INTRODUCTIONOnly the liver has the great capability ofregeneration in mammal.Few hepatocytes are inthe phase of division in the normal liver of an adultmammal (including human beings),but theremaining hepatocytes can ...INTRODUCTIONOnly the liver has the great capability ofregeneration in mammal.Few hepatocytes are inthe phase of division in the normal liver of an adultmammal (including human beings),but theremaining hepatocytes can be induced to proliferatequickly by partial hepatectomy (PH),and,to somedegree,they stop dividing and re-differentiate intocells functioning as hepatocytes.This shows展开更多
Severe fever with thrombocytopenia syndrome(SFTS)caused by the SFTS virus(SFTSV)is an emerging disease in East Asia with a fatality rate of up to 30%.However,the viral-host interaction of SFTSV remains largely unknown...Severe fever with thrombocytopenia syndrome(SFTS)caused by the SFTS virus(SFTSV)is an emerging disease in East Asia with a fatality rate of up to 30%.However,the viral-host interaction of SFTSV remains largely unknown.The heat-shock protein 90(Hsp90)family consists of highly conserved chaperones that fold and remodel proteins and has a broad impact on the infection of many viruses.Here,we showed that Hsp90 is an important host factor involved in SFTSV infection.Hsp90 inhibitors significantly reduced SFTSV replication,viral protein expression,and the formation of inclusion bodies consisting of nonstructural proteins(NSs).Among viral proteins,NSs appeared to be the most reduced when Hsp90 inhibitors were used,and further analysis showed that their translation was affected.Co-immunoprecipitation of NSs with four isomers of Hsp90 showed that Hsp90βspecifically interacted with them.Knockdown of Hsp90βexpression also inhibited replication of SFTSV.These results suggest that Hsp90βplays a critical role during SFTSV infection and could be a potential target for the development of drugs against SFTS.展开更多
喉癌是上呼吸道常见的恶性肿瘤,严重影响着人类的健康。研究表明热休克蛋白70(heat shock protein 70,HSP70)在正常组织中多不表达,但在包括头颈鳞状细胞癌在内的多种肿瘤组织却有异常表达。肿瘤细胞中高表达的HSP70,对细胞的增殖、侵...喉癌是上呼吸道常见的恶性肿瘤,严重影响着人类的健康。研究表明热休克蛋白70(heat shock protein 70,HSP70)在正常组织中多不表达,但在包括头颈鳞状细胞癌在内的多种肿瘤组织却有异常表达。肿瘤细胞中高表达的HSP70,对细胞的增殖、侵袭及转移、机体对肿瘤治疗耐受性的发生及肿瘤预后等方面均有很重要的作用[1]。RNA干扰(RNA interference,RNAi)技术是近年来发展起来的一项新的基因技术。展开更多
To study the endocytic activity of dendritic cells(DCs)by obtaining fusion protein HSP70-EGFP as exogenous antigen and loading it with DCs derived from human peripheral blood.Fusion protein HSP70-EGFP was prokaryotica...To study the endocytic activity of dendritic cells(DCs)by obtaining fusion protein HSP70-EGFP as exogenous antigen and loading it with DCs derived from human peripheral blood.Fusion protein HSP70-EGFP was prokaryotically expressed,isolated and purified.DCs were isolated and cultured from human peripheral blood.The DCs were divided into 3 groups in the endocytic experiment.There were 106 DCs in each group.Group 1 and 2 were respectively incubated for 30 min.with HSP70-EGFP and EGFP.Group 3 was incubated with HSP70 for 30 min,and then incubated for 30 min.with HSP70-EGFP.Subsequently,3 groups were placed in an incubator at 37℃for 0.5,1,2 and 24 h.Flow cytometry(FCM)was adopted to detect the amount of DCs with EGFP inside.IL-12 Eli-spot was adopted to detect the amount of DCs which secreted IL-12.There were 5 types in the experiment:LPS,inactive LPS,HSP70-EGFP,EGFP and no antigen.Fusion protein HSP70-EGFP was successfully obtained and its molecular weight was 97000.It accounted for 35.32%of the total protein.Under irradiation of an ultraviolet lamp,the protein solution sent out viridescent fluorescence.The result detected by FCM indicated that after incubation for 0.5 h at 37℃,the positive rate in group 1 was 63%,while the other 2 groups were negative.After incubation for 1,2 and 24 h at 37℃,the positive rates in the 3 groups were above 80%.The IL-12 Eli-spot examination shows that with HSP70-EGFP being loaded,the amount of DCs secreting IL-12 was 134.09±31.78/10^(5)cells,a little lower than that of DCs with LPS loaded(with the average point of 156.36±15.73).There was no significant difference between the 2 groups(P<0.01).By contrast,both of them were significantly higher than inactive LPS-(33.78±1.40)/10^(5)cells and EGFP-loaded(23.13±4.57)/105 cells DC groups in the amount of DCs secreting IL-12(P<0.01).The results suggest that receptor-mediated phagocytosis plays a main role in the preliminary stage of DCs internalizing HSP70-EGFP.With increasing incubation time,pinocytosis begins to dominate.HSP70-EGFP may promote DCs to secret cell factor IL-12.展开更多
