Pancreatic cancer is one of the deadliest cancers with a very poor prognosis. Recently, there has been a significant increase in research directed towards identifying potential biomarkers that can be used to diagnose ...Pancreatic cancer is one of the deadliest cancers with a very poor prognosis. Recently, there has been a significant increase in research directed towards identifying potential biomarkers that can be used to diagnose and provide prognostic information for pancreatic cancer. These markers can be used clinically to optimize and personalize therapy for individual patients. In this review, we focused on 3 biomarkers involved in the DNA damage response pathway and the necroptosis pathway: Chromodomainhelicase-DNA binding protein 5, chromodomain-helicaseDNA binding protein 7, and mixed lineage kinase domain-like protein. The aim of this article is to review present literature provided for these biomarkers and current studies in which their effectiveness as prognostic biomarkers are analyzed in order to determine their future use as biomarkers in clinical medicine. Based on the data presented, these biomarkers warrant further investigation,and should be validated in future studies.展开更多
PANoptosis is a newly identified type of regulated cell death that consists of pyroptosis,apoptosis,and nec roptosis,which simultaneously occur during the pathophysiological process of infectious and inflammatory dise...PANoptosis is a newly identified type of regulated cell death that consists of pyroptosis,apoptosis,and nec roptosis,which simultaneously occur during the pathophysiological process of infectious and inflammatory diseases.Although our previous lite rature mining study suggested that PANoptosis might occur in neuronal ischemia/repe rfusion injury,little experimental research has been reported on the existence of PANoptosis.In this study,we used in vivo and in vitro retinal neuronal models of ischemia/repe rfusion injury to investigate whether PAN optosis-like cell death(simultaneous occurrence of pyroptosis,apo ptosis,and necroptosis)exists in retinal neuronal ischemia/repe rfusion injury.Our results showed that ischemia/repe rfusion injury induced changes in morphological features and protein levels that indicate PANoptosis-like cell death in retinal neurons both in vitro and in vivo.Ischemia/repe rfusion inju ry also significantly upregulated caspase-1,caspase-8,and NLRP3 expression,which are important components of the PANoptosome.These results indicate the existence of PANoptosis-like cell death in ischemia/reperfusion injury of retinal neurons and provide preliminary experimental evidence for future study of this new type of regulated cell death.展开更多
坏死样凋亡是细胞调节性死亡方式之一,由受体相互作用蛋白(receptor interacting protein kinase,RIPK)1/RIPK3/混合系列蛋白激酶样结构域(mixed lineage kinase domain like protein,MLKL)信号通路介导。其中,MLKL对坏死样凋亡最终执...坏死样凋亡是细胞调节性死亡方式之一,由受体相互作用蛋白(receptor interacting protein kinase,RIPK)1/RIPK3/混合系列蛋白激酶样结构域(mixed lineage kinase domain like protein,MLKL)信号通路介导。其中,MLKL对坏死样凋亡最终执行分子有着不可替代的作用。RIPK1/RIPK3/MLKL坏死复合体的形成诱导MLKL磷酸化激活,活化的MLKL插入细胞膜双分子层形成膜孔,破坏膜完整性,从而导致细胞死亡。除参与坏死样凋亡外,MLKL也与其他细胞死亡方式[如中性粒细胞特殊死亡方式(NETosis),细胞焦亡和细胞自噬]密切相关。MLKL参与多种细胞死亡通路异常相关疾病(如心血管疾病、神经退行性疾病和癌症)的病理过程,有可能成为多种疾病的治疗靶点。了解MLKL在不同细胞死亡方式中的作用,可为多种MLKL相关疾病靶点的寻找奠定基础,也可为MLKL抑制剂的开发应用指明方向。展开更多
