Objectives Phenotypic switching of smooth muscle cells(SMCs) plays a critical role in the pathogenesis of atherosclerotic lesions such as coronary artery disease (CAD).Accumulating evidence demonstrates(hat a cellular...Objectives Phenotypic switching of smooth muscle cells(SMCs) plays a critical role in the pathogenesis of atherosclerotic lesions such as coronary artery disease (CAD).Accumulating evidence demonstrates(hat a cellular repressor of E1A-stimulated genes(CREG) plays a role in the maintenance of the mature phenotype of vascular SMCs. The purpose of the present study was to assess the possible association between CREG and CAD in the Han population of North China.Methods The promoter region of CREG by direct sequencing was conducted in 48 subjects.Then SNP rs2995073 and another 4 tagSNPs(rs4657669,rs3767443, rsl6859185,rs3753921) were selected for the association study.All five selected SNPs were determined in 1161 patients with angiographically proven CAD and 960 controls with normal coronary angiograms to investigate the possible involvement of CREG in CAD.Results Genotype frequencies of the five examined polymorphisms were similarly distributed between CAD group and controls(P】0.05).Further haplotype analysis also found no significant differences in the distributions between CAD group and controls(P】0.05). Conclusions This study did not show an association between common variants of CREG and CAD in the northern Chinese Han population.展开更多
Background Cellular Repressor of E1A-stimu-lated gene(CREG) is widely expressed in adult tissues such as the brain,heart,lung,liver,intestine and kidney in mice.It is not known whether tissue CREG is decreased in the ...Background Cellular Repressor of E1A-stimu-lated gene(CREG) is widely expressed in adult tissues such as the brain,heart,lung,liver,intestine and kidney in mice.It is not known whether tissue CREG is decreased in the common setting of myocardial infarction which may lead to heart failure.We studied the expression and protein localization of CREG and its main receptor(IFR2R) in a mouse model of myocardial infarction.Methods Male mice were randomized to proximal left anterior descending ligation.The animals were killed on day 1,3,7,14,and 28 after ligation to examine gene expression and protein production of CREG and IGF2R from the infarct,peri-infarct,and contralateral zones of infarcted heart.Results There was decreased CREG mRNA production throughout the myocardium at dav 1,and the expression gradually increased at day 28 after myocardial infarction.The decreased expression of this glycoprotein was not confined strictly to the infarct or peri-infarct zones but also expressed by cardiac myocytes within the myocardium in the contralateral normal zone.Levels of CREG protein in the infarct and peri-infarct zones declined to 1/3- to 1/2-fold of normal levels and declined to 1/2- to 2/3- fold in the contralateral zone.Finally,the expression of the IGF2R mRNA transcripts was downregulated at day 3 and 7 after ligation in the infarct and peri-infarct zones,suggesting that the signal transduction pathways necessary for CREG in the heart remain intact as CREG biosynthesis decreases. Conclusions CREG is constantly present in a model of large myocardial infarction and is decreased at the early stage within the myocardium.The decreased expression of this glycoprotein is not only confined strictly to the infarct or periinfarct zone but also is expressed by cardiac myocytes within the myocardium contralateral to the infarct.Therefore CREG production decreased due to myocardial stress response to injury.展开更多
目的探讨E1A激活基因阻遏子(CREG1)蛋白能否改善心肌纤维化小鼠的心功能。方法应用基因打靶方法建立广泛性基因敲除的CREG1杂合子小鼠和CREG1野生型小鼠模型。应用血管紧张素Ⅱ(AngⅡ)皮下埋泵方法建立小鼠心肌纤维化损伤模型,给予Ang...目的探讨E1A激活基因阻遏子(CREG1)蛋白能否改善心肌纤维化小鼠的心功能。方法应用基因打靶方法建立广泛性基因敲除的CREG1杂合子小鼠和CREG1野生型小鼠模型。应用血管紧张素Ⅱ(AngⅡ)皮下埋泵方法建立小鼠心肌纤维化损伤模型,给予AngⅡ刺激14d后,采用HE和Masson染色检测小鼠心肌纤维化情况。应用Western blotting和免疫组化染色技术检测给予AngⅡ前及3、7、14d后两组小鼠心肌中CREG1蛋白的表达,并于给予AngⅡ14d后应用小动物超声仪检测心功能情况。AngⅡ给药同时,以皮下埋泵方式分别给予15、30、60、300μg/(kg·d)的外源性重组CREG1蛋白(治疗组)和生理盐水(对照组)14d,检测心功能,并应用TUNEL染色和Western blotting检测心肌凋亡情况。结果 Western blotting和免疫组化检测结果显示,未给予AngⅡ刺激时杂合子小鼠心肌中CREG1蛋白表达明显低于野生型小鼠(P<0.05)。给予AngⅡ刺激3、7、14d时,野生型小鼠和杂合子小鼠心肌中CREG1蛋白表达均明显下降(P<0.05),但杂合子小鼠下降更为显著(P<0.01);HE和Masson染色显示杂合子小鼠心肌纤维化程度较野生型小鼠严重,二者心功能明显下降,且杂合子小鼠心功能下降更为明显(P<0.05)。给予外源性重组CREG1蛋白治疗后,治疗组心功能较对照组明显改善(P<0.05),心肌凋亡数量明显下降(P<0.05)。结论在AngⅡ引起的小鼠心肌纤维化模型中,CREG1蛋白减少可使小鼠心功能损伤加重,给予外源性重组CREG1蛋白可明显改善心功能。展开更多
