目的获得能够过表达OPTN和TAU-P301-L基因的重组腺相关病毒,检测其在体外对293T细胞的感染,并且为阿尔茨海默氏病(AD)研究提供新思路。方法从含有目的基因的大肠杆菌中提取包涵目的基因的质粒,利用PCR扩增获得目的基因,目的基因OPTN构建...目的获得能够过表达OPTN和TAU-P301-L基因的重组腺相关病毒,检测其在体外对293T细胞的感染,并且为阿尔茨海默氏病(AD)研究提供新思路。方法从含有目的基因的大肠杆菌中提取包涵目的基因的质粒,利用PCR扩增获得目的基因,目的基因OPTN构建到pAAV-IRES-ZsGreen1载体,目的基因TAU-P301L-V5构建到pAAV-CMV-mkate-IRES载体。鉴定质粒阳性克隆并测序。重组质粒pAAV-IRES-ZsGreen1-AAV9-OPTN和pAAV-CMV-mkate-IRES-AAV9-TAU-P301L-V5分别与包装质粒pAAV-RC9、辅助质粒pHelper通过Lipofiter^(TM)2000共转染293T细胞,获得AAV9病毒。利用相同方法,不使用目的基因构建对应绿色和红色AAV9对照病毒。qPCR法测定重组腺相关病毒载体滴度,病毒载体感染293T细胞进行病毒有效性及安全性鉴定,Western blotting和RT-PCR进行蛋白表达的鉴定,MTT检测OPTN蛋白与TAU-P301-L蛋白对293T细胞活力的影响。结果测序结果显示成功构建pAAV-IRES-ZsGreen1-AAV9-OPTN和pAAV-CMV-mkate-IRES-AAV9-TAU-P301L-V5腺相关病毒载体,qPCR测得病毒滴度为1.0×10^(12) vg/ml,病毒载体感染293T细胞转染效率为33.6%和20.5%, Western blot和RT-PCR显示蛋白过量表达。MTT结果显示OPTN组挽救了TAU-P301-L组的细胞凋亡。结论成功构建过表达OPTN和TAU-P301-L基因的腺相关病毒。OPTN蛋白可以减弱TAU-P301-L蛋白的细胞毒性。展开更多
器官获得和移植网(Organ Procure Transplantation Network,OPTN/United Network for Organ Sharing,UNOS)于2014年6月3日起负责监测全美的血管化复合组织(vascularized composite allograft,VCA)的获取与移植。截至2017年12月31日,美...器官获得和移植网(Organ Procure Transplantation Network,OPTN/United Network for Organ Sharing,UNOS)于2014年6月3日起负责监测全美的血管化复合组织(vascularized composite allograft,VCA)的获取与移植。截至2017年12月31日,美国共批准27家中心的61个VCA项目,15家中心的50例患者列入术前等待名单。14个项目组已开展28例VCA移植,包括10例上肢移植、10例子宫移植、5例颅面移植、1例头皮移植、1例腹壁移植和1例阴茎移植。展开更多
Background:The association between optineurin(OPTN)M98K variant and primary open angle glaucoma(POAG)has been widely investigated.However,the results remain controversial among published meta-analyses.Therefore,we con...Background:The association between optineurin(OPTN)M98K variant and primary open angle glaucoma(POAG)has been widely investigated.However,the results remain controversial among published meta-analyses.Therefore,we conducted an updated meta-analysis to further explore the association between M98K and POAG and its subgroups.Methods:PubMed,Embase,Web of Science,and China National Knowledge Infrastructure(CNKI)databases were searched to find all articles describing the relationships between the M98K variant and POAG,which were published from the inception to 31 December,2019.The associations between M98K and overall POAG,normal tension glaucoma(NTG),high tension glaucoma(HTG),POAG in Asian and non-Asian populations,juvenile open angle glaucoma(JOAG)and adult-onset POAG were evaluated by calculating the pooled odds ratio(OR)and 95%confidence interval(CI).The Bonferroni correction was used to determine the statistically significant genetic models.Results:A total of 34 eligible articles involving 7,310 POAG patients and 5,173 controls were identified in the present meta-analysis.The articles achieved an average of 6.21 stars for quality assessment by the Newcastle-Ottawa Scale(NOS).Evidence from the pooled results indicated significant association between M98K and overall POAG susceptibility under the dominant model(OR=1.30,95%CI,1.12-1.52;P<0.001).In the subgroup analyses,no significant associations were found between M98K and the risks of NTG,HTG,JOAG,adult-onset POAG,Asian POAG or non-Asian POAG.Conclusions:The updated meta-analysis revealed that OPTN M98K was significantly associated with the susceptibility to overall POAG.展开更多
