The ligand-binding domain of VLDL receptor contains eight imperfectly similar repeats. To discuss the contribution of each repeat to ligand binding, the RT-PCR technique was used to clone the VLDLR-cDNA from the heart...The ligand-binding domain of VLDL receptor contains eight imperfectly similar repeats. To discuss the contribution of each repeat to ligand binding, the RT-PCR technique was used to clone the VLDLR-cDNA from the heart muscle of Chinese people. Two recombinants were further constructed, which contained the full-length cDNA of VLDLR and the mutant lacking repeats 1-5. CHO cell line was transfected with two recombinants. The expression of VLDLR gene could be detected by RT-PCR from the CHO cells transfected with pCD-VR. The results of binding experiments showed that the ability of the CHO cells transfected with the full-length cDNA of VLDL-R binding DiI-labeled β-VLDL was higher than that of the CHO cells transfected with the mutant. Our findings indicated that human VLDL-R gene could be expressed effectively on CHO cells, and the receptor was almost inactivated when repeats1-5 were deleted.展开更多
Summary: The polyclonal antibodies against VLDL receptor were prepared and identified. Rabbits were immunized with polypeptide fragment of VLDL receptor as antigen. The collected blood serum of the immunized rabbits w...Summary: The polyclonal antibodies against VLDL receptor were prepared and identified. Rabbits were immunized with polypeptide fragment of VLDL receptor as antigen. The collected blood serum of the immunized rabbits was analyzed and identified by using ELISA and Western Blot. The results showed that the rabbit against mouse and human VLDL receptor antibodies were obtained with high titer and could recognize the natural VLDL receptors through Western blot. The prepared polyclonal antibodies against VLDL receptor provide a new tool to study the protein of VLDL receptor.展开更多
To elucidate the intracellular signaling pathways for VLDL-induced VLDLR transcription, Western blot analysis was used to examine phosphorylated ERK1/2 protein. It was found that that VLDL induced an increase in ERK1/...To elucidate the intracellular signaling pathways for VLDL-induced VLDLR transcription, Western blot analysis was used to examine phosphorylated ERK1/2 protein. It was found that that VLDL induced an increase in ERK1/2 activity in a protein kinase C (PKC)-dependent manner in murine RAW264. 7 macrophages. By using different protein kinases inhibitors or activators it was observed that the effect of VLDL-induced VLDL receptor transcription, which is monitored by RT-PCR analysis of VLDL receptor mRNA, was not affected by the inhibitor of p38 kinase and cAMP analog, but completely abolished by pretreatment of the cells with PD 98059, an inhibitor of MEK and GF 109203X, an inhibitor of PKC. These results demonstrated that the PKC/ERK1/2 cascade is the essential signaling pathway by which VLDL activates VLDL receptor mRNA expression.展开更多
To explore the intracellular signal pathways for β-VLDL induced very low density lipoprotein receptor (VLDL-R) transcription up-regulation and their effects on lipid accumulation in macrophages, Western Blot was used...To explore the intracellular signal pathways for β-VLDL induced very low density lipoprotein receptor (VLDL-R) transcription up-regulation and their effects on lipid accumulation in macrophages, Western Blot was used to examine phosphorylated ERK1/2 protein and regulated effects by different singal kinase inhibitants. It was found that β-VLDL induced an increase in ERK1/2 activity in a protein kinase C (PKC)-dependent manner in murine RAW264.7 macrophages. By using different protein kinases inhibitors or activators, it was observed that the effect of β-VLDL induced VLDL receptor transcription, which was monitored by RT-PCR analysis of VLDL receptor mRNA, was not affected by the inhibitor of p38 kinase and cAMP analog, but extremely abolished by pretreating cells with PD98059, an inhibitor of ERK and GF 109203X, an inhibitor of PKC. These results demonstrated that the PKC-ERK1/2 cascade is the essential signaling pathway by which β-VLDL activated VLDL-R mRNA expression. Inhibition of the ERK1/2 signaling cascade resulted in suppression of the cellular lipid accumulation induced by β-VLDL in macrophages.展开更多
缺血性心肌损伤的分子病理机制一直备受国际关注。该文应用C57BL/6品系的野生型(WT)小鼠、极低密度脂蛋白(Very Low Density Lipoprotein,VLDL)受体基因敲除(VLDLR-KO)小鼠、心脏特异性转基因(VLDLR-TG)小鼠,通过在体及离体实验研究VLD...缺血性心肌损伤的分子病理机制一直备受国际关注。该文应用C57BL/6品系的野生型(WT)小鼠、极低密度脂蛋白(Very Low Density Lipoprotein,VLDL)受体基因敲除(VLDLR-KO)小鼠、心脏特异性转基因(VLDLR-TG)小鼠,通过在体及离体实验研究VLDL受体对小鼠缺血性心肌损伤的影响,构建生物分子网络研究VLDL受体发挥生物学功能的关键分子及其关键信号通路。通过生物网络的功能模块计算,发现了VLDL受体→MAPKs、AKT、NF-κB信号通路→心肌损伤病理表型的因果关联性,阐明了VLDL受体影响缺血性心肌损伤的分子机制。展开更多
文摘The ligand-binding domain of VLDL receptor contains eight imperfectly similar repeats. To discuss the contribution of each repeat to ligand binding, the RT-PCR technique was used to clone the VLDLR-cDNA from the heart muscle of Chinese people. Two recombinants were further constructed, which contained the full-length cDNA of VLDLR and the mutant lacking repeats 1-5. CHO cell line was transfected with two recombinants. The expression of VLDLR gene could be detected by RT-PCR from the CHO cells transfected with pCD-VR. The results of binding experiments showed that the ability of the CHO cells transfected with the full-length cDNA of VLDL-R binding DiI-labeled β-VLDL was higher than that of the CHO cells transfected with the mutant. Our findings indicated that human VLDL-R gene could be expressed effectively on CHO cells, and the receptor was almost inactivated when repeats1-5 were deleted.
