的病毒载体。AAV定点整合的位点AAVS1 (the adeno-associated virus site 1)已经被证明是安全的基因整合位点。本实验以Rep蛋白介导基因定点整合至人T淋巴细胞基因组AAVS1位点,随后采用标准曲线定量的方法,对混合细胞克隆AAVS1位点的定点整合率进行定量。实验进行两轮PCR,首先通过常规PCR扩增包含ITR-AAVS1结合位点的序列,然后取标准品和检测样本第一轮PCR产物作为模板,进行第二轮定量PCR。通过本实验中的研究方法可以简单快速地定量分析AAVS1定点整合率。展开更多
为应对低空飞行器(low-altitude aircraft,LAA)日益增长的自主计算需求与有限机载能源之间的日益突出的矛盾,动态电压频率调整(dynamic voltage and frequency scaling,DVFS)技术作为平衡性能与功耗的关键,对提升LAA续航与任务能力具有...为应对低空飞行器(low-altitude aircraft,LAA)日益增长的自主计算需求与有限机载能源之间的日益突出的矛盾,动态电压频率调整(dynamic voltage and frequency scaling,DVFS)技术作为平衡性能与功耗的关键,对提升LAA续航与任务能力具有重要研究价值。在剖析LAA异构计算架构与动态负载特性的基础上,系统梳理了DVFS技术的演进路径,总结了从传统启发式、前瞻性预测式,到基于学习的自适应式与面向异构平台的系统级协同优化等主流方法,并对比分析了各类策略在实时性保障、动态响应与实现复杂度等方面的优劣。以水空跨介质飞行器(aerial-aquatic vehicles,AAVs)为例,重点探讨了极端工况对现有DVFS技术提出的严峻挑战。结合当前研究的不足,对未来融合模式感知、轻量化预测与多维度资源协同的整体式管理框架进行了展望。展开更多
Gene therapy is expected to fundamentally correct mutant genes to cure genetic diseases in vivo.As a delivery system for gene therapy,adenovirus-associated virus(AAV)vectors have achieved positive results in clinical ...Gene therapy is expected to fundamentally correct mutant genes to cure genetic diseases in vivo.As a delivery system for gene therapy,adenovirus-associated virus(AAV)vectors have achieved positive results in clinical and preclinical research,including the treatment of genetic diseases,such as those affecting the blood and eyes[1].展开更多
Retinitis pigmentosa is a group of inherited diseases that lead to retinal degeneration and photoreceptor cell death.However,there is no effective treatment for retinitis pigmentosa caused by PDE6B mutation.Adeno-asso...Retinitis pigmentosa is a group of inherited diseases that lead to retinal degeneration and photoreceptor cell death.However,there is no effective treatment for retinitis pigmentosa caused by PDE6B mutation.Adeno-associated virus(AAV)-mediated gene therapy is a promising strategy for treating retinitis pigmentosa.The aim of this study was to explore the molecular mechanisms by which AAV2-PDE6B rescues retinal function.To do this,we injected retinal degeneration 10(rd10)mice subretinally with AAV2-PDE6B and assessed the therapeutic effects on retinal function and structure using dark-and light-adapted electroretinogram,optical coherence tomography,and immunofluorescence.Data-independent acquisition-mass spectrometry-based proteomic analysis was conducted to investigate protein expression levels and pathway enrichment,and the results from this analysis were verified by real-time polymerase chain reaction and western blotting.AAV2-PDE6B injection significantly upregulated PDE6βexpression,preserved electroretinogram responses,and preserved outer nuclear layer thickness in rd10 mice.Differentially expressed proteins between wild-type and rd10 mice were closely related to visual perception,and treating rd10 mice with AAV2-PDE6B restored differentially expressed protein expression to levels similar to those seen in wild-type mice.Kyoto Encyclopedia of Genes and Genome analysis showed that the differentially expressed proteins whose expression was most significantly altered by AAV2-PDE6B injection were enriched in phototransduction pathways.Furthermore,the phototransductionrelated proteins Pde6α,Rom1,Rho,Aldh1a1,and Rbp1 exhibited opposite expression patterns in rd10 mice with or without AAV2-PDE6B treatment.Finally,Bax/Bcl-2,p-ERK/ERK,and p-c-Fos/c-Fos expression levels decreased in rd10 mice following AAV2-PDE6B treatment.Our data suggest that AAV2-PDE6B-mediated gene therapy promotes phototransduction and inhibits apoptosis by inhibiting the ERK signaling pathway and upregulating Bcl-2/Bax expression in retinitis pigmentosa.展开更多
