The asialoglycoprotein （ASGP） receptor is a wellcharacterized hepatic receptor that is recycled via the common cellular process of receptor-mediated endocytosis （RME）. The RME process plays an integral part in the...The asialoglycoprotein （ASGP） receptor is a wellcharacterized hepatic receptor that is recycled via the common cellular process of receptor-mediated endocytosis （RME）. The RME process plays an integral part in the proper trafficking and routing of receptors and ligands in the healthy cell. Thus, the missorting or altered transport of proteins during RME is thought to play a role in several diseases associated with hepatocyte and liver dysfunction. Previously, we examined in detail alterations that occur in hepatocellular RME and associated receptor functions as a result of one particular liver injury, alcoholic liver disease （ALD）. The studies revealed profound ethanol- mediated impairments to the ASGP receptor and the RME process, indicating the importance of this receptor and the maintenance of proper endocytic events in normal tissue. To further clarify these observations, studies were performed utilizing knockout mice （lacking a functional ASGP receptor） to which were administered several liver toxicants. In addition to alcohol, we examined the effects following administration of anti- Fas （CD95） antibody, carbon tetrachloride （CCh） and lipopolysaccharide （LPS）/galactosamine. The results of these studies demonstrated that the knockout mice sustained enhanced liver injury in response to all of the treatments, as shown by increased indices of liver damage, such as enhancement of serum enzyme levels, histopathological scores, as well as hepatocellular death. Overall, the work completed to date suggests a possible link between hepatic receptors and liver injury. In particular, adequate function and content of the ASGP receptor may provide protection against various toxinmediated liver diseases.展开更多
AIM: To investigate the existence and levels of sH2a, a soluble secreted form of the asialoglycoprotein receptor in human serum. METHODS: Production of recombinant sH2a and development of a monoclonal antibody and an ...AIM: To investigate the existence and levels of sH2a, a soluble secreted form of the asialoglycoprotein receptor in human serum. METHODS: Production of recombinant sH2a and development of a monoclonal antibody and an enzyme-linked immunosorbent assay (ELISA). This assay was used to determine the presence and concentration of sH2a in human sera of individuals of both sexes and a wide range of ages. RESULTS: The recombinant protein was produced successfully and a specific ELISA assay was developed. The levels of sH2a in sera from 62 healthy individuals variedminimally (147 ± 19 ng/mL). In contrast, 5 hepatitis C patients with cirrhosis showed much decreased sH2a levels (50 ± 9 ng/mL). CONCLUSION: Constant sH2a levels suggest constitutive secretion from hepatocytes in healthy individuals. This constant level and the decrease with cirrhosis suggest a diagnostic potential.展开更多
The objective of this study is to express the carbohydrate recognition domain （CRD） of the asialoglycoprotein receptor （ASGPR） H1 and H2 subunits of Marmota himalayan in vitro, and develop polyclonal antibodies ag...The objective of this study is to express the carbohydrate recognition domain （CRD） of the asialoglycoprotein receptor （ASGPR） H1 and H2 subunits of Marmota himalayan in vitro, and develop polyclonal antibodies against the recombinant proteins. RT-PCR was used to amplify ASGPR CRDH1 and CRDH2 from the liver tissue of Marmota himalayan. The products of amplification were subcloned into prokaryotic expression vector pRSET-B, and expressed in E.coli BL21（DE3）plysS. The recombinant proteins were purified using Ni-NTA spin column. The purified proteins were inoculated into BALB/c mice to develop polyclonal antibodies. The sensitivity and specificity of antibodies were evaluated by enzyme-linked immunosorbent assay （ELISA）, Western blotting and immunohistochemical staining （IHC）. The polyclonal antibodies showed high sensitivity and specificity against both denaturated and native ASGPR proteins. We successfully amplified and expressed the ASGPR CRDs of Marmota himalayan. The nucleic sequences of ASGPR CRDH1 and CRDH2 of Marmota himalayan have been submitted to Genbank and the sequence ID are DQ 845465 and DQ845466, respectively. The proteins and antibodies prepared can be used for targeting gene therapy in a new animal model-Marrnota himalayan—— for the research of infectious diseases of hepatitis viruses and liver cancer treatment.展开更多
Receptor mediated gene delivery is a new gene transfer strategy. Asialoglycoprotein receptor (ASGP-R), the receptor of asialoorosomucoid (Asor), is specially expressed on the surface of hepatocyte. In this paper, the ...Receptor mediated gene delivery is a new gene transfer strategy. Asialoglycoprotein receptor (ASGP-R), the receptor of asialoorosomucoid (Asor), is specially expressed on the surface of hepatocyte. In this paper, the nuclide 131I was combined with Asor to form a kind of soluble nuclide-protein complex, which can be specifically endocytosed into hepatocyte by ASGP-R. After intravenous injection of the complex into experimental animals, the deposition of Asor in vivo and the targeting quality of hepatocyte was detected by ECT. This research testified the feasibility of targeting Asor complex delivery to hepatocyte mediated by ASGP-R in vivo, and provided foundation for the genetic diagnosis and gene therapy of hepatic cell-related diseases.展开更多