OBJECTIVE: To examine if the ultra-highly diluted homeopathic remedy Thuja 30C can ameliorate benzo(a)pyrene (BaP)-induced DNA damage, stress and viability of perfused lung cells of Swiss albino mice in vitro. ME...OBJECTIVE: To examine if the ultra-highly diluted homeopathic remedy Thuja 30C can ameliorate benzo(a)pyrene (BaP)-induced DNA damage, stress and viability of perfused lung cells of Swiss albino mice in vitro. METHODS: Perfused normal lung cells from mice were cultured in 5% Roswell Park Memorial Institute medium and exposed to BaP, a potent carcinogen, at the half maximal inhibitory concentration dose (2.2 μmol/L) for 24 h. Thereafter, the intoxicated cells were either treated with Thuja 30C (used against tumor or cancer) or its vehicle media, succussed alcohol 30C. Relevant parameters of study involving reactive oxygen species (ROS) accumulation, total glutathione (GSH) content, and generations of heat shock protein (hsp)-90 were measured; the cell viability and other test parameters were measured after treatment with either Thuja 30C or its vehicle media. Circular dichroism spectroscopy was performed to examine if Thuja 30C directly interacted with calf thymus DNA as target. For ascertaining if DNA damaged by BaP could be partially repaired and restituted by the remedy, 4',6-diamidino-2-phenylindole staining was performed. RESULTS: Thuja 30C increased cell viability of BaP-intoxicated cells significantly, as compared to drug-untreated or drug-vehicle control. A minimal dose of Thuja 30C significantly inhibited BaP-induced stress level, by down-regulating ROS and hsp-90, and increasing GSH content. Thuja 30C itself had no DNA-damaging effect, and no direct drug-DNA interaction. However, it showed quite striking ability to repair DNA damage caused by BaP. CONCLUSION: Thuja 30C ameliorates BaP-induced toxicity, stress and DNA damage in perfused lung cells of mice and it apparently has no effect on normal lung cells.展开更多
The purpose of this work was to construct the plasmid that could direct the synthesis of siRNA-like transcripts and thus mediate strong and specific repression of human heat shock protein 90β(Hsp90β)gene expression ...The purpose of this work was to construct the plasmid that could direct the synthesis of siRNA-like transcripts and thus mediate strong and specific repression of human heat shock protein 90β(Hsp90β)gene expression and to compare the transfection efficiency of the plasmids in varying conditions of transfection.Three 64 nt oligos corresponding to different regions of the target gene were chemically synthesized and annealed and were then ligated with pSUPER EGFP1 plasmid and double-digested with HindIII and BglII.Recombinant plasmids were transformed into Escherichia coli,DH5a,and the colonies were picked and grown in the Amp-agarose.The presence of positive clones was checked by the means of endodigestion and sequencing.Three cell strains,HepG2,Human umbilicus vein endothelium cells(HUVEC)and HeK293,were cultured.Then the plasmids were transfected into the cells at different ratios of plasmid to Lipofectamine.The transfection efficiency was measured by detection of enhanced green fluorescence protein(EGFP).The presence of positive recombinant clones were verified by double-digestion and sequencing.The bases inserted into the plasmids were correct and the positive colonies were named pSuper-Hsp90β1,pSuper-Hsp90β2 and pSuper-Hsp90β3.After optimizing the ratio of plasmid to Lipofectamine,we achieved high transfection efficiency in HeK293 cells.Transfection efficiency was still low in the HepG2 cells.In conclusion,the si-RNA-synthesizing plasmids targeting Hsp90βwere constructed and transfected into cells with different transfection efficiency.展开更多
