Constant serum levels of secreted asialoglycoprotein receptor sH2a and decrease with cirrhosis

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.

Impact of asialoglycoprotein receptor deficiency on the development of liver injury

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.

Cloning, Expression and Polyclonal Antibody Preparation of the Asialoglycoprotein Receptor of Marmota Himalayan

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.

Synthesis and biological evaluation of ^(18)F-FB-NGA as a hepatic asialoglycoprotein receptor PET imaging agent

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.

Feasibility on Systemic Delivery of Asialoorosomucoid Complex to Hepatic Origin Cells Mediated by Asialoglycoprotein Receptor

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.

ASGP-R介导放射性标记肿瘤坏死因子防治肝癌的可行性

利用肝细胞膜表面的无唾液酸糖蛋白受体(ASGP-R)所能识别的配基作为载体,与放射性标记的肿瘤坏死因子(TNF)相结合,获得肝靶向放射性标记肿瘤坏死因子,使得肿瘤坏死因子选择性地导向肝脏,局部作用于肿瘤细胞,降低肿瘤坏死因子的毒副作用,从而提高靶与非靶比,促进肝癌细胞凋亡;防止肝癌术后复发,提高缓解率。因此,ASGP-R可能成为较理想的肝癌靶向放射性和生物治疗双效药物。

ASGP-R介导的乳糖酸-壳聚糖-SPIO纳米颗粒的制备及体外MR成像

目的:探讨去唾液酸糖蛋白受体(ASGP-R)介导的肝细胞靶向磁性纳米颗粒应用于磁共振分子成像的可行性。方法:乳糖酸与壳聚糖偶联修饰超顺磁性氧化铁纳米颗粒,采用透射电镜与动态光散射纳米粒度分析仪对LA-CS@SPION进行形貌表征;MTT法检测细胞毒性;利用体外及体内磁共振成像验证磁性纳米粒对肝细胞的靶向增强作用;普鲁士蓝染色观察肝细胞铁颗粒摄取情况。结果:LA-CS@SPION显示超顺磁性,T2弛豫率为456±5.8mM‐1S‐1。普鲁士蓝染色显示肝细胞浆内大量铁颗粒聚集,同时加入乳糖酸后肝细胞内未见铁颗粒。结论:ASGP-R介导的LA-CS@SPION对肝细胞具有特异靶向性,可作为肝脏MRI靶向对比剂。

肝癌细胞膜上转铁蛋白受体和去唾液酸糖蛋白受体数量的变化

目的:研究肝癌细胞膜摄取转铁蛋白受体的变化。方法：采用免疫组化及配体受体结合法分别测定正常肝脏、肝癌组织及癌旁肝组织转铁蛋白受体及去唾液酸糖蛋白受体的含量。结果：肝癌组织中转铁蛋白受体的含量高于癌旁及正常肝组织，而去唾液酸糖蛋白受体却明显低于癌旁及正常肝组织。结论：该变化有利于肝癌细胞对转铁蛋白的有效提取，也增加了肝癌细胞对高度唾液酸化的转铁蛋白的相对特异性摄取。

甲氨喋呤-乳糖酰基壳聚糖的制备及其体外实验

制备了不同乳糖酰基取代度的N-乳糖酰基壳聚糖(LCH),并通过红外光谱(IR)及核磁共振氢谱(1H NMR)对其进行了结构表征.体外放射性配基竞争结合实验结果显示,当乳糖酰基取代度为15·5%～38·9%时,LCH对肝实质细胞膜表面去唾液酸糖蛋白受体(ASGPR)具有特异亲和性,即具有潜在的肝靶向性.将甲氨喋呤(MTX)与壳聚糖以酰胺键偶联,制备MTX-壳聚糖(MTX-CH),再与乳糖酸反应生成目标产物MTX-乳糖酰基壳聚糖(MTX-LCH),并通过紫外分光光度法(UV)检测MTX的取代度为5·6%,乳糖酰基取代度为33·8%.溶解性实验结果显示,MTX-LCH溶于pH=1～14的水溶液;体外释放实验结果表明,MTX-LCH性质稳定,能明显延缓MTX的释放.