AIM To validate the effects of receptor interacting protein kinase-3(RIP3) deletion in non-alcoholic fatty liver disease(NAFLD) and to clarify the mechanism of action.METHODS Wild-type(WT) and RIP3 knockout(KO) mice w...AIM To validate the effects of receptor interacting protein kinase-3(RIP3) deletion in non-alcoholic fatty liver disease(NAFLD) and to clarify the mechanism of action.METHODS Wild-type(WT) and RIP3 knockout(KO) mice werefed normal chow and high fat(HF) diets for 12 wk. The body weight was assessed once weekly. After 12 wk, the liver and serum samples were extracted. The liver tissue expression levels of RIP3, microsomal triglyceride transfer protein, protein disulfide isomerase, apolipoprotein-B, X-box binding protein-1, sterol regulatory element-binding protein-1c, fatty acid synthase, cluster of differentiation-36, diglyceride acyltransferase, peroxisome proliferator-activated receptor alpha, tumor necrosis factor-alpha(TNF-α), and interleukin-6 were assessed. Oleic acid treated primary hepatocytes from WT and RIP3 KO mice were stained with Nile red. The expression of inflammatory cytokines, including chemokine(C-X-C motif) ligand(CXCL) 1, CXCL2, and TNF-α, in monocytes was evaluated.RESULTS RIP3 KO HF diet fed mice showed a significant gain in body weight, and liver weight, liver to body weight ratio, and liver triglycerides were increased in HF diet fed RIP3 KO mice compared to HF diet fed WT mice. RIP3 KO primary hepatocytes also had increased intracellular fat droplets compared to WT primary hepatocytes after oleic acid treatment. RIP3 overexpression decreased hepatic fat content. Quantitative real-time polymerase chain reaction analysis showed that the expression of very-low-density lipoproteins secretion markers(microsomal triglyceride transfer protein, protein disulfide isomerase, and apolipoprotein-B) was significantly suppressed in RIP3 KO mice. The overall NAFLD Activity Score was the same between WT and RIP3 KO mice; however, RIP3 KO mice had increased fatty change and decreased lobular inflammation compared to WT mice. Inflammatory signals(CXCL1/2, TNF-α, and interleukin-6) increased after lipopolysaccharide and pancaspase inhibitor(necroptotic condition) treatment in monocytes. Neutrophil chemokines(CXCL1, and CXCL2) were decreased, and TNF-α was increased after RIP3 inhibitor treatment in monocytes.CONCLUSION RIP3 deletion exacerbates steatosis, and partially inhibits inflammation in the HF diet induced NAFLD model.展开更多
OBJECTIVE To explore the effect of ligustroflavone on ischemic brain injury in stroke rat and the under⁃lying mechanisms.METHODS A rat model of ischemic stroke was established by middle cerebral artery occlusion(MCAO)...OBJECTIVE To explore the effect of ligustroflavone on ischemic brain injury in stroke rat and the under⁃lying mechanisms.METHODS A rat model of ischemic stroke was established by middle cerebral artery occlusion(MCAO).Administration of ligustroflavone(10,30,60 mg·kg-1,ig)15 min before ischemia,after which neurological deficit score and infarct volume were detected by longa score and TTC stain.The cell viability and necrosis rate of hypoxia-cultured PC12 cells(O2/N2/CO2,1:94:5,8 h)were evaluated by MTS and LDH release rate.Flow cytometry further verified the mortality rate of PC12 cells.Necroptosis-associated proteins(RIPK1,RIPK3 and MLKL/p-MLKL)were detected by Western blotting.The interaction between RIPK3 and RIPK1 or MLKL were confirmed by immunoprecipitation.Operating