Gefitinib,a well-known epidermal growth factor receptor(EGFR)tyrosine kinase inhibitor for the targeted therapy of lung cancer,induces autophagy in association with drug resistance.However,it remains unclear whether g...Gefitinib,a well-known epidermal growth factor receptor(EGFR)tyrosine kinase inhibitor for the targeted therapy of lung cancer,induces autophagy in association with drug resistance.However,it remains unclear whether gefitinib treatment can affect the selective form of autophagy(i.e.,mitophagy)and be beneficial for the treatment of human diseases with decreased autophagy,such as neurodegenerative diseases.Here,we show that gefitinib treatment promotes PINK1/Parkin-mediated mitophagy in both nonneuronal and neuronal cells,and this effect is independent of EGFR.Moreover,we found that gefitinib treatment increases the recruitment of the autophagy receptor optineurin(OPTN)to damaged mitochondria,which is a downstream signaling event in PINK1/Parkin-mediated mitophagy.In addition,gefitinib treatment significantly alleviated neuronal damage in TBK1-deficient neurons,resulting in impeded mitophagy.In conclusion,our study suggests that gefitinib promotes PINK1/Parkin-mediated mitophagy via OPTN and may be beneficial for the treatment of neurodegenerative diseases that are associated with defective mitophagy.展开更多
文摘目的获得能够过表达OPTN和TAU-P301-L基因的重组腺相关病毒,检测其在体外对293T细胞的感染,并且为阿尔茨海默氏病(AD)研究提供新思路。方法从含有目的基因的大肠杆菌中提取包涵目的基因的质粒,利用PCR扩增获得目的基因,目的基因OPTN构建到pAAV-IRES-ZsGreen1载体,目的基因TAU-P301L-V5构建到pAAV-CMV-mkate-IRES载体。鉴定质粒阳性克隆并测序。重组质粒pAAV-IRES-ZsGreen1-AAV9-OPTN和pAAV-CMV-mkate-IRES-AAV9-TAU-P301L-V5分别与包装质粒pAAV-RC9、辅助质粒pHelper通过Lipofiter^(TM)2000共转染293T细胞,获得AAV9病毒。利用相同方法,不使用目的基因构建对应绿色和红色AAV9对照病毒。qPCR法测定重组腺相关病毒载体滴度,病毒载体感染293T细胞进行病毒有效性及安全性鉴定,Western blotting和RT-PCR进行蛋白表达的鉴定,MTT检测OPTN蛋白与TAU-P301-L蛋白对293T细胞活力的影响。结果测序结果显示成功构建pAAV-IRES-ZsGreen1-AAV9-OPTN和pAAV-CMV-mkate-IRES-AAV9-TAU-P301L-V5腺相关病毒载体,qPCR测得病毒滴度为1.0×10^(12) vg/ml,病毒载体感染293T细胞转染效率为33.6%和20.5%, Western blot和RT-PCR显示蛋白过量表达。MTT结果显示OPTN组挽救了TAU-P301-L组的细胞凋亡。结论成功构建过表达OPTN和TAU-P301-L基因的腺相关病毒。OPTN蛋白可以减弱TAU-P301-L蛋白的细胞毒性。
文摘器官获得和移植网(Organ Procure Transplantation Network,OPTN/United Network for Organ Sharing,UNOS)于2014年6月3日起负责监测全美的血管化复合组织(vascularized composite allograft,VCA)的获取与移植。截至2017年12月31日,美国共批准27家中心的61个VCA项目,15家中心的50例患者列入术前等待名单。14个项目组已开展28例VCA移植,包括10例上肢移植、10例子宫移植、5例颅面移植、1例头皮移植、1例腹壁移植和1例阴茎移植。
基金This work was supported by Major Project of National Natural Science Foundation of China(NSFC)-Guangdong Province Joint Fund(grant number 3030902113080)the Science and Technology Planning Project of Guangdong Province(grant number 303090100502050-18)+2 种基金Guangzhou Science and Technology Plan Project(grant number 2018-1202-SF-0019)Research Funds of the State Key Laboratory of Ophthalmology(grant number 30306020240020153,30306020240020192,3030902113058,3030902113118,PT1001022)Fundamental Research Funds of Sun Yat-sen University(grant number 16ykjc31).
