BACKGROUND： Glucose-regulated protein 78 （GRP78）, a marker of endoplasmic reticulum stress, can prolong cell survival. Alternatively, CCAAT enhancer-binding protein homologous protein （CHOP）, a transcription fact...BACKGROUND： Glucose-regulated protein 78 （GRP78）, a marker of endoplasmic reticulum stress, can prolong cell survival. Alternatively, CCAAT enhancer-binding protein homologous protein （CHOP）, a transcription factor specific for endoplasmic reticulum stress, can cause cell cycle arrest and cell apoptosis. OBJECTIVE： To study the protective effects of serum containing natural cerebrolysin on endoplasmic reticulum stress in tunicamycin-induced neuronal PC12 cells, and analyze the influence on GRP78 and CHOP expressions. DESIGN, TIME AND SETTING： A parallel controlled study was performed at the Institute of Integrated Western and Traditional Chinese Medicine, Shenzhen Hospital, Southern Medical University, between March 2006 and August 2008. MATERIALS： Adult Sprague-Dawley rats were perfused with natural Cerebrolysin aqueous extract （0.185 g/kg/d） to produce serum containing natural Cerebrolysin. Physiological saline was used to produce blank serum. PC12 cell line was provided by Shanghai Institute of Cell Biology, Chinese Academy of Science. Tunicamycin was provided by Sigma （St. Louis, USA）, and natural Cerebrolysin, containing ginseng, rhizoma gastrodiae, and gingko leaf （1：2：2）, by Shengzhen Institute of Integrated Western and Traditional Chinese Medicine. METHODS： PC12 cells were treated with DMEM culture media containing 10% blank serum （normal control group）, tunicamycin （1 μg/mL; model group）, and 5%, 10%, and 15% serum containing natural cerebrolysin and tunicamycin （1 μ g/mL; low-, moderate-, and high-dose serum containing natural cerebrotysin groups）, for 2 hours. MAIN OUTCOME MEASURES： PC12 cells were treated with tunicamycin for 48 hours after which apoptosis was measured using the TUNEL method to calculate apoptotic index. GRP78 expression was detected using immunocytochemistry. After 24 hours of treatment with tunicamycin, GRP78 and CHOP mRNA expressions were measured using RT-PCR. RESULTS： The apoptotic index and CHOP mRNA expression were in the model group and three cerebrolysin groups were significantly increased when compared to the normal control group （P 〈 0.05）. In contrast, GRP78 mRNA and protein expressions were significantly decreased （P 〈 0.05）. CONCLUSION： Serum containing natural cerebrolysin significantly reduced apoptosis in neuronal PC12 cells following tunicamycin-induced endoplasmic reticulum stress. These results may be related to an up-regulation of GRP78 expression and down-regulation of CHOP expression, both of which displayed dose-dependent effects.展开更多
BACKGROUND Chronic biliary obstruction results in ischemia and hypoxia of hepatocytes,and leads to apoptosis.Apoptosis is very important in regulating the homeostasis of the hepatobiliary system.Endoplasmic reticulum(...BACKGROUND Chronic biliary obstruction results in ischemia and hypoxia of hepatocytes,and leads to apoptosis.Apoptosis is very important in regulating the homeostasis of the hepatobiliary system.Endoplasmic reticulum(ER)stress is one of the signaling pathways that induce apoptosis.Moreover,the protein kinase RNA-like endoplasmic reticulum kinase(PERK)-induced apoptotic pathway is the main way;but its role in liver injury remains unclear.Yinchenhao decoction(YCHD)is a traditional Chinese medicine formula that alleviates liver injury and apoptosis,yet its mechanism is unknown.We undertook this study to investigate the effects of YCHD on the expression of ER stress proteins and hepatocyte apoptosis in rats with obstructive jaundice(OJ).AIM To investigate whether YCHD can attenuate OJ-induced liver injury and hepatocyte apoptosis by inhibiting the PERK-CCAAT/enhancer-binding protein homologous protein(CHOP)-growth arrest and DNA damage-inducible