Asialoglycoprotein receptor(ASGP-R)is a hepatic membrane receptor that uniquely exists on the surface of mammalian hepatocytes,and has been used as target of liver functional imaging agents for many years.We labeled t...Asialoglycoprotein receptor(ASGP-R)is a hepatic membrane receptor that uniquely exists on the surface of mammalian hepatocytes,and has been used as target of liver functional imaging agents for many years.We labeled the Galactosyl-neoglycoalbumin(NGA)with 18F to get a PET molecular probe 18F-FB-NGA and evaluated its ability as a liver functional PET imaging agent.The 18F-FB-NGA was prepared with NGA by conjugation with Nsuccinimidyl-4-18F-fluorobenzoate(18F-SFB)and purified with PD-10 desalting column.The radiolabeling yield and radiochemical purity of 18F-FB-NGA were determined by radio-HPLC.Starting with 18F-F–,the total time for 18F-FB-NGA was about 120±10 min.The decay-corrected radiochemical yield is about 25–30%.The radiochemical purity of purified 18F-FB-NGA was more than 98%.Labeled with 185–1850 MBq 18F-SFB,the specific activity of 18F-FBNGA was estimated to be 7.83–78.3 TBq/mmol.Biodistribution of 18F-FB-NGA in normal mice was investigated after injection through the tail vein.The results showed that the liver accumulated 39.47±3.42 and 12.12±6.11%ID/g at 10 and 30 min after injection,respectively.Dynamic MicroPET images in mice were acquired with and without block after injection of the radiotracer,respectively.High liver activity accumulation was observed at 5 min after injection in normal group.On the contrary,the liver accumulation was significantly lower after block,indicating the specific binding to ASGP-R.18F-FB-NGA is probably a potential PET liver imaging agent.展开更多
Gene transfer into primary rat hepatocytes was performed by employing cationic liposome as DNA carrier and the specific ligand of hepatic asialoglycoprotein receptor (ASGPR), asialofetuin, as liver-targeting ligand. T...Gene transfer into primary rat hepatocytes was performed by employing cationic liposome as DNA carrier and the specific ligand of hepatic asialoglycoprotein receptor (ASGPR), asialofetuin, as liver-targeting ligand. The results showed that asialofetuin, when added to the gene transfer complexes, could significantly increase the hepatocyte transfection efficiency, and alleviate the cellular toxicity of Lipofectin. Several synthetic ligands of ASGPR (galactosyl albumin) could also increase the transfection efficiency of hepatocyte like asialofetuin. It was proved that ASGPR and cationic liposome could synergistically mediate the gene transfer into primary rat hepatocytes. This novel gene delivery system provided a safer, more simple and efficient gene transfer method for primary hepatocytes, and showed prospecting application in hepatic gene therapy.展开更多
基金Supported by The National Institute on Alcohol Abuse and Alcoholismthe Department of Veterans Affairs
文摘The asialoglycoprotein （ASGP） receptor is a wellcharacterized hepatic receptor that is recycled via the common cellular process of receptor-mediated endocytosis （RME）. The RME process plays an integral part in the proper trafficking and routing of receptors and ligands in the healthy cell. Thus, the missorting or altered transport of proteins during RME is thought to play a role in several diseases associated with hepatocyte and liver dysfunction. Previously, we examined in detail alterations that occur in hepatocellular RME and associated receptor functions as a result of one particular liver injury, alcoholic liver disease （ALD）. The studies revealed profound ethanol- mediated impairments to the ASGP receptor and the RME process, indicating the importance of this receptor and the maintenance of proper endocytic events in normal tissue. To further clarify these observations, studies were performed utilizing knockout mice （lacking a functional ASGP receptor） to which were administered several liver toxicants. In addition to alcohol, we examined the effects following administration of anti- Fas （CD95） antibody, carbon tetrachloride （CCh） and lipopolysaccharide （LPS）/galactosamine. The results of these studies demonstrated that the knockout mice sustained enhanced liver injury in response to all of the treatments, as shown by increased indices of liver damage, such as enhancement of serum enzyme levels, histopathological scores, as well as hepatocellular death. Overall, the work completed to date suggests a possible link between hepatic receptors and liver injury. In particular, adequate function and content of the ASGP receptor may provide protection against various toxinmediated liver diseases.
基金Supported by The Ramot research fundthe Israel Science Foundationthe Israel Cancer Association and the Israeli Ministry of Health (to Lederkremer GZ)
文摘AIM: To investigate the existence and levels of sH2a, a soluble secreted form of the asialoglycoprotein receptor in human serum. METHODS: Production of recombinant sH2a and development of a monoclonal antibody and an enzyme-linked immunosorbent assay (ELISA). This assay was used to determine the presence and concentration of sH2a in human sera of individuals of both sexes and a wide range of ages. RESULTS: The recombinant protein was produced successfully and a specific ELISA assay was developed. The levels of sH2a in sera from 62 healthy individuals variedminimally (147 ± 19 ng/mL). In contrast, 5 hepatitis C patients with cirrhosis showed much decreased sH2a levels (50 ± 9 ng/mL). CONCLUSION: Constant sH2a levels suggest constitutive secretion from hepatocytes in healthy individuals. This constant level and the decrease with cirrhosis suggest a diagnostic potential.