OBJECTIVE: To investigate the effects of extracts from Renshen(Radix Ginseng), Sanqi(Radix Notoginseng), and Chuanxiong(Rhizoma Chuanxiong) on the endothelial actin cytoskeleton in senescent human cardiac microvascula...OBJECTIVE: To investigate the effects of extracts from Renshen(Radix Ginseng), Sanqi(Radix Notoginseng), and Chuanxiong(Rhizoma Chuanxiong) on the endothelial actin cytoskeleton in senescent human cardiac microvascular endothelial cells(HCMECs), and to propose the possible mechanism underlying the actions.METHODS: Lentiviral mediated RNA interference was applied to a replicative senescent HCMEC model by knocking down heat shock protein 27(HSP27)gene. Cells were treated with extracts from Renshen(Radix Ginseng), Sanqi(Radix Notoginseng), and Chuanxiong(Rhizoma Chuanxiong) at final concentrations of 50, 100, and 200 mg/L, respectively and with 10 μM resveratrol for 48 h. Untreated cells were used as controls. Senescence was detected by senescence β-galactosidase staining and cell proliferation was analyzed by cell counting kit-8 assays.Secreted nitric oxide levels were detected by nitrate reductase. Morphological changes of F-actin and G-actin were observed by laser scanning confocal microscopy. Protein and gene expression of Factin and HSP27 was detected by western blotting.RESULTS: Compared with the control group, the proportion of senescent HSP27 shRNA cells treated with the extracts was decreased and their proliferation was increased. In the extract intervention group, F-actin around the cell periphery became irregular and jagged fractures formed gradually and then dissipated. Moreover, some dynamic actin stress fiber filaments appeared. The G-actin stretched to the cell periphery and punctate staining was scattered in the cytoplasm. In addition, the mean optical density value of F/G-actin was decreased significantly and the protein expression of F-actin was downregulated.CONCLUSION: The extracts delayed microvascular endothelial cell senescence by downregulating the expression of F-actin through HSP27.展开更多
Violence against the neck can result in a range of macromorphological and micromorphological findings.However,the forensic relevance of the carotid sinus in cases of violence against the neck remains controversial.In ...Violence against the neck can result in a range of macromorphological and micromorphological findings.However,the forensic relevance of the carotid sinus in cases of violence against the neck remains controversial.In this follow-up study of 22 cases of suicidal and accidental strangulations,carotid bifurcations were examined histologically for morphological changes implying direct trauma,including haemorrhage and immunohistochemical expression of heat-shock proteins 27,60,and 70 and aquaporin-3.These cases were compared with a control group(82 cases)without neck compression or head trauma and with variable causes of death.No relevant histopathological findings implying direct trauma of the carotid bifurcation were found.No cases showed positive aquaporin-3 staining and only five cases showed positive heat-shock protein-27 staining,all of which were hangings.Without massive trauma of the carotid bifurcation,histological alterations cannot be expected.Without signs of rapid death,findings of acute circulatory failure,macromorphological and micromorphological findings of neck compression,and reliable markers indicating relevant impact on the carotid bifurcation the diagnosis of a lethal reflex cannot be verified.展开更多
基金China-France Scientific end Technical Cooperation (No.1996-134)Bioengineering Key Laboratory of Henan Province
文摘INTRODUCTIONOnly the liver has the great capability ofregeneration in mammal.Few hepatocytes are inthe phase of division in the normal liver of an adultmammal (including human beings),but theremaining hepatocytes can be induced to proliferatequickly by partial hepatectomy (PH),and,to somedegree,they stop dividing and re-differentiate intocells functioning as hepatocytes.This shows
基金supported by grants from the National Natural Science Foundation of China(31900146)the key Biosafety Science and Technology Program of Hubei Jiangxia Laboratory(JXBS001)+1 种基金the Hubei Natural Science Foundation for Distinguished Young Scholars(2021CFA050)the Creative Research Group Program of Natural Science Foundation of Hubei Province(2022CFA021).