protein 34(GADD34)pathway and B cell lymphoma/leukemia-2 related X protein(Bax)/B cell lymphoma/leukemia-2(Bcl-2)ratio.METHODS For in vivo experiments,30 rats were divided into three groups:control group,OJ model group,and YCHD-treated group.Blood was collected to detect the indicators of liver function,and liver tissues were used for histological analysis.For in vitro experiments,30 rats were divided into three groups:G1,G2,and G3.The rats in group G1 had their bile duct exposed without ligation,the rats in group G2 underwent total bile duct ligation,and the rats in group G3 were given a gavage of YCHD.According to the serum pharmacology,serum was extracted and centrifuged from the rat blood to cultivate the BRL-3A cells.Terminal deoxynucleotidyl transferase mediated dUTP nick end-labelling(TUNEL)assay was used to detect BRL-3A hepatocyte apoptosis.Alanine aminotransferase(ALT)and aspartate transaminase(AST)levels in the medium were detected.Western blot and quantitative real-time polymerase chain reaction(qRT-PCR)analyses were used to detect protein and gene expression levels of PERK,CHOP,GADD34,Bax,and Bcl-2 in the liver tissues and BRL-3A cells.RESULTS Biochemical assays and haematoxylin and eosin staining suggested severe liver function injury and liver tissue structure damage in the OJ model group.The TUNEL assay showed that massive BRL-3A rat hepatocyte apoptosis was induced by OJ.Elevated ALT and AST levels in the medium also demonstrated that hepatocytes could be destroyed by OJ.Western blot or qRT-PCR analyses showed that the protein and mRNA expression levels of PERK,CHOP,and GADD34 were significantly increased both in the rat liver tissue and BRL-3A rat hepatocytes by OJ.The Bax and Bcl-2 levels were increased,and the Bax/Bcl-2 ratio was also increased.When YCHD was used,the PERK,CHOP,GADD34,and Bax levels quickly decreased,while the Bcl-2 levels increased,and the Bax/Bcl-2 ratio decreased.CONCLUSION OJ-induced liver injury and hepatocyte apoptosis are associated with the activation of the PERK-CHOP-GADD34 pathway and increased Bax/Bcl-2 ratio.YCHD can attenuate these changes.展开更多
Polycomb group proteins represent a global silencing system involved in development regulation.In specific,they regulate the transition from proliferation to differentiation,contributing to stem-cell maintenance and i...Polycomb group proteins represent a global silencing system involved in development regulation.In specific,they regulate the transition from proliferation to differentiation,contributing to stem-cell maintenance and inhibiting an inappropriate activation of differentiation programs.Enhancer of Zeste Homolog 2(EZH2) is the catalytic subunit of Polycomb repressive complex 2,which induces transcriptional inhibition through the tri-methylation of histone H3,an epigenetic change associated with gene silencing.EZH2 expression is high in precursor cells while its level decreases in differentiated cells.EZH2 is upregulated in various cancers with high levels associated with metastatic cancer and poor prognosis.Indeed,aberrant expression of EZH2 causes the inhibition of several tumor suppressors and differentiation genes,resulting in an uncontrolled proliferation and tumor formation.This editorial explores the role of Polycomb repressive complex 2 in cancer,focusing in particular on EZH2.The canonical function of EZH2 in gene silencing,the non-canonical activities as the methylation of other proteins and the role in gene transcriptional activation,were summarized.Moreover,mutations of EZH2,responsible for an increased methyltransferase activity in cancer,were recapitulated.Finally,various drugs able to inhibit EZH2 with different mechanism were described,specifically underscoring the effects in several cancers,in order to clarify the role of EZH2 and understand if EZH2 blockade could be a new strategy for developing specific therapies or a way to increase sensitivity of cancer cells to standard therapies.展开更多