基金This project was supported by grants from National Natural Sciences Foundation of China (No 30571646)the Na-tional Basic Research Program (Program 973, No 2005CB522901)
文摘The objective of this study is to express the carbohydrate recognition domain （CRD） of the asialoglycoprotein receptor （ASGPR） H1 and H2 subunits of Marmota himalayan in vitro, and develop polyclonal antibodies against the recombinant proteins. RT-PCR was used to amplify ASGPR CRDH1 and CRDH2 from the liver tissue of Marmota himalayan. The products of amplification were subcloned into prokaryotic expression vector pRSET-B, and expressed in E.coli BL21（DE3）plysS. The recombinant proteins were purified using Ni-NTA spin column. The purified proteins were inoculated into BALB/c mice to develop polyclonal antibodies. The sensitivity and specificity of antibodies were evaluated by enzyme-linked immunosorbent assay （ELISA）, Western blotting and immunohistochemical staining （IHC）. The polyclonal antibodies showed high sensitivity and specificity against both denaturated and native ASGPR proteins. We successfully amplified and expressed the ASGPR CRDs of Marmota himalayan. The nucleic sequences of ASGPR CRDH1 and CRDH2 of Marmota himalayan have been submitted to Genbank and the sequence ID are DQ 845465 and DQ845466, respectively. The proteins and antibodies prepared can be used for targeting gene therapy in a new animal model-Marrnota himalayan—— for the research of infectious diseases of hepatitis viruses and liver cancer treatment.
基金This project was supported by a grant fromthe Hubei Natural Sciences Foundation (No .2001ABA004) .
文摘Receptor mediated gene delivery is a new gene transfer strategy. Asialoglycoprotein receptor (ASGP-R), the receptor of asialoorosomucoid (Asor), is specially expressed on the surface of hepatocyte. In this paper, the nuclide 131I was combined with Asor to form a kind of soluble nuclide-protein complex, which can be specifically endocytosed into hepatocyte by ASGP-R. After intravenous injection of the complex into experimental animals, the deposition of Asor in vivo and the targeting quality of hepatocyte was detected by ECT. This research testified the feasibility of targeting Asor complex delivery to hepatocyte mediated by ASGP-R in vivo, and provided foundation for the genetic diagnosis and gene therapy of hepatic cell-related diseases.
基金Supported by Jiangsu Province’s Key Medical Talents Program(No.RC2007097)Natural Science Foundation of Jiangsu Province,China(No.BK2010154)Science Foundation of Health Department of Jiangsu Province(No.H201226)
文摘Asialoglycoprotein receptor(ASGP-R)is a hepatic membrane receptor that uniquely exists on the surface of mammalian hepatocytes,and has been used as target of liver functional imaging agents for many years.We labeled the Galactosyl-neoglycoalbumin(NGA)with 18F to get a PET molecular probe 18F-FB-NGA and evaluated its ability as a liver functional PET imaging agent.The 18F-FB-NGA was prepared with NGA by conjugation with Nsuccinimidyl-4-18F-fluorobenzoate(18F-SFB)and purified with PD-10 desalting column.The radiolabeling yield and radiochemical purity of 18F-FB-NGA were determined by radio-HPLC.Starting with 18F-F–,the total time for 18F-FB-NGA was about 120±10 min.The decay-corrected radiochemical yield is about 25–30%.The radiochemical purity of purified 18F-FB-NGA was more than 98%.Labeled with 185–1850 MBq 18F-SFB,the specific activity of 18F-FBNGA was estimated to be 7.83–78.3 TBq/mmol.Biodistribution of 18F-FB-NGA in normal mice was investigated after injection through the tail vein.The results showed that the liver accumulated 39.47±3.42 and 12.12±6.11%ID/g at 10 and 30 min after injection,respectively.Dynamic MicroPET images in mice were acquired with and without block after injection of the radiotracer,respectively.High liver activity accumulation was observed at 5 min after injection in normal group.On the contrary,the liver accumulation was significantly lower after block,indicating the specific binding to ASGP-R.18F-FB-NGA is probably a potential PET liver imaging agent.
文摘Gene transfer into primary rat hepatocytes was performed by employing cationic liposome as DNA carrier and the specific ligand of hepatic asialoglycoprotein receptor (ASGPR), asialofetuin, as liver-targeting ligand. The results showed that asialofetuin, when added to the gene transfer complexes, could significantly increase the hepatocyte transfection efficiency, and alleviate the cellular toxicity of Lipofectin. Several synthetic ligands of ASGPR (galactosyl albumin) could also increase the transfection efficiency of hepatocyte like asialofetuin. It was proved that ASGPR and cationic liposome could synergistically mediate the gene transfer into primary rat hepatocytes. This novel gene delivery system provided a safer, more simple and efficient gene transfer method for primary hepatocytes, and showed prospecting application in hepatic gene therapy.