Erratum to:J Huazhong Univ Sci Technol[Med Sci]36(4):548–553,2016 https://doi.org/10.1007/s11596-016-1623-6 In the originally published article(https://doi.org/10.1007/s11596-016-1623-6),the immunofluorescence images...Erratum to:J Huazhong Univ Sci Technol[Med Sci]36(4):548–553,2016 https://doi.org/10.1007/s11596-016-1623-6 In the originally published article(https://doi.org/10.1007/s11596-016-1623-6),the immunofluorescence images in shRNA group in Fig.3 were accidentally used rather than the final,formal experiments.To retain consistency,the entire Fig.3 is replaced here with original images of the experiments.The authors declare that this correction will not affect the conclusion of the study.展开更多
目的 构建 、鉴定自身互补双链DNA的腺相关病毒（scAAV）重组载体，使其分泌表达Exendin-4，检测其转导效率并观察在糖尿病大鼠模型中的治疗作用。 方法 应用基因工程方法改建穿梭质粒pSSHG-CMV，插入外源性基因Exendin-...目的 构建 、鉴定自身互补双链DNA的腺相关病毒（scAAV）重组载体，使其分泌表达Exendin-4，检测其转导效率并观察在糖尿病大鼠模型中的治疗作用。 方法 应用基因工程方法改建穿梭质粒pSSHG-CMV，插入外源性基因Exendin-4，构建重组scAAV载体，感染HEK293细胞，ELISA检测转染NIH3T3细胞上清Exendin-4滴度，链佐霉素诱导20只6周龄体质量180～220 g SD成年雄性大鼠为糖尿病鼠模型，逆向注射重组scAAV于糖尿病大鼠颌下腺，检测其血糖及胰岛素分泌水平。 结果 重组scpSSHG/exn4可有效包装和复制，病毒滴度为2.5×1011pfu/mL，转染细胞上清分泌Exendin-4浓度可达到4.53 ng/mL，scAAV治疗组血糖浓度在2、4周及8周均低于对照组[分别为（639.17±27.89）vs （396.00±34.00），（657.02±39.87） vs （315.62±42.56），（215.6±24.7） vs （458.6±19.7） mg/dL]，胰岛素浓度均高于对照组[分别为（156.8±24.5） vs （535.9±35.6），（236.5±12.3） vs （495.3±18.6），（620.43±46.90） vs （381.56±21.78） pg/mL]，二者比较有显著统计学差异（P〈0.05）。 结论 成功构建重组双链腺伴病毒scAAV-Ex-4，具有高效转导能力，对糖尿病大鼠模型具有控制血糖及增加胰岛素分泌作用。展开更多
文摘基因疗法是将基因导入靶细胞内,以纠正缺陷和异常基因引起的疾病的治疗方法。目前,多个基因治疗药物已经进入临床研究阶段或已上市,为癌症、罕见病、神经性疾病等多个领域的疾病治疗带来了希望。基因治疗中最关键的步骤就是如何将外源基因准确安全地导入宿主细胞。腺相关病毒(adeno-associated virus, AAV)载体是当前最受欢迎的人类基因治疗载体之一,它是唯一能定点整合到人类基因组特定位点的病毒载体。AAV定点整合的位点AAVS1 (the adeno-associated virus site 1)已经被证明是安全的基因整合位点。本实验以Rep蛋白介导基因定点整合至人T淋巴细胞基因组AAVS1位点,随后采用标准曲线定量的方法,对混合细胞克隆AAVS1位点的定点整合率进行定量。实验进行两轮PCR,首先通过常规PCR扩增包含ITR-AAVS1结合位点的序列,然后取标准品和检测样本第一轮PCR产物作为模板,进行第二轮定量PCR。通过本实验中的研究方法可以简单快速地定量分析AAVS1定点整合率。
文摘为应对低空飞行器(low-altitude aircraft,LAA)日益增长的自主计算需求与有限机载能源之间的日益突出的矛盾,动态电压频率调整(dynamic voltage and frequency scaling,DVFS)技术作为平衡性能与功耗的关键,对提升LAA续航与任务能力具有重要研究价值。在剖析LAA异构计算架构与动态负载特性的基础上,系统梳理了DVFS技术的演进路径,总结了从传统启发式、前瞻性预测式,到基于学习的自适应式与面向异构平台的系统级协同优化等主流方法,并对比分析了各类策略在实时性保障、动态响应与实现复杂度等方面的优劣。以水空跨介质飞行器(aerial-aquatic vehicles,AAVs)为例,重点探讨了极端工况对现有DVFS技术提出的严峻挑战。结合当前研究的不足,对未来融合模式感知、轻量化预测与多维度资源协同的整体式管理框架进行了展望。
基金supported by the National Natural Science Foundation of China(81930121 and U1602224)the National Key Research and Development Program of China(2018YFA0107902,2017YFC1001902,and 2018YFA0801403)+1 种基金the Yunnan Fundamental Research Projects(2019FY002 and 2018FB114)the Major Basic Research Project of Science and Technology of Yunnan(202001BC070001)。
文摘Gene therapy is expected to fundamentally correct mutant genes to cure genetic diseases in vivo.As a delivery system for gene therapy,adenovirus-associated virus(AAV)vectors have achieved positive results in clinical and preclinical research,including the treatment of genetic diseases,such as those affecting the blood and eyes[1].
