为探讨病毒如何利用宿主细胞自噬机制进行复制和传播提供研究模型,本研究利用CRISPR/Cas9系统构建ATG5基因敲除细胞系。首先针对非洲绿猴肾细胞(Vero)的ATG5基因设计靶向sgRNA序列,并将其连接到慢病毒载体中,包装得到重组慢病毒,用其感...为探讨病毒如何利用宿主细胞自噬机制进行复制和传播提供研究模型,本研究利用CRISPR/Cas9系统构建ATG5基因敲除细胞系。首先针对非洲绿猴肾细胞(Vero)的ATG5基因设计靶向sgRNA序列,并将其连接到慢病毒载体中,包装得到重组慢病毒,用其感染Vero-SN细胞,经过筛选得到ATG5基因敲除的亚克隆细胞系。利用TCID_(50)和Western-blot技术检测小反刍兽疫病毒(peste des petits ruminants virus,PPRV)分别在Vero-SN细胞和ATG5基因敲除细胞系中的复制能力。结果表明,本研究成功建立了VeroSN-ATG5KO细胞系,且抑制细胞自噬不利于病毒复制。该基因敲除细胞系的建立为后续探究病毒与自噬蛋白的相互作用及分子机制提供了试验材料。展开更多
目的:探讨自噬相关基因4A(autophagy related 4A,ATG4A)及自噬相关基因16L1(autophagy related 16 like 1,ATG16L1)的多态性位点与中国西南地区人群肺结核易感性的关系。方法:采用聚合酶链反应-限制性片段长度多态性技术检测218例肺结...目的:探讨自噬相关基因4A(autophagy related 4A,ATG4A)及自噬相关基因16L1(autophagy related 16 like 1,ATG16L1)的多态性位点与中国西南地区人群肺结核易感性的关系。方法:采用聚合酶链反应-限制性片段长度多态性技术检测218例肺结核患者及248例健康对照者ATG4A基因rs807185位点及ATG16L1基因rs2241880位点的基因型,用logistic回归分析上述2个位点与肺结核病易感的相关性。结果:2组人群中rs807185位点的A等位基因、AT基因型及显性模型AA+AT vs.TT的频率分布存在统计学差异(P<0.05),且在女性中差异更突出。而rs2241880位点的各指标频率分布差异均不明显(P>0.05)。结论:ATG4A基因rs807185位点多态性与中国西南地区人群肺结核易感性呈负相关,而ATG16L1基因rs2241880位点多态性则与该地区人群肺结核易感性无明显相关。展开更多
BACKGROUND Type 2 diabetes mellitus(T2DM)is a severe global health problem that causes prolonged disease exposure and an elevated risk for chronic complications,posing a substantial health burden.Although therapies,su...BACKGROUND Type 2 diabetes mellitus(T2DM)is a severe global health problem that causes prolonged disease exposure and an elevated risk for chronic complications,posing a substantial health burden.Although therapies,such as GLP-1 receptor agonists and SGLT2 inhibitors,have been successfully developed,new therapeutic options are still expected to offer better blood glucose control and decrease complications.AIM To elucidate the mechanism by which TERT/FOXO1 affects high glucose(HG)-induced dysfunction in isletβ-cells via the regulation of ATG9A-mediated autophagy.METHODS High-fat diet(HFD)-fed/streptozotocin(STZ)-treated mice or HG-treated MIN6 cells were used to establish T2DM models.Fasting blood glucose(FBG)and insulin levels in mice,as well as morphological changes in islet tissues,were assessed.Cell proliferation and the apoptosis rate were measured via EdU assays and flow cytometry,respectively.The expression levels of TERT,FOXO1,ATG9A and autophagy-related proteins(LC3B,p62)were analyzed via western blotting.The relationship between FOXO1 and ATG9A was assessed using dual-luciferase reporter gene assays and ChIP assays.RESULTS T2DM modeling in HFD-fed/STZ-treated mice and HG-treated MIN6 cells led to elevated TERT and FOXO1 expression and reduced ATG9A expression.Mice with T2DM were found to have decreased body weight,worsened morphology,elevated FBG and suppressed insulin levels.HG-treated MIN6 cells presented decreased viability and LC3B expression,in addition to increased p62 expression and apoptosis rates.FOXO1 knockdown both in vitro and in vivo protected mice and cells against isletβ-cell dysfunction via the activation of autophagy.The molecular mechanism involved the suppression of ATG9A expression by TERT through FOXO1 transcription activation.CONCLUSION Our results suggested that TERT/FOXO1 inhibits ATG9A expression to decrease isletβ-cell function in T2DM.展开更多