文摘Background:The association between optineurin(OPTN)M98K variant and primary open angle glaucoma(POAG)has been widely investigated.However,the results remain controversial among published meta-analyses.Therefore,we conducted an updated meta-analysis to further explore the association between M98K and POAG and its subgroups.Methods:PubMed,Embase,Web of Science,and China National Knowledge Infrastructure(CNKI)databases were searched to find all articles describing the relationships between the M98K variant and POAG,which were published from the inception to 31 December,2019.The associations between M98K and overall POAG,normal tension glaucoma(NTG),high tension glaucoma(HTG),POAG in Asian and non-Asian populations,juvenile open angle glaucoma(JOAG)and adult-onset POAG were evaluated by calculating the pooled odds ratio(OR)and 95%confidence interval(CI).The Bonferroni correction was used to determine the statistically significant genetic models.Results:A total of 34 eligible articles involving 7,310 POAG patients and 5,173 controls were identified in the present meta-analysis.The articles achieved an average of 6.21 stars for quality assessment by the Newcastle-Ottawa Scale(NOS).Evidence from the pooled results indicated significant association between M98K and overall POAG susceptibility under the dominant model(OR=1.30,95%CI,1.12-1.52;P<0.001).In the subgroup analyses,no significant associations were found between M98K and the risks of NTG,HTG,JOAG,adult-onset POAG,Asian POAG or non-Asian POAG.Conclusions:The updated meta-analysis revealed that OPTN M98K was significantly associated with the susceptibility to overall POAG.
基金supported by the National Natural Science Foundation of China(Grant Nos.82022022,31771117,and 82071274)a Project Funded by the Suzhou Science and Technology Program(Grant No.SYS2019068)+2 种基金a Project Funded by the Clinical Research Program of the WuJieping Medical Foundation(Grant No.320.6750.19092-32)a Project Funded by Jiangsu Key Laboratory of Neuropsychiatric Diseases(Grant No.BM2013003)a Project Funded by the Priority Academic Program Development of the Jiangsu Higher Education Institutes(PAPD).
文摘Gefitinib,a well-known epidermal growth factor receptor(EGFR)tyrosine kinase inhibitor for the targeted therapy of lung cancer,induces autophagy in association with drug resistance.However,it remains unclear whether gefitinib treatment can affect the selective form of autophagy(i.e.,mitophagy)and be beneficial for the treatment of human diseases with decreased autophagy,such as neurodegenerative diseases.Here,we show that gefitinib treatment promotes PINK1/Parkin-mediated mitophagy in both nonneuronal and neuronal cells,and this effect is independent of EGFR.Moreover,we found that gefitinib treatment increases the recruitment of the autophagy receptor optineurin(OPTN)to damaged mitochondria,which is a downstream signaling event in PINK1/Parkin-mediated mitophagy.In addition,gefitinib treatment significantly alleviated neuronal damage in TBK1-deficient neurons,resulting in impeded mitophagy.In conclusion,our study suggests that gefitinib promotes PINK1/Parkin-mediated mitophagy via OPTN and may be beneficial for the treatment of neurodegenerative diseases that are associated with defective mitophagy.