Histone lysine methyltransferase EZH2 has been reported to be frequently overexpressed in hepatocellular carcinoma(HCC) tissues and associated with hepatocarcinogenesis.However,the exact mechanism of EZH2 up-regulatio...Histone lysine methyltransferase EZH2 has been reported to be frequently overexpressed in hepatocellular carcinoma(HCC) tissues and associated with hepatocarcinogenesis.However,the exact mechanism of EZH2 up-regulation in HCC has not been determined.In this study,we used murine hepatocyte AML12 cells to investigate the role of hepatitis B virus X protein(HBx) in regulating the expression of mEZH2.Western blot analysis demonstrated that the expression level of mEZH2 protein in AML12 cells was up-regulated by HBx in a dose-dependent manner.To further investigate the mechanism of mEZH2 overexpression,the 2500 bp regulatory sequence upstream from the first exon of the mEZH2 gene was amplified from AML12 genomic DNA and constructed into a luciferase reporter plasmid.The luciferase activity of the mEZH2 promoter significantly increased in AML12 cells co-transfected with HBx plasmid,and deleting the-486/-214 promoter region decreased HBx-induced mEZH2 promoter activation by nearly 50%.The-486/-214 region was then analyzed in the TRANSFAC 6.0 database and a typical E2F1-binding site was found.Mutation of this E2F1-binding site or knockdown of E2F1 expression by RNAi led to a dramatic decrease in HBx-induced activation of the mEZH2 promoter and mEZH2 overexpression in AML12 cells.These results provide evidence that HBx up-regulates mEZH2 expression by transactivating the mEZH2 promoter through E2F1 transcription factor,thereby providing new epigenetic evidence for the carcinogenic effect of HBx.展开更多
目的观察血压正常大鼠和自发性高血压大鼠全脑缺血后海马区CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein homologous protein,CHOP)的表达变化,探讨其在高血压加重脑缺血神经损伤中的作用。方法分别取血压正常大鼠和...目的观察血压正常大鼠和自发性高血压大鼠全脑缺血后海马区CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein homologous protein,CHOP)的表达变化,探讨其在高血压加重脑缺血神经损伤中的作用。方法分别取血压正常大鼠和自发性高血压大鼠,应用改良的Pulsineli 4血管阻断(4-VO)法制作全脑缺血再灌注模型。分别在脑缺血6、24、48h应用HE染色观察海马区神经细胞形态变化、免疫组织化学法和免疫印迹法检测海马区CHOP表达;在48h应用八臂迷宫检测动物行为学的变化。结果与血压正常脑缺血组比较,高血压脑缺血组各时间点存活神经细胞数量减少,6h和24hCHOP表达增多,48h减少;八臂迷宫检测动物总错误次数、参考记忆错误次数和工作记忆错误次数增多。结论高血压可加重脑缺血后神经损伤,其机制与加重CHOP表达增加有关。展开更多
目的观察CCAAT/增强子结合蛋白同源蛋白(CHOP)及钙联蛋白(CNX)在内侧颞叶癫痫小鼠海马区域中表达的时间和空间分布。方法采用海人酸(KA)诱导内侧颞叶癫痫小鼠模型,免疫印迹和免疫荧光技术检测CHOP和CNX在急性期(12、24 h)小鼠海马CA3区...目的观察CCAAT/增强子结合蛋白同源蛋白(CHOP)及钙联蛋白(CNX)在内侧颞叶癫痫小鼠海马区域中表达的时间和空间分布。方法采用海人酸(KA)诱导内侧颞叶癫痫小鼠模型,免疫印迹和免疫荧光技术检测CHOP和CNX在急性期(12、24 h)小鼠海马CA3区的表达量及分布差异,并与注射PBS的正常小鼠进行对照。结果免疫印迹检测结果显示,KA注射后12 h小鼠海马中的CHOP(F=1.136,P=0.4069)和CNX表达量(F=2.378,P=0.2087)与对照组差异没有统计学意义,KA注射后24 h注射侧海马中的CHOP(F=8.510,P=0.0362)和CNX表达量(F=6.968,P=0.0497)明显高于对照组。免疫荧光结果显示,KA注射后12 h CHOP的表达主要集中于CA3区,注射后24 h在CA1和CA3区表达水平均升高;KA注射后24 h CHOP蛋白(F=24.480,P=0.0057)和CNX蛋白(F=7.149,P=0.0478)的表达量显著高于对照组。结论伴随着癫痫发作的产生,CHOP蛋白表达量上升,提示神经元内质网应激水平不断增加,可能需要更多CNX作为分子伴侣帮助更多未折叠蛋白完成折叠过程。展开更多
To label embryonic stem (ES) cells with enhanced green fluorescent protein (EGF P) on the hypoxanthineguanine phosphoribosyl transferase (HPRT) gene locus for t he first time to provide a convenient and efficient way ...To label embryonic stem (ES) cells with enhanced green fluorescent protein (EGF P) on the hypoxanthineguanine phosphoribosyl transferase (HPRT) gene locus for t he first time to provide a convenient and efficient way for cell tracking and ma nipulation in the studies of transplantation and stem cell therapy Methods Homologous fragments were obtained by polymerase chain reaction (PCR), from whic h the gene targeting vector pHPRT EGFP was constructed The linearized vector was introduced into ES cells by electroporation The G418 r6TG r cell clones were obtained after selection with G418 and 6TG media The integration patterns of these resistant cell clones were identified with Southern blotting Results EGFP