肝靶向抗病毒药NGA－ACV的制备及其趋肝性

以无唾液酸糖蛋白受体（ａｓｉａｌｏｇｌｙｃｏｐｒｏｔｅｉｎｒｅｃｅｐｔｏｒ，ＡＳＧＰＲ）的特异性配体半乳糖基拟糖白蛋白（ｎｅｏｇｌｙｃｏａｌｂｕｍｉｎ，ＮＧＡ）为载体，通过丁二酰基桥将抗病毒药无环鸟苷（ａｃｙｃｌｏｖｉｒ，ＡＣＶ）与ＮＧＡ偶联，得到肝靶向抗病毒药ＮＧＡＡＣＶ。差热分析和高效液相色谱分析结果表明，ＮＧＡＡＣＶ是共价键偶联物，且在血液中稳定性很好。将偶联物用１３１Ｉ标记后进行家兔放射性显像比较研究。结果，高、低药密度ＮＧＡＡＣＶ的肝脏放射性分别是全身放射性的８１．６％和８６．６％，其趋肝性无明显差别。研究小鼠体内高药密度１３１ＩＮＧＡＡＣＶ的分布，在５ｍｉｎ时肝脏放射性达到峰值，为注入量的８１．７±１０．４％。受体竞争抑制实验表明ＮＧＡＡＣＶ的肝靶向机理为受体介导的主动靶向过程。初步体外抗乙肝病毒比较研究表明，ＮＧＡＡＣＶ较ＡＣＶ的抗病毒剂量有明显降低。

肝靶向抗病毒药Lac-PLL-ACV的制备及其趋肝性研究

目的 减少抗病毒药阿昔洛韦 (Acyclovir ,ACV)在治疗乙型肝炎中的肝外毒副作用 ,获得肝靶向ACV偶联物。方法 多聚赖氨酸 (Poly L lysine ,PLL)在氰硼酸钠的催化作用下与乳糖反应生成乳糖化多聚赖氨酸 ,乳糖化多聚赖氨酸与咪唑化阿昔洛韦在 3 7℃pH 9 5的条件下反应合成偶联物乳糖化多聚赖氨酸 阿昔洛韦 (Lactosaminatedpoly L lysine acyclovir ,Lac PLL ACV)。偶联物经尾静脉注射进入小鼠体内后 ,收集小鼠血浆及肝脏样品 ,用高效液相色谱法测定药物浓度 ,研究偶联物的体内分布和药代动力学。结果 HPLC检测证实Lac PLL ACV在血浆中十分稳定 ,不易解离出ACV。偶联物的肝最大摄取率约为 60 % ,是ACV组的 10倍以上 ,偶联物肝中的T1/ 2 ,AUC ,CL分别为ACV的 4 2倍 ,5 6 6倍和 1/ 5 7,具有明显的肝靶向性。结论 乳糖化多聚赖氨酸作为肝去唾液酸糖蛋白受体 (Asialoglycoproteinreceptor ,ASGPR)介导的一种药物载体 。

肝靶向配体半乳糖基白蛋白和多聚谷氨酸

化学合成两类去唾液酸糖蛋白受体（ＡＳＧＰＲ）的人工配体———半乳糖基白蛋白（ＧａｌｎＨＳＡ）和半乳糖基多聚Ｌ谷氨酸（ＧａｌｎＰＬＧＡ），并以１２５Ｉ标记的去唾液酸胎球蛋白（ＡＳＦ）为标准配体，测定了合成配体抑制１２５ＩＡＳＦ与大鼠肝细胞膜ＡＳＧＰＲ结合的ＩＣ５０值．结果表明，Ｇａｌ１２ＨＳＡ、Ｇａｌ１５ＨＳＡ、Ｇａｌ２６ＨＳＡ、Ｇａｌ３０ＨＳＡ和Ｇａｌ３４ＰＬＧＡ均能够有效地抑制１２５ＩＡＳＦ与ＡＳＧＰＲ的结合，且前者与ＡＳＧＰＲ的亲和力随半乳糖基化程度的增加而增加．这些合成配体来源丰富、制备简单，适合于作为药物或基因肝靶向运送的导向配体．

脂质体的乳糖化修饰及其肝脏靶向性研究

目的探讨乳糖化白蛋白－脂质体交联物（简称交联物）作为肝脏靶向性药物载体的可行性。方法应用生物化学技 术合成交联物并对大鼠进行体内实验，了解交联物在动物肝脏的聚集量以及预先注射乳糖化白蛋白对交联物肝脏聚集 性的抑制作用；比较交联物、普通脂质体分别包裹的 α－干扰素及游离 α－干扰素在 ２．２．１５细胞中的抗乙型肝炎病毒效 应。结果交联物在肝脏的聚集量是脾脏的２倍以上，是其他脏器的４－９倍；预先注射乳糖化白蛋白后交联物的肝脏聚 集性减弱，与脾脏聚集量无显著差异；交联物包裹干扰素后抗乙肝病毒效应较普通脂质体包裹的干扰素及游离干扰素 显著提高，相当于普通脂质体干扰素１／４剂量、游离干扰素１／８剂量的抗病毒效应。结论文联物通过去唾液酸糖蛋白受 体实现肝脏聚集性，可望成为理想的肝脏靶向性药物载体。