目的探讨E1A激活基因阻遏子(CREG)在动脉粥样硬化(AS)血管中的表达及其与炎症因子的关系。方法 Apo E(-/-)小鼠6周龄断奶后给予高脂喂养。8周后取主动脉血管,采用HE染色观察血管的形态学变化;采用免疫荧光染色观察CREG、SMα-actin和巨...目的探讨E1A激活基因阻遏子(CREG)在动脉粥样硬化(AS)血管中的表达及其与炎症因子的关系。方法 Apo E(-/-)小鼠6周龄断奶后给予高脂喂养。8周后取主动脉血管,采用HE染色观察血管的形态学变化;采用免疫荧光染色观察CREG、SMα-actin和巨噬细胞标志物Mac-3的表达变化。取高脂喂养的1~8周小鼠血管,采用Western blot方法检测AS血管中CREG及核转录因子(NF)-κB的表达的变化。结果在Apo E(-/-)小鼠高脂喂养8周,主动脉血管AS斑块明显形成,斑块内有胆固醇结晶,斑块凸凹不平,管腔明显狭窄。免疫荧光显示AS血管中,CREG表达明显下降,同时SMα-actin表达也下降,而Mac-3表达增高。Apo E(-/-)小鼠高脂喂养2~8周,取动脉血管行蛋白定量分析,结果显示高脂喂养2周时,CREG在动脉血管中的表达不是降低,而是明显升高,高脂喂养第4周,CREG表达急剧下降,而后又逐渐上升,但不能升至正常水平。同时NF-κB随着时间的推移,表达逐渐升高。结论在AS进程中,血管中膜的VSMCs由收缩表型向合成表型转化,细胞增生活跃、分化减弱,CREG作为维持VSMCs分化的蛋白,在血管中的表达与AS进展密切相关,同时伴随着炎症因子表达逐渐升高。展开更多
Objectives The cellular repressor of E1A-activated genes (CREG), a novel gene, was recently found to play a role in inhibiting cell growth and promoting cell differentiation. The purpose of this study was to obtain an...Objectives The cellular repressor of E1A-activated genes (CREG), a novel gene, was recently found to play a role in inhibiting cell growth and promoting cell differentiation. The purpose of this study was to obtain antibody against CREG protein and to study the expression of CREG protein in human internal thoracic artery cells (HITASY) which express different patterns of differentiation markers after serum withdrawal. Methods The open reading frame of CREG gene sequence was amplified by PCR and cloned into the pGEX-4T-1 vector. Glutathione-S-transferase (GST)-CREG fusion protein was expressed in E. Coli BL21 and purified from inclusion bodies by Sephacryl S-200 chromatography. Rabbits were immunized with the purified GST-CREG protein. Western blot examined with immunohistochemistry staining and the protein expression level was analyzed by Western blot in HITASY cells after serum removal. Results It was confirmed by using endonuclease digesting and DNA sequencing that the PCR product of CREG was correctly inserted into the vector. The GST-CREG protein was purified with gel filtration chromatography. Polyclonal antibody against GST-CREG was obtained from rabbits. CREG protein immunohistochemistry staining displayed a perinuclear distribution in the cytoplasm of HITASY cells. Results from Western blot suggested that comparing with the untreated cells upregulation of CREG polyclonal antibody against CREG was comfirmed. Using this antibody, the changes of CREG protein expression was observed in the process of phenotypic modulation of HITASY cells. These results provide basic understanding on the relationship of CREG gene with the cell phenotypic conversion.展开更多
文摘Objectives Phenotypic switching of smooth muscle cells(SMCs) plays a critical role in the pathogenesis of atherosclerotic lesions such as coronary artery disease (CAD).Accumulating evidence demonstrates(hat a cellular repressor of E1A-stimulated genes(CREG) plays a role in the maintenance of the mature phenotype of vascular SMCs. The purpose of the present study was to assess the possible association between CREG and CAD in the Han population of North China.Methods The promoter region of CREG by direct sequencing was conducted in 48 subjects.Then SNP rs2995073 and another 4 tagSNPs(rs4657669,rs3767443, rsl6859185,rs3753921) were selected for the association study.All five selected SNPs were determined in 1161 patients with angiographically proven CAD and 960 controls with normal coronary angiograms to investigate the possible involvement of CREG in CAD.Results Genotype frequencies of the five examined polymorphisms were similarly distributed between CAD group and controls(P】0.05).Further haplotype analysis also found no significant differences in the distributions between CAD group and controls(P】0.05). Conclusions This study did not show an association between common variants of CREG and CAD in the northern Chinese Han population.