基金supported by the National Natural Science Foundation of China,Nos.82071008(to BL)and 82004001(to XJ)Medical Science and Technology Program of Health Commission of Henan Province,No.LHGJ20210072(to RQ)Science and Technology Department of Henan Province,No.212102310307(to XJ)。
文摘Retinitis pigmentosa is a group of inherited diseases that lead to retinal degeneration and photoreceptor cell death.However,there is no effective treatment for retinitis pigmentosa caused by PDE6B mutation.Adeno-associated virus(AAV)-mediated gene therapy is a promising strategy for treating retinitis pigmentosa.The aim of this study was to explore the molecular mechanisms by which AAV2-PDE6B rescues retinal function.To do this,we injected retinal degeneration 10(rd10)mice subretinally with AAV2-PDE6B and assessed the therapeutic effects on retinal function and structure using dark-and light-adapted electroretinogram,optical coherence tomography,and immunofluorescence.Data-independent acquisition-mass spectrometry-based proteomic analysis was conducted to investigate protein expression levels and pathway enrichment,and the results from this analysis were verified by real-time polymerase chain reaction and western blotting.AAV2-PDE6B injection significantly upregulated PDE6βexpression,preserved electroretinogram responses,and preserved outer nuclear layer thickness in rd10 mice.Differentially expressed proteins between wild-type and rd10 mice were closely related to visual perception,and treating rd10 mice with AAV2-PDE6B restored differentially expressed protein expression to levels similar to those seen in wild-type mice.Kyoto Encyclopedia of Genes and Genome analysis showed that the differentially expressed proteins whose expression was most significantly altered by AAV2-PDE6B injection were enriched in phototransduction pathways.Furthermore,the phototransductionrelated proteins Pde6α,Rom1,Rho,Aldh1a1,and Rbp1 exhibited opposite expression patterns in rd10 mice with or without AAV2-PDE6B treatment.Finally,Bax/Bcl-2,p-ERK/ERK,and p-c-Fos/c-Fos expression levels decreased in rd10 mice following AAV2-PDE6B treatment.Our data suggest that AAV2-PDE6B-mediated gene therapy promotes phototransduction and inhibits apoptosis by inhibiting the ERK signaling pathway and upregulating Bcl-2/Bax expression in retinitis pigmentosa.
文摘Erratum to:J Huazhong Univ Sci Technol[Med Sci]36(4):548–553,2016 https://doi.org/10.1007/s11596-016-1623-6 In the originally published article(https://doi.org/10.1007/s11596-016-1623-6),the immunofluorescence images in shRNA group in Fig.3 were accidentally used rather than the final,formal experiments.To retain consistency,the entire Fig.3 is replaced here with original images of the experiments.The authors declare that this correction will not affect the conclusion of the study.
文摘为了建立一种快速、特异、灵敏的检测I群禽腺病毒的液相基因芯片方法。根据Hexon基因序列设计特异引物,上游引物5'端加TAG序列,下游引物5'端加生物素,进行PCR扩增,将扩增产物与链霉素亲和蛋白、磁珠37℃孵育30 min,通过Luminex200仪器分析。用所建立的液相基因芯片方法进行特异性、灵敏度、重复性及病料样品的检测。结果该方法的特异性强;检测的敏感性可达1×102copies/μL;批内批间的变异系数都在5%以下;检测结果与SYBR Green I荧光PCR方法 100%相符。表明建立的液相基因芯片方法特异性好、灵敏高、重复性好,可用于I群禽腺病毒的检测。
文摘目的 构建 、鉴定自身互补双链DNA的腺相关病毒（scAAV）重组载体，使其分泌表达Exendin-4，检测其转导效率并观察在糖尿病大鼠模型中的治疗作用。 方法 应用基因工程方法改建穿梭质粒pSSHG-CMV，插入外源性基因Exendin-4，构建重组scAAV载体，感染HEK293细胞，ELISA检测转染NIH3T3细胞上清Exendin-4滴度，链佐霉素诱导20只6周龄体质量180～220 g SD成年雄性大鼠为糖尿病鼠模型，逆向注射重组scAAV于糖尿病大鼠颌下腺，检测其血糖及胰岛素分泌水平。 结果 重组scpSSHG/exn4可有效包装和复制，病毒滴度为2.5×1011pfu/mL，转染细胞上清分泌Exendin-4浓度可达到4.53 ng/mL，scAAV治疗组血糖浓度在2、4周及8周均低于对照组[分别为（639.17±27.89）vs （396.00±34.00），（657.02±39.87） vs （315.62±42.56），（215.6±24.7） vs （458.6±19.7） mg/dL]，胰岛素浓度均高于对照组[分别为（156.8±24.5） vs （535.9±35.6），（236.5±12.3） vs （495.3±18.6），（620.43±46.90） vs （381.56±21.78） pg/mL]，二者比较有显著统计学差异（P〈0.05）。 结论 成功构建重组双链腺伴病毒scAAV-Ex-4，具有高效转导能力，对糖尿病大鼠模型具有控制血糖及增加胰岛素分泌作用。