Environ⁃ment(MOE)program demonstrated the possible combination of ligustroflavone with RIPK1,RIPK3 and MLKL.RESULTS Ischemic injury(increase in neurological deficit score and infarct volume)and upregulation of necroptosis-associated proteins were showed in rat MCAO model.Administration of ligustroflavone(30 mg·kg^-1,ig)evidently improved neurological func⁃tion,reduced infarct volume,and decreased the levels of necroptosis-associated proteins except the RIPK1.Consistently,hypoxia-cultured PC12 cells caused cellular injury(LDH release and necroposis)concomitant with up-regulation of necroptosis-associated proteins,and these phenomena were blocked in the presence of ligustroflavone(25μmol·L^-1)except the elevated RIPK1 levels.Using the Molecular Operating Environment(MOE)program,we identified RIPK1,RIPK3,and MLKL as potential targets of ligustroflavone.Further studies showed that the interaction between RIPK3 and RIPK1 or MLKL was significantly enhanced,which was blocked in the presence of ligustroflavone.CONCLUSION Ligus⁃troflavone protects rat brain from ischemic injury,and its beneficial effect is related to the prevention of necroptosis through a mechanism involving targeting RIPK1,RIPK3,and/or MLKL.展开更多
Methamphetamine is one of the most prevalent drugs abused in the world.Methamphetamine abusers usually present with hyperpyrexia (39℃),hallucination and other psychiatric symptoms.However,the detailed mechanism under...Methamphetamine is one of the most prevalent drugs abused in the world.Methamphetamine abusers usually present with hyperpyrexia (39℃),hallucination and other psychiatric symptoms.However,the detailed mechanism underlying its neurotoxic action remains elusive.This study investigated the effects of methamphetamine + 39℃ on primary cortical neurons from the cortex of embryonic Sprague-Dawley rats.Primary cortex neurons were exposed to 1 mM methamphetamine + 39℃.Propidium iodide staining and lactate dehydrogenase release detection showed that methamphetamine + 39℃ triggered obvious necrosis-like death in cultured primary cortical neurons,which could be partially inhibited by receptor-interacting protein-1 (RIP1) inhibitor Necrostatin-1 partially.Western blot assay results showed that there were increases in the expressions of receptor-interacting protein-3 (RIP3) and mixed lineage kinase domain-like protein (MLKL) in the primary cortical neurons treated with 1 mM methamphetamine + 39℃ for 3 hours.After pre-treatment with RIP3 inhibitor GSK’872,propidium iodide staining and lactate dehydrogenase release detection showed that neuronal necrosis rate was significantly decreased;RIP3 and MLKL protein expression significantly decreased.Immunohistochemistry staining results also showed that the expressions of RIP3 and MLKL were up-regulated in brain specimens from humans who had died of methamphetamine abuse.Taken together,the above results suggest that methamphetamine + 39℃ can induce RIP3/MLKL regulated necroptosis,thereby resulting in neurotoxicity.The study protocol was approved by the Medical Ethics Committee of the Third Xiangya Hospital of Central South University,China (approval numbers: 2017-S026 and 2017-S033) on March 7,2017.展开更多