expressing ES cells on the locus of HPRT were successfu lly generated They have normal properties, such as karyotype, viability and di fferentiation ability The green fluorescence of EGFP expressing cells was main tained in propagation of the ES cells for more than 30 passages and in different iated cells Cultured in suspension, the 'green' ES cells aggregated and forme d embryoid bodies, retaining the green fluorescence at varying developmental sta ges The 'green' embryoid bodies could expand and differentiate into various t ypes of cells, exhibiting ubiquitous green fluorescence Conclusions This generation of 'green' targeted ES cells is described in an efficient proto col for obtaining the homologous fragments by PCR Introducing the marker gene in the genome of ES cells, we should be able to manipulate them in vitro and use them as vehicles in cell replacement therapy as well as for other biomedical a nd research purposes展开更多
目的:对Tribbles相关蛋白3(Tribbles related protein 3,TRB3)及CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein homologous protein,CHOP)在高脂高糖饮食诱导的大鼠非酒精性脂肪肝(nonalcoholic fatty liver disease,NA...目的:对Tribbles相关蛋白3(Tribbles related protein 3,TRB3)及CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein homologous protein,CHOP)在高脂高糖饮食诱导的大鼠非酒精性脂肪肝(nonalcoholic fatty liver disease,NAFLD)中的表达变化进行研究并探讨他们在NAFLD中的作用.方法:将30只Wistar大鼠随机分为正常组和NAFLD模型组,每组各15只.模型组大鼠采用高脂高糖饮食诱导NAFLD,正常组大鼠则给予普通饲料喂养,造模时间总计为16 wk.血清中总胆固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)、高密度脂蛋白(highdensity lipoprotein,HDL)和低密度脂蛋白(lowdensity lipoprotein,LDL)的含量采用全自动生化分析仪进行检测;应用PCR技术对肝脏中TRB3及CHOP m RNA水平的改变进行检测;应用免疫组织化学技术对肝脏中TRB3及CHOP蛋白水平的改变进行检测;采用流式细胞仪检测细胞凋亡改变.结果:模型组大鼠血清中TC、TG和LDL含量较正常组大鼠显著升高(P<0.05),而HDL则明显低于正常组(P<0.05);与正常组相比,模型组大鼠肝脏中TRB3及CHOP m RNA水平均显著增加(P<0.05或P<0.01);免疫组织化学结果显示模型组大鼠肝脏中TRB3及CHOP蛋白表达水平较正常组大鼠显著升高(P<0.01);此外流式细胞仪对大鼠肝细胞凋亡进行检测发现,模型组大鼠肝细胞凋亡与正常组相比明显增多.结论:TRB3和CHOP在基因及蛋白水平表达上调可能与高脂高糖诱导的NAFLD的发生发展有关.展开更多
基金Supported by:Scientific and Technological Foundation of the National Administration of Traditional Chinese Medicine of China,No.02-03LP41the Scientific and Technological Key Project of Guangdong Province,No. 2006B35630007
文摘BACKGROUND： Glucose-regulated protein 78 （GRP78）, a marker of endoplasmic reticulum stress, can prolong cell survival. Alternatively, CCAAT enhancer-binding protein homologous protein （CHOP）, a transcription factor specific for endoplasmic reticulum stress, can cause cell cycle arrest and cell apoptosis. OBJECTIVE： To study the protective effects of serum containing natural cerebrolysin on endoplasmic reticulum stress in tunicamycin-induced neuronal PC12 cells, and analyze the influence on GRP78 and CHOP expressions. DESIGN, TIME AND SETTING： A parallel controlled study was performed at the Institute of Integrated Western and Traditional Chinese Medicine, Shenzhen Hospital, Southern Medical University, between March 2006 and August 2008. MATERIALS： Adult Sprague-Dawley rats were perfused with natural Cerebrolysin aqueous extract （0.185 g/kg/d） to produce serum containing natural Cerebrolysin. Physiological saline was used to produce blank serum. PC12 cell line was provided by Shanghai Institute of Cell Biology, Chinese Academy of Science. Tunicamycin was provided by Sigma （St. Louis, USA）, and natural Cerebrolysin, containing ginseng, rhizoma gastrodiae, and gingko leaf （1：2：2）, by Shengzhen Institute of Integrated Western and Traditional Chinese Medicine. METHODS： PC12 cells were treated with DMEM culture media containing 10% blank serum （normal control group）, tunicamycin （1 μg/mL; model group）, and 5%, 10%, and 15% serum containing natural cerebrolysin and tunicamycin （1 μ g/mL; low-, moderate-, and high-dose serum containing natural cerebrotysin groups）, for 2 hours. MAIN OUTCOME MEASURES： PC12 cells were treated with tunicamycin for 48 hours after which apoptosis was measured using the TUNEL method to calculate apoptotic