【作者】 刘嘉；

【导师】 杨东亮；

【作者基本信息】 华中科技大学 ， 免疫学， 2009， 博士

【摘要】 目的1.研究肝脏去唾液酸糖蛋白受体（asialoglycoprotein receptor,ASGPR）大亚基异构体的产生机制、表达水平及其编码蛋白的特性,为进一步阐明ASGPR的生物学功能及以其为靶标的肝脏靶向治疗奠定基础。2.筛选获得能够特异性高亲和力结合肝脏特异性去唾液酸糖蛋白受体的RNA适配子,为开发诊断和治疗肝脏疾病的靶向性试剂和药物奠定基础。方法1.从正常人肝组织和HepG2细胞中克隆ASGPR大亚基H1的两种异构体H1a和H1b的cDNA序列,测序并比对分析H1b的产生机制;实时荧光定量PCR检测正常人肝组织和HepG2细胞中H1a和H1b的表达比例,以及在HBV、HCV感染情况下和肝癌组织中H1b表达水平的变化.2.合成H1b特异性多肽并与钥孔戚血蓝素（KLH）偶联,免疫小鼠制备H1b特异性多克隆抗体,鉴定抗体的效价和特异性;亲和柱层析的方法从人血清和HepG2细胞培养上清中分离并纯化可溶性ASGPR,采用特异性抗体通过Western blot鉴定可溶性ASGPR的组成;免疫组织化学检测H1b在肝组织中表达和定位。3.合成一个长度为115nt含有25个随机序列的单链DNA随机文库,通过体外转录构建出单链RNA适配子随机文库,从肝组织中分离纯化ASGPR大亚基作为靶蛋白,采用SELEX（systematic evolution of ligands by exponential enrichment）技术筛选高亲和力的ASGPR特异性RNA适配子;测序分析筛选适配子的序列,预测并分析其二级结构特点。4.同位素³²p标记适配子,通过膜结合测定实验、凝胶阻滞实验鉴定筛选适配子对靶蛋白的特异性和亲和力;绿色荧光FITC标记适配子,鉴定其与肝细胞系HepG2和Huh7的特异性结合。结果1.人肝组织和肝细胞系HepG2中普遍表达ASGPR大亚基异构体H1a和H1b,H1b的cDNA较H1a缺失了一段117 nt的序列,该序列是ASGPR大亚基编码基因的第二个外显子,其两端具有典型的AG/GT序列,提示H1的两种异构体产生于对ASGPR mRNA的选择性剪接。2.正常肝组织和HepG2细胞中H1a和H1b的表达比例分别为5.2:1和2.6:1,而且H1b的表达水平在HBV和HCV感染细胞中下降约60%,在肝癌组织中下降90%以上。3.在从正常人血清和HepG2细胞培养上清中纯化的可溶性ASGPR蛋白中,可以检测到H1b蛋白单体以及H1b和H2构成的多聚体;对肝组织进行的免疫组织化学检测显示H1b不能定位至细胞膜,而主要存在于细胞质中。4.经过12轮SELEX筛选,适配子文库中与靶蛋白结合的适配子得到明显富集;对第12轮文库中随机挑选的48个适配子进行测序并预测其二级结构,发现文库中适配子从结构上主要由两个家族构成,其占总适配子的比例分别为45.8%和33.3%;并找到一个适配子H1-A25与靶蛋白具有很高的亲和力,Kd值为48.79nM。5、在膜结合测定实验和凝胶阻滞实验中,反应体系中不添加靶蛋白,或用无关蛋白替代靶蛋白,则不能检测到明显的适配子H1-A25和蛋白结合;而在反应体系中加入过量未标记适配子H1-A25,则能明显阻断放射标记适配子H1-A25与H1蛋白的结合。6、FITC标记的适配子H1-A25能结合至肝癌细胞系HepG2和HuH-7,但不能结合不表达ASGPR的HeLa细胞;加入ASGPR的多克隆抗体可部分阻断荧光标记适配子H1-A25与HepG2细胞或HuH-7细胞结合,而加入过量未标记适配子H1-A25则几乎可以完全阻断荧光标记适配子H1-A25结合HepG2细胞的荧光信号。结论1.人肝组织及肝细胞系HepG2和Huh7中普遍表达ASGPR大亚基异构体H1a和H1b。2.大亚基异构体H1b编码的蛋白为分泌型H1。3.血清中的可溶性ASGPR是由分泌型的H1和H2构成的功能性复合物。4.成功地筛选出了具有高亲和力的肝脏ASGPR特异性RNA适配子H1-A25。5.适配子H1-A25能特异性靶向结合至肝细胞。本研究的创新点及意义1.首次发现人类肝脏去唾液酸糖蛋白受体大亚基H1存在剪接异构体H1b,并证明此剪接异构体编码的分泌型蛋白参与构成功能性的可溶性ASGPR,从而完善了对可溶性ASGPR的认识,并为进一步全面系统探讨ASGPR的生物学功能奠定了坚实的基础。2.本研究首次筛选得到了肝细胞特异性的RNA适配子并鉴定了其功能,而对其进一步的改进和修饰将使其有望成为新的诊断和治疗肝脏疾病的靶向性试剂和药物载体。更多还原