The mixed lineage kinase domain-like(MLKL)protein is a key factor in tumor necrosis factor-induced necroptosis.Recent studies on necroptosis execution revealed a commitment role of MLKL in membrane disruption.However,...The mixed lineage kinase domain-like(MLKL)protein is a key factor in tumor necrosis factor-induced necroptosis.Recent studies on necroptosis execution revealed a commitment role of MLKL in membrane disruption.However,our knowledge of how MLKL functions on membrane remains very limited.Here we demonstrate that MLKL forms cation channels that are permeable preferential y to Mg2+rather than Ca2+in the presence of Na+and K+.Moreover,the N-terminal domain containing six helices(H1-H6)is sufficient to form channels.Using the substituted cysteine accessibility method,we further determine that helix H1,H2,H3,H5 and H6 are transmembrane segments,while H4 is located in the cytoplasm.Finally,MLKL-induced membrane depolarization and cell death exhibit a positive correlation to its channel activity.The Mg2+-preferred permeability and five transmembrane segment topology distinguish MLKL from previously identified Mg2+-permeable channels and thus establish MLKL as a novel class of cation channels.展开更多
目的构建呼吸道合胞病毒(RSV)感染气液相界面(ALI)培养的人支气管上皮细胞(hBEC)模型,为深入研究呼吸道病毒致病机制提供更接近体内环境的细胞模型;通过分析RSV感染对高迁移率族蛋白1(HMGB1)和磷酸化混合系列蛋白激酶样结构域(pMLKL)表...目的构建呼吸道合胞病毒(RSV)感染气液相界面(ALI)培养的人支气管上皮细胞(hBEC)模型,为深入研究呼吸道病毒致病机制提供更接近体内环境的细胞模型;通过分析RSV感染对高迁移率族蛋白1(HMGB1)和磷酸化混合系列蛋白激酶样结构域(pMLKL)表达的影响,探讨RSV感染损伤支气管上皮的致病机制。方法将hBEC接种到Transwell膜上,进行液体浸没式培养,细胞汇合度达100%后转ALI培养,倒置显微镜观察细胞生长状态。细胞分化成熟后按照以下分组分别感染RSV病毒:6 h对照组,6 h感染复数(MOI)1.0组,6 h MOI 3.0组,24 h对照组,24 h MOI 1.0组,24 h MOI 3.0组。每组3个复孔。通过免疫荧光染色确认RSV感染效果和RSV感染对细胞核蛋白HMGB1和pMLKL表达的影响。结果经细胞扩展期液体浸没式培养4~10 d后,细胞汇合度达100%。转ALI培养约4周后,细胞条索状分布越来越清晰,且分泌肉眼可见的黏液,形成黏液层,将Transwell膜石蜡包埋切片,得到典型的假复层上皮HE染色结果。经抗RSV抗体免疫荧光染色确认,MOI为3.0感染24 h RSV病毒成功感染细胞;抗HMGB1和抗pMLKL免疫荧光双染色结果显示,该感染条件下,细胞核内出现粉色荧光[为蓝色4',6-二脒基-2-苯基吲哚(DAPI)和红色HMGB1荧光Merge结果],证实RSV感染后出现HMGB1核表达;而RSV感染前后均未见pMLKL表达。结论通过在Transwell膜上液体浸没式扩展培养和ALI分化培养,可获得分化良好的hBEC,且能较长时间维持其形态及功能,可为呼吸道病毒感染及其他常见呼吸道疾病研究提供更接近体内环境的细胞模型。在MOI 3.024 h条件下,RSV成功感染该细胞模型,并引起损伤相关分子蛋白HMGB1核表达。展开更多
基金Supported by The National Center for Advancing Translational Sciences of the National Institutes of Health under award numbers ULl TR000454 previously awarded to Dr.Colbert and Dr.Fisher and TLlT R000456 to Dr.ColbertPancreatic Cancer Action Network(Pan-CAN)&sol American Association for Cancer Research(AACR)award 16982+1 种基金Department of Defense(DOD)/Peer Reviewed Cancer Research Program(PRCRP)award CA110535Georgia Cancer Coalition award 11072,all to Dr.Yu
文摘Pancreatic cancer is one of the deadliest cancers with a very poor prognosis. Recently, there has been a significant increase in research directed towards identifying potential biomarkers that can be used to diagnose and provide prognostic information for pancreatic cancer. These markers can be used clinically to optimize and personalize therapy for individual patients. In this review, we focused on 3 biomarkers involved in the DNA damage response pathway and the necroptosis pathway: Chromodomainhelicase-DNA binding protein 5, chromodomain-helicaseDNA binding protein 7, and mixed lineage kinase domain-like protein. The aim of this article is to review present literature provided for these biomarkers and current studies in which their effectiveness as prognostic biomarkers are analyzed in order to determine their future use as biomarkers in clinical medicine. Based on the data presented, these biomarkers warrant further investigation,and should be validated in future studies.