文摘Severe fever with thrombocytopenia syndrome(SFTS)caused by the SFTS virus(SFTSV)is an emerging disease in East Asia with a fatality rate of up to 30%.However,the viral-host interaction of SFTSV remains largely unknown.The heat-shock protein 90(Hsp90)family consists of highly conserved chaperones that fold and remodel proteins and has a broad impact on the infection of many viruses.Here,we showed that Hsp90 is an important host factor involved in SFTSV infection.Hsp90 inhibitors significantly reduced SFTSV replication,viral protein expression,and the formation of inclusion bodies consisting of nonstructural proteins(NSs).Among viral proteins,NSs appeared to be the most reduced when Hsp90 inhibitors were used,and further analysis showed that their translation was affected.Co-immunoprecipitation of NSs with four isomers of Hsp90 showed that Hsp90βspecifically interacted with them.Knockdown of Hsp90βexpression also inhibited replication of SFTSV.These results suggest that Hsp90βplays a critical role during SFTSV infection and could be a potential target for the development of drugs against SFTS.
文摘喉癌是上呼吸道常见的恶性肿瘤,严重影响着人类的健康。研究表明热休克蛋白70(heat shock protein 70,HSP70)在正常组织中多不表达,但在包括头颈鳞状细胞癌在内的多种肿瘤组织却有异常表达。肿瘤细胞中高表达的HSP70,对细胞的增殖、侵袭及转移、机体对肿瘤治疗耐受性的发生及肿瘤预后等方面均有很重要的作用[1]。RNA干扰(RNA interference,RNAi)技术是近年来发展起来的一项新的基因技术。
基金The study was supported by the General Program of National Natural Science Foundation of China(Grant No.30400167).
文摘To study the endocytic activity of dendritic cells(DCs)by obtaining fusion protein HSP70-EGFP as exogenous antigen and loading it with DCs derived from human peripheral blood.Fusion protein HSP70-EGFP was prokaryotically expressed,isolated and purified.DCs were isolated and cultured from human peripheral blood.The DCs were divided into 3 groups in the endocytic experiment.There were 106 DCs in each group.Group 1 and 2 were respectively incubated for 30 min.with HSP70-EGFP and EGFP.Group 3 was incubated with HSP70 for 30 min,and then incubated for 30 min.with HSP70-EGFP.Subsequently,3 groups were placed in an incubator at 37℃for 0.5,1,2 and 24 h.Flow cytometry(FCM)was adopted to detect the amount of DCs with EGFP inside.IL-12 Eli-spot was adopted to detect the amount of DCs which secreted IL-12.There were 5 types in the experiment:LPS,inactive LPS,HSP70-EGFP,EGFP and no antigen.Fusion protein HSP70-EGFP was successfully obtained and its molecular weight was 97000.It accounted for 35.32%of the total protein.Under irradiation of an ultraviolet lamp,the protein solution sent out viridescent fluorescence.The result detected by FCM indicated that after incubation for 0.5 h at 37℃,the positive rate in group 1 was 63%,while the other 2 groups were negative.After incubation for 1,2 and 24 h at 37℃,the positive rates in the 3 groups were above 80%.The IL-12 Eli-spot examination shows that with HSP70-EGFP being loaded,the amount of DCs secreting IL-12 was 134.09±31.78/10^(5)cells,a little lower than that of DCs with LPS loaded(with the average point of 156.36±15.73).There was no significant difference between the 2 groups(P<0.01).By contrast,both of them were significantly higher than inactive LPS-(33.78±1.40)/10^(5)cells and EGFP-loaded(23.13±4.57)/105 cells DC groups in the amount of DCs secreting IL-12(P<0.01).The results suggest that receptor-mediated phagocytosis plays a main role in the preliminary stage of DCs internalizing HSP70-EGFP.With increasing incubation time,pinocytosis begins to dominate.HSP70-EGFP may promote DCs to secret cell factor IL-12.