index. GRP78 expression was detected using immunocytochemistry. After 24 hours of treatment with tunicamycin, GRP78 and CHOP mRNA expressions were measured using RT-PCR. RESULTS： The apoptotic index and CHOP mRNA expression were in the model group and three cerebrolysin groups were significantly increased when compared to the normal control group （P 〈 0.05）. In contrast, GRP78 mRNA and protein expressions were significantly decreased （P 〈 0.05）. CONCLUSION： Serum containing natural cerebrolysin significantly reduced apoptosis in neuronal PC12 cells following tunicamycin-induced endoplasmic reticulum stress. These results may be related to an up-regulation of GRP78 expression and down-regulation of CHOP expression, both of which displayed dose-dependent effects.
基金Supported by the National Natural Science Foundation of China,No.81273952
文摘BACKGROUND Chronic biliary obstruction results in ischemia and hypoxia of hepatocytes,and leads to apoptosis.Apoptosis is very important in regulating the homeostasis of the hepatobiliary system.Endoplasmic reticulum(ER)stress is one of the signaling pathways that induce apoptosis.Moreover,the protein kinase RNA-like endoplasmic reticulum kinase(PERK)-induced apoptotic pathway is the main way;but its role in liver injury remains unclear.Yinchenhao decoction(YCHD)is a traditional Chinese medicine formula that alleviates liver injury and apoptosis,yet its mechanism is unknown.We undertook this study to investigate the effects of YCHD on the expression of ER stress proteins and hepatocyte apoptosis in rats with obstructive jaundice(OJ).AIM To investigate whether YCHD can attenuate OJ-induced liver injury and hepatocyte apoptosis by inhibiting the PERK-CCAAT/enhancer-binding protein homologous protein(CHOP)-growth arrest and DNA damage-inducible protein 34(GADD34)pathway and B cell lymphoma/leukemia-2 related X protein(Bax)/B cell lymphoma/leukemia-2(Bcl-2)ratio.METHODS For in vivo experiments,30 rats were divided into three groups:control group,OJ model group,and YCHD-treated group.Blood was collected to detect the indicators of liver function,and liver tissues were used for histological analysis.For in vitro experiments,30 rats were divided into three groups:G1,G2,and G3.The rats in group G1 had their bile duct exposed without ligation,the rats in group G2 underwent total bile duct ligation,and the rats in group G3 were given a gavage of YCHD.According to the serum pharmacology,serum was extracted and centrifuged from the rat blood to cultivate the BRL-3A cells.Terminal deoxynucleotidyl transferase mediated dUTP nick end-labelling(TUNEL)assay was used to detect BRL-3A hepatocyte apoptosis.Alanine aminotransferase(ALT)and aspartate transaminase(AST)levels in the medium were detected.Western blot and quantitative real-time polymerase chain reaction(qRT-PCR)analyses were used to detect protein and gene expression levels of PERK,CHOP,GADD34,Bax,and Bcl-2 in the liver tissues and BRL-3A cells.RESULTS Biochemical assays and haematoxylin and eosin staining suggested severe liver function injury and liver tissue structure damage in the OJ model group.The TUNEL assay showed that massive BRL-3A rat hepatocyte apoptosis was induced by OJ.Elevated ALT and AST levels in the medium also demonstrated that hepatocytes could be destroyed by OJ.Western blot or qRT-PCR analyses showed that the protein and mRNA expression levels of PERK,CHOP,and GADD34 were significantly increased both in the rat liver tissue and BRL-3A rat hepatocytes by OJ.The Bax and Bcl-2 levels were increased,and the Bax/Bcl-2 ratio was also increased.When YCHD was used,the PERK,CHOP,GADD34,and Bax levels quickly decreased,while the Bcl-2 levels increased,and the Bax/Bcl-2 ratio decreased.CONCLUSION OJ-induced liver injury and hepatocyte apoptosis are associated with the activation of the PERK-CHOP-GADD34 pathway and increased Bax/Bcl-2 ratio.YCHD can attenuate these changes.