文摘Background Cellular Repressor of E1A-stimu-lated gene(CREG) is widely expressed in adult tissues such as the brain,heart,lung,liver,intestine and kidney in mice.It is not known whether tissue CREG is decreased in the common setting of myocardial infarction which may lead to heart failure.We studied the expression and protein localization of CREG and its main receptor(IFR2R) in a mouse model of myocardial infarction.Methods Male mice were randomized to proximal left anterior descending ligation.The animals were killed on day 1,3,7,14,and 28 after ligation to examine gene expression and protein production of CREG and IGF2R from the infarct,peri-infarct,and contralateral zones of infarcted heart.Results There was decreased CREG mRNA production throughout the myocardium at dav 1,and the expression gradually increased at day 28 after myocardial infarction.The decreased expression of this glycoprotein was not confined strictly to the infarct or peri-infarct zones but also expressed by cardiac myocytes within the myocardium in the contralateral normal zone.Levels of CREG protein in the infarct and peri-infarct zones declined to 1/3- to 1/2-fold of normal levels and declined to 1/2- to 2/3- fold in the contralateral zone.Finally,the expression of the IGF2R mRNA transcripts was downregulated at day 3 and 7 after ligation in the infarct and peri-infarct zones,suggesting that the signal transduction pathways necessary for CREG in the heart remain intact as CREG biosynthesis decreases. Conclusions CREG is constantly present in a model of large myocardial infarction and is decreased at the early stage within the myocardium.The decreased expression of this glycoprotein is not only confined strictly to the infarct or periinfarct zone but also is expressed by cardiac myocytes within the myocardium contralateral to the infarct.Therefore CREG production decreased due to myocardial stress response to injury.
文摘目的探讨E1A激活基因阻遏子(CREG1)蛋白能否改善心肌纤维化小鼠的心功能。方法应用基因打靶方法建立广泛性基因敲除的CREG1杂合子小鼠和CREG1野生型小鼠模型。应用血管紧张素Ⅱ(AngⅡ)皮下埋泵方法建立小鼠心肌纤维化损伤模型,给予AngⅡ刺激14d后,采用HE和Masson染色检测小鼠心肌纤维化情况。应用Western blotting和免疫组化染色技术检测给予AngⅡ前及3、7、14d后两组小鼠心肌中CREG1蛋白的表达,并于给予AngⅡ14d后应用小动物超声仪检测心功能情况。AngⅡ给药同时,以皮下埋泵方式分别给予15、30、60、300μg/(kg·d)的外源性重组CREG1蛋白(治疗组)和生理盐水(对照组)14d,检测心功能,并应用TUNEL染色和Western blotting检测心肌凋亡情况。结果 Western blotting和免疫组化检测结果显示,未给予AngⅡ刺激时杂合子小鼠心肌中CREG1蛋白表达明显低于野生型小鼠(P<0.05)。给予AngⅡ刺激3、7、14d时,野生型小鼠和杂合子小鼠心肌中CREG1蛋白表达均明显下降(P<0.05),但杂合子小鼠下降更为显著(P<0.01);HE和Masson染色显示杂合子小鼠心肌纤维化程度较野生型小鼠严重,二者心功能明显下降,且杂合子小鼠心功能下降更为明显(P<0.05)。给予外源性重组CREG1蛋白治疗后,治疗组心功能较对照组明显改善(P<0.05),心肌凋亡数量明显下降(P<0.05)。结论在AngⅡ引起的小鼠心肌纤维化模型中,CREG1蛋白减少可使小鼠心功能损伤加重,给予外源性重组CREG1蛋白可明显改善心功能。
基金The work was supported by grant from The National Natural Sciences Foundation of China (No.30070280)
文摘Objectives The cellular repressor of E1A-activated genes (CREG), a novel gene, was recently found to play a role in inhibiting cell growth and promoting cell differentiation. The purpose of this study was to obtain antibody against CREG protein and to study the expression of CREG protein in human internal thoracic artery cells (HITASY) which express different patterns of differentiation markers after serum withdrawal. Methods The open reading frame of CREG gene sequence was amplified by PCR and cloned into the pGEX-4T-1 vector. Glutathione-S-transferase (GST)-CREG fusion protein was expressed in E. Coli BL21 and purified from inclusion bodies by Sephacryl S-200 chromatography. Rabbits were immunized with the purified GST-CREG protein. Western blot examined with immunohistochemistry staining and the protein expression level was analyzed by Western blot in HITASY cells after serum removal. Results It was confirmed by using endonuclease digesting and DNA sequencing that the PCR product of CREG was correctly inserted into the vector. The GST-CREG protein was purified with gel filtration chromatography. Polyclonal antibody against GST-CREG was obtained from rabbits. CREG protein immunohistochemistry staining displayed a perinuclear distribution in the cytoplasm of HITASY cells. Results from Western blot suggested that comparing with the untreated cells upregulation of CREG polyclonal antibody against CREG was comfirmed. Using this antibody, the changes of CREG protein expression was observed in the process of phenotypic modulation of HITASY cells. These results provide basic understanding on the relationship of CREG gene with the cell phenotypic conversion.