基金financially supported by a grant sanctioned to Prof.A.R.Khuda-Bukhsh,Department of Zoology, University of Kalyani,India,by Boiron Laboratories,Lyon, France
文摘OBJECTIVE: To examine if the ultra-highly diluted homeopathic remedy Thuja 30C can ameliorate benzo(a)pyrene (BaP)-induced DNA damage, stress and viability of perfused lung cells of Swiss albino mice in vitro. METHODS: Perfused normal lung cells from mice were cultured in 5% Roswell Park Memorial Institute medium and exposed to BaP, a potent carcinogen, at the half maximal inhibitory concentration dose (2.2 μmol/L) for 24 h. Thereafter, the intoxicated cells were either treated with Thuja 30C (used against tumor or cancer) or its vehicle media, succussed alcohol 30C. Relevant parameters of study involving reactive oxygen species (ROS) accumulation, total glutathione (GSH) content, and generations of heat shock protein (hsp)-90 were measured; the cell viability and other test parameters were measured after treatment with either Thuja 30C or its vehicle media. Circular dichroism spectroscopy was performed to examine if Thuja 30C directly interacted with calf thymus DNA as target. For ascertaining if DNA damaged by BaP could be partially repaired and restituted by the remedy, 4',6-diamidino-2-phenylindole staining was performed. RESULTS: Thuja 30C increased cell viability of BaP-intoxicated cells significantly, as compared to drug-untreated or drug-vehicle control. A minimal dose of Thuja 30C significantly inhibited BaP-induced stress level, by down-regulating ROS and hsp-90, and increasing GSH content. Thuja 30C itself had no DNA-damaging effect, and no direct drug-DNA interaction. However, it showed quite striking ability to repair DNA damage caused by BaP. CONCLUSION: Thuja 30C ameliorates BaP-induced toxicity, stress and DNA damage in perfused lung cells of mice and it apparently has no effect on normal lung cells.
基金The study was supported by grants from the National Excellent Doctorate Dissertation Author Specific Foundation Program of China(No.200156)the National Natural Science Foundation of China(Grant No.30470988).
文摘The purpose of this work was to construct the plasmid that could direct the synthesis of siRNA-like transcripts and thus mediate strong and specific repression of human heat shock protein 90β(Hsp90β)gene expression and to compare the transfection efficiency of the plasmids in varying conditions of transfection.Three 64 nt oligos corresponding to different regions of the target gene were chemically synthesized and annealed and were then ligated with pSUPER EGFP1 plasmid and double-digested with HindIII and BglII.Recombinant plasmids were transformed into Escherichia coli,DH5a,and the colonies were picked and grown in the Amp-agarose.The presence of positive clones was checked by the means of endodigestion and sequencing.Three cell strains,HepG2,Human umbilicus vein endothelium cells(HUVEC)and HeK293,were cultured.Then the plasmids were transfected into the cells at different ratios of plasmid to Lipofectamine.The transfection efficiency was measured by detection of enhanced green fluorescence protein(EGFP).The presence of positive recombinant clones were verified by double-digestion and sequencing.The bases inserted into the plasmids were correct and the positive colonies were named pSuper-Hsp90β1,pSuper-Hsp90β2 and pSuper-Hsp90β3.After optimizing the ratio of plasmid to Lipofectamine,we achieved high transfection efficiency in HeK293 cells.Transfection efficiency was still low in the HepG2 cells.In conclusion,the si-RNA-synthesizing plasmids targeting Hsp90βwere constructed and transfected into cells with different transfection efficiency.