文摘Polycomb group proteins represent a global silencing system involved in development regulation.In specific,they regulate the transition from proliferation to differentiation,contributing to stem-cell maintenance and inhibiting an inappropriate activation of differentiation programs.Enhancer of Zeste Homolog 2(EZH2) is the catalytic subunit of Polycomb repressive complex 2,which induces transcriptional inhibition through the tri-methylation of histone H3,an epigenetic change associated with gene silencing.EZH2 expression is high in precursor cells while its level decreases in differentiated cells.EZH2 is upregulated in various cancers with high levels associated with metastatic cancer and poor prognosis.Indeed,aberrant expression of EZH2 causes the inhibition of several tumor suppressors and differentiation genes,resulting in an uncontrolled proliferation and tumor formation.This editorial explores the role of Polycomb repressive complex 2 in cancer,focusing in particular on EZH2.The canonical function of EZH2 in gene silencing,the non-canonical activities as the methylation of other proteins and the role in gene transcriptional activation,were summarized.Moreover,mutations of EZH2,responsible for an increased methyltransferase activity in cancer,were recapitulated.Finally,various drugs able to inhibit EZH2 with different mechanism were described,specifically underscoring the effects in several cancers,in order to clarify the role of EZH2 and understand if EZH2 blockade could be a new strategy for developing specific therapies or a way to increase sensitivity of cancer cells to standard therapies.
文摘Histone lysine methyltransferase EZH2 has been reported to be frequently overexpressed in hepatocellular carcinoma(HCC) tissues and associated with hepatocarcinogenesis.However,the exact mechanism of EZH2 up-regulation in HCC has not been determined.In this study,we used murine hepatocyte AML12 cells to investigate the role of hepatitis B virus X protein(HBx) in regulating the expression of mEZH2.Western blot analysis demonstrated that the expression level of mEZH2 protein in AML12 cells was up-regulated by HBx in a dose-dependent manner.To further investigate the mechanism of mEZH2 overexpression,the 2500 bp regulatory sequence upstream from the first exon of the mEZH2 gene was amplified from AML12 genomic DNA and constructed into a luciferase reporter plasmid.The luciferase activity of the mEZH2 promoter significantly increased in AML12 cells co-transfected with HBx plasmid,and deleting the-486/-214 promoter region decreased HBx-induced mEZH2 promoter activation by nearly 50%.The-486/-214 region was then analyzed in the TRANSFAC 6.0 database and a typical E2F1-binding site was found.Mutation of this E2F1-binding site or knockdown of E2F1 expression by RNAi led to a dramatic decrease in HBx-induced activation of the mEZH2 promoter and mEZH2 overexpression in AML12 cells.These results provide evidence that HBx up-regulates mEZH2 expression by transactivating the mEZH2 promoter through E2F1 transcription factor,thereby providing new epigenetic evidence for the carcinogenic effect of HBx.