基金supported by the National Natural Science Foundation of China,Nos.81772134,81971891,82172196,81571939(ail to KX)the Key Laboratory of Emergency and Trauma(Hainan Medical University)of Ministry of Education,No.KLET-202108(to KX)+1 种基金the Fundamental Research Funds for the Central Universities of Central South University of China,No.2020zzts218(to WTY)Hunan Provincial Innovation Foundation for Postgraduate of China,No.CX20200116(to WTY)。
文摘PANoptosis is a newly identified type of regulated cell death that consists of pyroptosis,apoptosis,and nec roptosis,which simultaneously occur during the pathophysiological process of infectious and inflammatory diseases.Although our previous lite rature mining study suggested that PANoptosis might occur in neuronal ischemia/repe rfusion injury,little experimental research has been reported on the existence of PANoptosis.In this study,we used in vivo and in vitro retinal neuronal models of ischemia/repe rfusion injury to investigate whether PAN optosis-like cell death(simultaneous occurrence of pyroptosis,apo ptosis,and necroptosis)exists in retinal neuronal ischemia/repe rfusion injury.Our results showed that ischemia/repe rfusion injury induced changes in morphological features and protein levels that indicate PANoptosis-like cell death in retinal neurons both in vitro and in vivo.Ischemia/repe rfusion inju ry also significantly upregulated caspase-1,caspase-8,and NLRP3 expression,which are important components of the PANoptosome.These results indicate the existence of PANoptosis-like cell death in ischemia/reperfusion injury of retinal neurons and provide preliminary experimental evidence for future study of this new type of regulated cell death.
文摘坏死样凋亡是细胞调节性死亡方式之一,由受体相互作用蛋白(receptor interacting protein kinase,RIPK)1/RIPK3/混合系列蛋白激酶样结构域(mixed lineage kinase domain like protein,MLKL)信号通路介导。其中,MLKL对坏死样凋亡最终执行分子有着不可替代的作用。RIPK1/RIPK3/MLKL坏死复合体的形成诱导MLKL磷酸化激活,活化的MLKL插入细胞膜双分子层形成膜孔,破坏膜完整性,从而导致细胞死亡。除参与坏死样凋亡外,MLKL也与其他细胞死亡方式[如中性粒细胞特殊死亡方式(NETosis),细胞焦亡和细胞自噬]密切相关。MLKL参与多种细胞死亡通路异常相关疾病(如心血管疾病、神经退行性疾病和癌症)的病理过程,有可能成为多种疾病的治疗靶点。了解MLKL在不同细胞死亡方式中的作用,可为多种MLKL相关疾病靶点的寻找奠定基础,也可为MLKL抑制剂的开发应用指明方向。
基金Supported by National Research Foundation of Korea(NRF)funded by the South Korean Government,No.NRF-2017M3A9C8028794
文摘AIM To validate the effects of receptor interacting protein kinase-3(RIP3) deletion in non-alcoholic fatty liver disease(NAFLD) and to clarify the mechanism of action.METHODS Wild-type(WT) and RIP3 knockout(KO) mice werefed normal chow and high fat(HF) diets for 12 wk. The body weight was assessed once weekly. After 12 wk, the liver and serum samples were extracted. The liver tissue expression levels of RIP3, microsomal triglyceride transfer protein, protein disulfide isomerase, apolipoprotein-B, X-box binding protein-1, sterol regulatory element-binding protein-1c, fatty acid synthase, cluster of differentiation-36, diglyceride acyltransferase, peroxisome proliferator-activated receptor alpha, tumor necrosis factor-alpha(TNF-α), and interleukin-6 were assessed. Oleic acid treated primary hepatocytes from WT and RIP3 KO mice were stained with Nile red. The expression of inflammatory cytokines, including chemokine(C-X-C motif) ligand(CXCL) 1, CXCL2, and TNF-α, in monocytes was evaluated.RESULTS RIP3 KO HF diet fed mice showed a significant gain in body weight, and liver weight, liver to body weight ratio, and liver triglycerides were increased in HF diet fed RIP3 KO mice compared to HF diet fed WT mice. RIP3 KO primary hepatocytes also had increased intracellular fat droplets compared to WT primary hepatocytes after oleic acid treatment. RIP3 overexpression decreased hepatic fat content. Quantitative real-time polymerase chain reaction analysis showed that the expression of very-low-density lipoproteins secretion markers(microsomal triglyceride transfer protein, protein disulfide isomerase, and apolipoprotein-B) was significantly suppressed in RIP3 KO mice. The overall NAFLD Activity Score was the same between WT and RIP3 KO mice; however, RIP3 KO mice had increased fatty change and decreased lobular inflammation compared to WT mice. Inflammatory signals(CXCL1/2, TNF-α, and interleukin-6) increased after lipopolysaccharide and pancaspase inhibitor(necroptotic condition) treatment in monocytes. Neutrophil chemokines(CXCL1, and CXCL2) were decreased, and TNF-α was increased after RIP3 inhibitor treatment in monocytes.CONCLUSION RIP3 deletion exacerbates steatosis, and partially inhibits inflammation in the HF diet induced NAFLD model.