基金Supported by the National Natural Science Foundation of China(the Study on Intervention and Mechanism of Chinese Herbal Medicines for Nourishing Qi and Resolving Blood Stasis on Cytoskeletal Protein of Vascular Aging(No.81273976)the Mechanism of Exploration about Formula for Reinforcing Qi,Warming Yang,Activating Blood and Promoting Diuresis in the Prevention and Treatment of Heart Failure through mT ORC1-Mitophagy Pathway(No.8170-3865)+2 种基金Independent Research Program of the China Academy of Chinese Medical Sciences(the Study on Mechanism of Chinese Herbal Medicines for Nourishing Qi and Resolving Blood Stasis Delaying Vascular Aging(No.zz2013002)the Mechanism of Exploration about Formula for Reinforcing Qi,Warming Yang,Activating Blood and Promoting Diuresis in the Prevention and Treatment of Heart Failure through Nrf2-iron Death Pathway(No.zz13-YQ-034)Beijing Science and Technology Bureau(Clinical study about Formula for Reinforcing Qi,Warming Yang,Activating Blood and Promoting Diuresis to Improve the Survival Status of Patients with Chronic Heart Failure(No.Z171100001017108)。
文摘OBJECTIVE: To investigate the effects of extracts from Renshen(Radix Ginseng), Sanqi(Radix Notoginseng), and Chuanxiong(Rhizoma Chuanxiong) on the endothelial actin cytoskeleton in senescent human cardiac microvascular endothelial cells(HCMECs), and to propose the possible mechanism underlying the actions.METHODS: Lentiviral mediated RNA interference was applied to a replicative senescent HCMEC model by knocking down heat shock protein 27(HSP27)gene. Cells were treated with extracts from Renshen(Radix Ginseng), Sanqi(Radix Notoginseng), and Chuanxiong(Rhizoma Chuanxiong) at final concentrations of 50, 100, and 200 mg/L, respectively and with 10 μM resveratrol for 48 h. Untreated cells were used as controls. Senescence was detected by senescence β-galactosidase staining and cell proliferation was analyzed by cell counting kit-8 assays.Secreted nitric oxide levels were detected by nitrate reductase. Morphological changes of F-actin and G-actin were observed by laser scanning confocal microscopy. Protein and gene expression of Factin and HSP27 was detected by western blotting.RESULTS: Compared with the control group, the proportion of senescent HSP27 shRNA cells treated with the extracts was decreased and their proliferation was increased. In the extract intervention group, F-actin around the cell periphery became irregular and jagged fractures formed gradually and then dissipated. Moreover, some dynamic actin stress fiber filaments appeared. The G-actin stretched to the cell periphery and punctate staining was scattered in the cytoplasm. In addition, the mean optical density value of F/G-actin was decreased significantly and the protein expression of F-actin was downregulated.CONCLUSION: The extracts delayed microvascular endothelial cell senescence by downregulating the expression of F-actin through HSP27.
文摘Violence against the neck can result in a range of macromorphological and micromorphological findings.However,the forensic relevance of the carotid sinus in cases of violence against the neck remains controversial.In this follow-up study of 22 cases of suicidal and accidental strangulations,carotid bifurcations were examined histologically for morphological changes implying direct trauma,including haemorrhage and immunohistochemical expression of heat-shock proteins 27,60,and 70 and aquaporin-3.These cases were compared with a control group(82 cases)without neck compression or head trauma and with variable causes of death.No relevant histopathological findings implying direct trauma of the carotid bifurcation were found.No cases showed positive aquaporin-3 staining and only five cases showed positive heat-shock protein-27 staining,all of which were hangings.Without massive trauma of the carotid bifurcation,histological alterations cannot be expected.Without signs of rapid death,findings of acute circulatory failure,macromorphological and micromorphological findings of neck compression,and reliable markers indicating relevant impact on the carotid bifurcation the diagnosis of a lethal reflex cannot be verified.