文摘目的观察血压正常大鼠和自发性高血压大鼠全脑缺血后海马区CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein homologous protein,CHOP)的表达变化,探讨其在高血压加重脑缺血神经损伤中的作用。方法分别取血压正常大鼠和自发性高血压大鼠,应用改良的Pulsineli 4血管阻断(4-VO)法制作全脑缺血再灌注模型。分别在脑缺血6、24、48h应用HE染色观察海马区神经细胞形态变化、免疫组织化学法和免疫印迹法检测海马区CHOP表达;在48h应用八臂迷宫检测动物行为学的变化。结果与血压正常脑缺血组比较,高血压脑缺血组各时间点存活神经细胞数量减少,6h和24hCHOP表达增多,48h减少;八臂迷宫检测动物总错误次数、参考记忆错误次数和工作记忆错误次数增多。结论高血压可加重脑缺血后神经损伤,其机制与加重CHOP表达增加有关。
文摘目的观察CCAAT/增强子结合蛋白同源蛋白(CHOP)及钙联蛋白(CNX)在内侧颞叶癫痫小鼠海马区域中表达的时间和空间分布。方法采用海人酸(KA)诱导内侧颞叶癫痫小鼠模型,免疫印迹和免疫荧光技术检测CHOP和CNX在急性期(12、24 h)小鼠海马CA3区的表达量及分布差异,并与注射PBS的正常小鼠进行对照。结果免疫印迹检测结果显示,KA注射后12 h小鼠海马中的CHOP(F=1.136,P=0.4069)和CNX表达量(F=2.378,P=0.2087)与对照组差异没有统计学意义,KA注射后24 h注射侧海马中的CHOP(F=8.510,P=0.0362)和CNX表达量(F=6.968,P=0.0497)明显高于对照组。免疫荧光结果显示,KA注射后12 h CHOP的表达主要集中于CA3区,注射后24 h在CA1和CA3区表达水平均升高;KA注射后24 h CHOP蛋白(F=24.480,P=0.0057)和CNX蛋白(F=7.149,P=0.0478)的表达量显著高于对照组。结论伴随着癫痫发作的产生,CHOP蛋白表达量上升,提示神经元内质网应激水平不断增加,可能需要更多CNX作为分子伴侣帮助更多未折叠蛋白完成折叠过程。
文摘To label embryonic stem (ES) cells with enhanced green fluorescent protein (EGF P) on the hypoxanthineguanine phosphoribosyl transferase (HPRT) gene locus for t he first time to provide a convenient and efficient way for cell tracking and ma nipulation in the studies of transplantation and stem cell therapy Methods Homologous fragments were obtained by polymerase chain reaction (PCR), from whic h the gene targeting vector pHPRT EGFP was constructed The linearized vector was introduced into ES cells by electroporation The G418 r6TG r cell clones were obtained after selection with G418 and 6TG media The integration patterns of these resistant cell clones were identified with Southern blotting Results EGFP expressing ES cells on the locus of HPRT were successfu lly generated They have normal properties, such as karyotype, viability and di fferentiation ability The green fluorescence of EGFP expressing cells was main tained in propagation of the ES cells for more than 30 passages and in different iated cells Cultured in suspension, the 'green' ES cells aggregated and forme d embryoid bodies, retaining the green fluorescence at varying developmental sta ges The 'green' embryoid bodies could expand and differentiate into various t ypes of cells, exhibiting ubiquitous green fluorescence Conclusions This generation of 'green' targeted ES cells is described in an efficient proto col for obtaining the homologous fragments by PCR Introducing the marker gene in the genome of ES cells, we should be able to manipulate them in vitro and use them as vehicles in cell replacement therapy as well as for other biomedical a nd research purposes
文摘目的:对Tribbles相关蛋白3(Tribbles related protein 3,TRB3)及CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein homologous protein,CHOP)在高脂高糖饮食诱导的大鼠非酒精性脂肪肝(nonalcoholic fatty liver disease,NAFLD)中的表达变化进行研究并探讨他们在NAFLD中的作用.方法:将30只Wistar大鼠随机分为正常组和NAFLD模型组,每组各15只.模型组大鼠采用高脂高糖饮食诱导NAFLD,正常组大鼠则给予普通饲料喂养,造模时间总计为16 wk.血清中总胆固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)、高密度脂蛋白(highdensity lipoprotein,HDL)和低密度脂蛋白(lowdensity lipoprotein,LDL)的含量采用全自动生化分析仪进行检测;应用PCR技术对肝脏中TRB3及CHOP m RNA水平的改变进行检测;应用免疫组织化学技术对肝脏中TRB3及CHOP蛋白水平的改变进行检测;采用流式细胞仪检测细胞凋亡改变.结果:模型组大鼠血清中TC、TG和LDL含量较正常组大鼠显著升高(P<0.05),而HDL则明显低于正常组(P<0.05);与正常组相比,模型组大鼠肝脏中TRB3及CHOP m RNA水平均显著增加(P<0.05或P<0.01);免疫组织化学结果显示模型组大鼠肝脏中TRB3及CHOP蛋白表达水平较正常组大鼠显著升高(P<0.01);此外流式细胞仪对大鼠肝细胞凋亡进行检测发现,模型组大鼠肝细胞凋亡与正常组相比明显增多.结论:TRB3和CHOP在基因及蛋白水平表达上调可能与高脂高糖诱导的NAFLD的发生发展有关.