基金National Natural Science Foundation of China(8157343081872873)Hunan Provincial Natural Science Foundation of China(2015JJ2156)
文摘OBJECTIVE To explore the effect of ligustroflavone on ischemic brain injury in stroke rat and the under⁃lying mechanisms.METHODS A rat model of ischemic stroke was established by middle cerebral artery occlusion(MCAO).Administration of ligustroflavone(10,30,60 mg·kg-1,ig)15 min before ischemia,after which neurological deficit score and infarct volume were detected by longa score and TTC stain.The cell viability and necrosis rate of hypoxia-cultured PC12 cells(O2/N2/CO2,1:94:5,8 h)were evaluated by MTS and LDH release rate.Flow cytometry further verified the mortality rate of PC12 cells.Necroptosis-associated proteins(RIPK1,RIPK3 and MLKL/p-MLKL)were detected by Western blotting.The interaction between RIPK3 and RIPK1 or MLKL were confirmed by immunoprecipitation.Operating Environ⁃ment(MOE)program demonstrated the possible combination of ligustroflavone with RIPK1,RIPK3 and MLKL.RESULTS Ischemic injury(increase in neurological deficit score and infarct volume)and upregulation of necroptosis-associated proteins were showed in rat MCAO model.Administration of ligustroflavone(30 mg·kg^-1,ig)evidently improved neurological func⁃tion,reduced infarct volume,and decreased the levels of necroptosis-associated proteins except the RIPK1.Consistently,hypoxia-cultured PC12 cells caused cellular injury(LDH release and necroposis)concomitant with up-regulation of necroptosis-associated proteins,and these phenomena were blocked in the presence of ligustroflavone(25μmol·L^-1)except the elevated RIPK1 levels.Using the Molecular Operating Environment(MOE)program,we identified RIPK1,RIPK3,and MLKL as potential targets of ligustroflavone.Further studies showed that the interaction between RIPK3 and RIPK1 or MLKL was significantly enhanced,which was blocked in the presence of ligustroflavone.CONCLUSION Ligus⁃troflavone protects rat brain from ischemic injury,and its beneficial effect is related to the prevention of necroptosis through a mechanism involving targeting RIPK1,RIPK3,and/or MLKL.
基金funded by the National Natural Science Foundation of China,No.81971891(to KX),81571939(to KX),81772134(to KX),81772024(to JY),and 81860781(to FXL)the Key Research and Development Program of Hunan Province of China,No.2018SK2091(to KX)+1 种基金the Natural Science Foundation of Hunan Province of China,No.2017JJ2339(to JY)the Wu Jie-Ping Medical Foundation of the Minister of Health of China,No.320.6750.14118(to KX)
文摘Methamphetamine is one of the most prevalent drugs abused in the world.Methamphetamine abusers usually present with hyperpyrexia (39℃),hallucination and other psychiatric symptoms.However,the detailed mechanism underlying its neurotoxic action remains elusive.This study investigated the effects of methamphetamine + 39℃ on primary cortical neurons from the cortex of embryonic Sprague-Dawley rats.Primary cortex neurons were exposed to 1 mM methamphetamine + 39℃.Propidium iodide staining and lactate dehydrogenase release detection showed that methamphetamine + 39℃ triggered obvious necrosis-like death in cultured primary cortical neurons,which could be partially inhibited by receptor-interacting protein-1 (RIP1) inhibitor Necrostatin-1 partially.Western blot assay results showed that there were increases in the expressions of receptor-interacting protein-3 (RIP3) and mixed lineage kinase domain-like protein (MLKL) in the primary cortical neurons treated with 1 mM methamphetamine + 39℃ for 3 hours.After pre-treatment with RIP3 inhibitor GSK’872,propidium iodide staining and lactate dehydrogenase release detection showed that neuronal necrosis rate was significantly decreased;RIP3 and MLKL protein expression significantly decreased.Immunohistochemistry staining results also showed that the expressions of RIP3 and MLKL were up-regulated in brain specimens from humans who had died of methamphetamine abuse.Taken together,the above results suggest that methamphetamine + 39℃ can induce RIP3/MLKL regulated necroptosis,thereby resulting in neurotoxicity.The study protocol was approved by the Medical Ethics Committee of the Third Xiangya Hospital of Central South University,China (approval numbers: 2017-S026 and 2017-S033) on March 7,2017.