基金ThisprojectwassupportedbyagrantfromNationalNaturalSciencesFoundationofChina (No .39970 30 7)
文摘Summary: The polyclonal antibodies against VLDL receptor were prepared and identified. Rabbits were immunized with polypeptide fragment of VLDL receptor as antigen. The collected blood serum of the immunized rabbits was analyzed and identified by using ELISA and Western Blot. The results showed that the rabbit against mouse and human VLDL receptor antibodies were obtained with high titer and could recognize the natural VLDL receptors through Western blot. The prepared polyclonal antibodies against VLDL receptor provide a new tool to study the protein of VLDL receptor.
基金This project was supported by a grant from National Natural Sciences Foundation of China(Serial No.39970307).
文摘To elucidate the intracellular signaling pathways for VLDL-induced VLDLR transcription, Western blot analysis was used to examine phosphorylated ERK1/2 protein. It was found that that VLDL induced an increase in ERK1/2 activity in a protein kinase C (PKC)-dependent manner in murine RAW264. 7 macrophages. By using different protein kinases inhibitors or activators it was observed that the effect of VLDL-induced VLDL receptor transcription, which is monitored by RT-PCR analysis of VLDL receptor mRNA, was not affected by the inhibitor of p38 kinase and cAMP analog, but completely abolished by pretreatment of the cells with PD 98059, an inhibitor of MEK and GF 109203X, an inhibitor of PKC. These results demonstrated that the PKC/ERK1/2 cascade is the essential signaling pathway by which VLDL activates VLDL receptor mRNA expression.
文摘To explore the intracellular signal pathways for β-VLDL induced very low density lipoprotein receptor (VLDL-R) transcription up-regulation and their effects on lipid accumulation in macrophages, Western Blot was used to examine phosphorylated ERK1/2 protein and regulated effects by different singal kinase inhibitants. It was found that β-VLDL induced an increase in ERK1/2 activity in a protein kinase C (PKC)-dependent manner in murine RAW264.7 macrophages. By using different protein kinases inhibitors or activators, it was observed that the effect of β-VLDL induced VLDL receptor transcription, which was monitored by RT-PCR analysis of VLDL receptor mRNA, was not affected by the inhibitor of p38 kinase and cAMP analog, but extremely abolished by pretreating cells with PD98059, an inhibitor of ERK and GF 109203X, an inhibitor of PKC. These results demonstrated that the PKC-ERK1/2 cascade is the essential signaling pathway by which β-VLDL activated VLDL-R mRNA expression. Inhibition of the ERK1/2 signaling cascade resulted in suppression of the cellular lipid accumulation induced by β-VLDL in macrophages.
文摘缺血性心肌损伤的分子病理机制一直备受国际关注。该文应用C57BL/6品系的野生型(WT)小鼠、极低密度脂蛋白(Very Low Density Lipoprotein,VLDL)受体基因敲除(VLDLR-KO)小鼠、心脏特异性转基因(VLDLR-TG)小鼠,通过在体及离体实验研究VLDL受体对小鼠缺血性心肌损伤的影响,构建生物分子网络研究VLDL受体发挥生物学功能的关键分子及其关键信号通路。通过生物网络的功能模块计算,发现了VLDL受体→MAPKs、AKT、NF-κB信号通路→心肌损伤病理表型的因果关联性,阐明了VLDL受体影响缺血性心肌损伤的分子机制。