去唾液酸糖蛋白受体特异性单链抗体的优化表达及亲和常数的测定

目的:表达及纯化抗去唾液酸糖蛋白受体(ASG-PR)的单链抗体的可溶性,并测定其亲和常数。方法:用噬菌体C1克隆感染E.coliHB2151,挑取单个菌落接种于2×TY培养基中,于37℃震荡培养过夜。将培养物作1∶100稀释并转种后,用终浓度为0.25、0.5、1.0mmol/L的IPTG,分别在37℃、25℃和20℃下诱导表达过夜。取其培养上清,用饱和硫酸铵沉淀后,以120g/LSDS-PAGE分析。另外,将饱和硫酸铵沉淀物用30mLPBS重新溶解、透析除盐后,用Ni2+螯合柱进行纯化,再以120g/LSDS-PAGE鉴定纯化scFvC1的纯度。用非竞争酶免疫法测定scFv的亲和常数。结果:用0.5mmol/LIPTG在25℃诱导过夜,表达的scFvC1的量较多,其相对分子质量(Mr)约为28000,以可溶性的形式存在于培养基中。通过Ni2+亲和柱纯化后scFvC1的纯度在95%以上,产量约为0.8mg/L。scFv的亲和常数为(2.31±0.36)×10-7mol/L。结论:以筛选的C1噬菌体感染E.coliHB2151后可表达低亲和力的可溶性scFv,对肝癌的基因治疗具有潜在的应用价值。

半乳糖基新糖白蛋白的制备及其对肝特异性受体结合试验的研究

作者用半乳糖经乙酰化、溴代、缩合、置换及加成反应制得2-亚氨基-2-甲氧基乙基-1-硫代-β-D-半乳吡喃糖苷,最后与人血清白蛋白偶连后得不同糖密度的半乳糖基新糖白蛋白(NGA),并用^(90)mTc标记后进行了动物及临床试验。结果^(99m)Tc-NGA-21的肝最大摄取率为82.6％,肝显像清晰。

^99mTc-DTPA-半乳糖人血清白蛋白在不同小鼠肝损伤模型中肝功能显像的应用

目的明确肝显像剂99mTc-DTPA-半乳糖人血清白蛋白(99mTc-GSA)在3种不同类型肝细胞损伤小鼠模型中不同的摄取和生物分布情况。方法3种小鼠模型分别为肝硬化、淤胆性肝损伤和肝肿瘤模型。肝硬化模型用腹腔内注射CCl4完成(每48小时腹腔注射0.4 ml 10%CCl4,共48 d);淤胆性肝损伤模型以结扎胆总管72 h建立;肝肿瘤模型用H22肿瘤细胞株种植肝包膜下10 d建立。各模型组小鼠和正常对照组小鼠均以0.1 ml(0.37 MBq)99mTc-GSA(2μg)注射尾静脉,5 min后断头处死,取出各重要器官或组织(肝脏、心脏、肺脏、肾脏、脾脏、胃、血液、骨骼、肌肉、肠道)称量,测定其放射性计数。同时血清学及肝脏病理学检查验证肝脏损伤。结果3个模型组和对照组中99mTc-GSA在小鼠肝脏均有显著浓聚(均>40%ID.g-1),但与对照组(90.05±10.55)%ID.g-1相比,肝损伤模型组的浓聚度均显著下降(P<0.001)。病理检查结果和建立模型时预期的病变相符;血清肝功能显示肝硬化模型组的损伤[天冬氨酸转氨酶为(235.3±14.7)U/L]轻于淤胆性肝损伤模型以及肝肿瘤模型[天冬氨酸转氨酶分别为(841.3±68.7)和(1 060.3±208.3)U/L];但肝硬化模型的浓聚度(72.20±2.13)%ID.g-1较淤胆性肝损伤模型(56.72±5.92)%ID.g-1及肝肿瘤模型(42.80±6.05)%ID.g-1显著增高(P<0.001)。结论99mTc-GSA在肝脏有显著浓聚,其浓聚程度与肝功能受损情况呈负相关。99mTc-GSA可能成为临床上反映肝功能的显像剂,如与三维扫描技术相结合,有希望建立评估肝脏区段功能的三维成像系统。