文摘The mixed lineage kinase domain-like(MLKL)protein is a key factor in tumor necrosis factor-induced necroptosis.Recent studies on necroptosis execution revealed a commitment role of MLKL in membrane disruption.However,our knowledge of how MLKL functions on membrane remains very limited.Here we demonstrate that MLKL forms cation channels that are permeable preferential y to Mg2+rather than Ca2+in the presence of Na+and K+.Moreover,the N-terminal domain containing six helices(H1-H6)is sufficient to form channels.Using the substituted cysteine accessibility method,we further determine that helix H1,H2,H3,H5 and H6 are transmembrane segments,while H4 is located in the cytoplasm.Finally,MLKL-induced membrane depolarization and cell death exhibit a positive correlation to its channel activity.The Mg2+-preferred permeability and five transmembrane segment topology distinguish MLKL from previously identified Mg2+-permeable channels and thus establish MLKL as a novel class of cation channels.
文摘目的构建呼吸道合胞病毒(RSV)感染气液相界面(ALI)培养的人支气管上皮细胞(hBEC)模型,为深入研究呼吸道病毒致病机制提供更接近体内环境的细胞模型;通过分析RSV感染对高迁移率族蛋白1(HMGB1)和磷酸化混合系列蛋白激酶样结构域(pMLKL)表达的影响,探讨RSV感染损伤支气管上皮的致病机制。方法将hBEC接种到Transwell膜上,进行液体浸没式培养,细胞汇合度达100%后转ALI培养,倒置显微镜观察细胞生长状态。细胞分化成熟后按照以下分组分别感染RSV病毒:6 h对照组,6 h感染复数(MOI)1.0组,6 h MOI 3.0组,24 h对照组,24 h MOI 1.0组,24 h MOI 3.0组。每组3个复孔。通过免疫荧光染色确认RSV感染效果和RSV感染对细胞核蛋白HMGB1和pMLKL表达的影响。结果经细胞扩展期液体浸没式培养4~10 d后,细胞汇合度达100%。转ALI培养约4周后,细胞条索状分布越来越清晰,且分泌肉眼可见的黏液,形成黏液层,将Transwell膜石蜡包埋切片,得到典型的假复层上皮HE染色结果。经抗RSV抗体免疫荧光染色确认,MOI为3.0感染24 h RSV病毒成功感染细胞;抗HMGB1和抗pMLKL免疫荧光双染色结果显示,该感染条件下,细胞核内出现粉色荧光[为蓝色4',6-二脒基-2-苯基吲哚(DAPI)和红色HMGB1荧光Merge结果],证实RSV感染后出现HMGB1核表达;而RSV感染前后均未见pMLKL表达。结论通过在Transwell膜上液体浸没式扩展培养和ALI分化培养,可获得分化良好的hBEC,且能较长时间维持其形态及功能,可为呼吸道病毒感染及其他常见呼吸道疾病研究提供更接近体内环境的细胞模型。在MOI 3.024 h条件下,RSV成功感染该细胞模型,并引起损伤相关分子蛋白HMGB1核表达。