文摘为探讨病毒如何利用宿主细胞自噬机制进行复制和传播提供研究模型,本研究利用CRISPR/Cas9系统构建ATG5基因敲除细胞系。首先针对非洲绿猴肾细胞(Vero)的ATG5基因设计靶向sgRNA序列,并将其连接到慢病毒载体中,包装得到重组慢病毒,用其感染Vero-SN细胞,经过筛选得到ATG5基因敲除的亚克隆细胞系。利用TCID_(50)和Western-blot技术检测小反刍兽疫病毒(peste des petits ruminants virus,PPRV)分别在Vero-SN细胞和ATG5基因敲除细胞系中的复制能力。结果表明,本研究成功建立了VeroSN-ATG5KO细胞系,且抑制细胞自噬不利于病毒复制。该基因敲除细胞系的建立为后续探究病毒与自噬蛋白的相互作用及分子机制提供了试验材料。
文摘目的:探讨自噬相关基因4A(autophagy related 4A,ATG4A)及自噬相关基因16L1(autophagy related 16 like 1,ATG16L1)的多态性位点与中国西南地区人群肺结核易感性的关系。方法:采用聚合酶链反应-限制性片段长度多态性技术检测218例肺结核患者及248例健康对照者ATG4A基因rs807185位点及ATG16L1基因rs2241880位点的基因型,用logistic回归分析上述2个位点与肺结核病易感的相关性。结果:2组人群中rs807185位点的A等位基因、AT基因型及显性模型AA+AT vs.TT的频率分布存在统计学差异(P<0.05),且在女性中差异更突出。而rs2241880位点的各指标频率分布差异均不明显(P>0.05)。结论:ATG4A基因rs807185位点多态性与中国西南地区人群肺结核易感性呈负相关,而ATG16L1基因rs2241880位点多态性则与该地区人群肺结核易感性无明显相关。
基金Supported by National Natural Science Foundation of China,No.82000792General Project of Chongqing Natural Science Foundation,No.CSTB2023NSCQ-MSX0246 and No.CSTB2022NSCQ-MSX1271+1 种基金Research Project of the State Administration of Traditional Chinese Medicine on Collaborative Chronic Disease Management of Traditional Chinese Medicine and Western Medicine,No.CXZH2024087Science and Health Joint Project of Dazu District Science and Technology Bureau,No.DZKJ2024JSYJ-KWXM1002.
文摘BACKGROUND Type 2 diabetes mellitus(T2DM)is a severe global health problem that causes prolonged disease exposure and an elevated risk for chronic complications,posing a substantial health burden.Although therapies,such as GLP-1 receptor agonists and SGLT2 inhibitors,have been successfully developed,new therapeutic options are still expected to offer better blood glucose control and decrease complications.AIM To elucidate the mechanism by which TERT/FOXO1 affects high glucose(HG)-induced dysfunction in isletβ-cells via the regulation of ATG9A-mediated autophagy.METHODS High-fat diet(HFD)-fed/streptozotocin(STZ)-treated mice or HG-treated MIN6 cells were used to establish T2DM models.Fasting blood glucose(FBG)and insulin levels in mice,as well as morphological changes in islet tissues,were assessed.Cell proliferation and the apoptosis rate were measured via EdU assays and flow cytometry,respectively.The expression levels of TERT,FOXO1,ATG9A and autophagy-related proteins(LC3B,p62)were analyzed via western blotting.The relationship between FOXO1 and ATG9A was assessed using dual-luciferase reporter gene assays and ChIP assays.RESULTS T2DM modeling in HFD-fed/STZ-treated mice and HG-treated MIN6 cells led to elevated TERT and FOXO1 expression and reduced ATG9A expression.Mice with T2DM were found to have decreased body weight,worsened morphology,elevated FBG and suppressed insulin levels.HG-treated MIN6 cells presented decreased viability and LC3B expression,in addition to increased p62 expression and apoptosis rates.FOXO1 knockdown both in vitro and in vivo protected mice and cells against isletβ-cell dysfunction via the activation of autophagy.The molecular mechanism involved the suppression of ATG9A expression by TERT through FOXO1 transcription activation.CONCLUSION Our results suggested that TERT/FOXO1 inhibits ATG9A expression to decrease isletβ-cell function in T2DM.