半乳糖配体介导多烯紫杉醇脂质体靶向性研究

目的:研究经半乳糖配体修饰后的多烯紫杉醇脂质体(docetaxel liposomes modified with galactose ligand,Gal-DOC-L)在小鼠体内的组织分布,探讨Gal-DOC-L对肝脏的靶向作用。方法:采用高效液相色谱法(high performance liquid chromatogra-phy,HPLC)测定各组织中多烯紫杉醇的药物浓度,以多烯紫杉醇普通脂质体(docetaxel liposomes,DOC-L)为对比,分别采用相对摄取率re、峰浓度比Ce和靶向效率te作为评价参数,对Gal-DOC-L的肝靶向性进行评价。结果:DOC-L和Gal-DOC-L的re分别为2.80和4.34,Ce分别为1.67和2.38,te分别为28.44%和38.91%。结论:药物经脂质体包封后对肝脏具有一定的靶向效应,而半乳糖配体的引入可进一步提高脂质体对肝脏的靶向性。

肝细胞表面去唾液酸糖蛋白受体的流式细胞分析

建立肝细胞表面去唾液酸糖蛋白受体 (ASGPR)的流式细胞分析方法 (FCM ) ,对正常及损伤鼠肝细胞、肝癌细胞 (BEL 740 2 )表面的ASGPR作同步比较分析 .以异硫氰酸荧光素标记的新半乳糖白蛋白 (FITC NGA)为ASGPR的特异性配体 ,以培养的正常肝细胞 (L 0 2 )为靶细胞 ,建立肝细胞表面ASGPR的FCM .测定并计算正常及损伤鼠肝细胞 ,BEL 740 2细胞与同一浓度的FITC NGA同步反应后的平均荧光强度 (MIF)值 .FITC NGA与L 0 2细胞表面ASGPR趋近饱和结合的浓度为 0 4mg/L ,该浓度下正常及损伤鼠肝细胞 ,BEL 740 2细胞的MIF值分别为 2 2 8 7、 5 81、 1 13.该结合可以被至少 5 0倍于FITC NGA的NGA或10mmol/L的EDTA完全抑制 .FCM能够良好地揭示FITC NGA同ASGPR之间的受配体结合特性 .该方法证实BEL 740 2细胞表面几乎没有ASGPR ,损伤鼠肝细胞表面ASGPR的数量较正常鼠肝细胞显著减少 .

肝靶向抗疟药NGA－PQ的合成及其抗疟作用

以肝细胞表面的无唾液酸糖蛋白受体的特异性配基──半乳糖基拟糖白蛋白（Ｎｅｏｇｌｙｃｏａｌｂｕｍｉｎ．ＮＧＡ）作为载体，通过丁二酰胺桥，成功地与抗疟药伯氨喹（ＰＱ）偶联，制备了肝靶向抗疟药ＮＧＡ－ＰＱ，药密度达到１６．８±０．８５，糖密度达到２１．７±１．７３．经药效学试验，ＮＧＡ－ＰＱ的抗疟作用明显优于同剂量的ＰＱ。

双糖密度半乳糖基胰岛素的合成及肝靶向性初步研究

为有效提高半乳糖基化合物的糖密度以增加其趋肝性 ,作者采用分枝化合物 3,5 -二羟基苯甲酸为偶联桥 ,将半乳糖与胰岛素偶联 ,得到糖密度较单链桥法高一倍的双糖密度半乳糖基胰岛素 ,而后用 1 31 分别标记双糖密度半乳糖基胰岛素、单链桥偶联半乳糖基胰岛素和普通胰岛素 ,通过家兔体内示踪实验对比研究它们的肝靶向性。结果显示 :双糖密度偶联物的趋肝性最大 ,单糖偶联物次之 ,普通胰岛素最差 ,其肝最大摄取率分别为5 9.5 %、43.8%和 2 1 .5 %。而 3种标记物的鸡全身显像皆未见肝脏特异性浓聚 ,从而证明以分枝化合物为偶联桥 ,可以有效地提高半乳糖基化合物的糖密度 ,大幅度提高其趋肝性。