【Abstract】 Objective1. To study the occurrence mechanism, expression level and protein characteristics of the major subunit variants of human asialoglycoprotein receptor, and lay a foundation for further study of the function of ASGPR and for hepatic targeting therapy through ASGPR.2. To obtain RNA aptamer with high affinity and specificity to the human liver specific asialoglycoprotein receptor （ASGPR） and lay a foundation for developing new reagents or drugs for the diagnosis and the targeting treatment of liver diseases.Methods1. Two major subunit variants of ASGPR （H1a and H1b） were cloned from normal human liver tissues and HepG2 cells, the occurrence mechanism of variant H1b was analyzed by sequences comparison. The expression levels of H1a and H1b in normal liver tissues and HepG2 cells were determined by real-time fluorescence quantitative PCR, as well as the level changes in HBV or HCV infected cells and HCC tissues.2. H1b specific peptide was synthesized and coupled with keyhole limpet hemocyanin （KLH） for immunization. Then H1b-KLH conjugation was injected into mouse subcutaneously to produce polyclonal antibody. The titer and specificity of the antibody were confirmed. Soluble ASGPR （sASGPR） in normal human sera or HepG2 cell supernatants were purified by lactose-agarose affinity chromatography, and the compositions of sASGPR were analyed by specific antibodies. The expression and location of protein H1b in liver tissues were also determined by immunohistochemistry. 3. A single-stranded 115 nucleotides （nt） random DNA library containing 25 random oligonucleotides was synthesized in vitro, then random RNA aptamers library was constructed by in vitro transcription. Aptamers that can specifically bind to human hepatic asialoglycoprotein receptor was isolated from the RNA random library by using the SELEX （systematic evolution of ligands by exponential enrichment） procedure. Selected aptamers were analyzed by sequencing, and the secondary structures of these aptamers were prediced and analyzed by using RNA Structure Program.4. Aptamers were labeled by ³²P, then filter biding assay and gel shift assay were performed to determine the specificity and affinity of the selected aptamers. One apatamer was also labeled by FITC, and the binding between the aptamer and the human hepatoma cell lines HepG2 and HuH-7 were identified by fluorescent staining.Results1. Two variants of ASGPR H1, designated H1a and H1b, are extensively expressed in both human liver tissues and in human hepatoma cell lines HepG2 and Huh7. Cloning of ASGPR H1 splice variant cDNAs and subsequent sequence analysis revealed that they differ only in the presence of 117 nt. This 117 nt segment corresponds to exon 2 in the genomic sequence of ASGPR, and the typical AG/GT 5’ consensus splice donor and acceptor sites were found at each end of the 117 nt segment, strongly suggesting that ASGPR H1b is generated by alternative splicing of ASGPR mRNA.2. The ratio of ASGPR H1a to H1b was about 2.6:1 in normal human liver tissue and 5.2:1 in HepG2 cells. H1b expression were decreased by approximately 60% in HBV or HCV infected cells, and were decreased more than 90% in HCC tissues.3. Both the H1b protein and a functional soluble ASGPR （sASGPR） composed of H1b and H2 were identified in both normal human sera and in the supernatant of HepG2 cells. Immunohistochemistry analysis shows that protein H1b in liver tissues can not accumulate at the cellular membrane, but in the cytoplasm.4. Twelve rounds of selection and amplification were performed, and the aptamers which can bind to H1 were significantly enriched. Round 12 RNAs were amplified by RT-PCR and cloned. Forty-eight randomly picked plasmid clones were sequenced. The secondary structures of these aptamers were predicted with RNA Structure Program. Most aptamers of Round 12 RNAs were represented by only two kinds of structures, and their percentages of composition of Round 12 RNAs were 45.8% and 33.3%, respectively. One aptamer, H1-A25, was found to have high affinity to protein H1. the dissociation constant for the aptamer:H1 complex was estimated to be 48.79 nM.5. While P³²-labeled H1-A25 significantly bound to H1 protein on the nitrocellulose filter （determined by nitrocellulose filter binding assay）, its binding to HBsAg, the HBV S antigen that served as a negative control, was similar to that of probe alone. Furthermore, gel mobility shift assays also confirmed the specific binding of aptamer H1-A25 to H1 protein, as only recombinant H1 but not HBsAg, could cause the retardation of mobility of P³²-labeled H1-A25 probe. The addition of 100-fold excess unlabeled aptamer to the reaction system abolished the aptamer-H1 interaction.6. FITC labeling H1-A25 specifically bound to the ASGPR-expressing hepatoma cell lines HepG2 and Huh-7. However, no binding was shown to the ASGPR-negative cell line HeLa. The fluorescence signal was significantly reduced by preincubating HepG2 cells with anti-H1 polyclonal antibody and can be nearly blocked by adding 100-fold excess unlabeled H1-A25.Conclusions1. Two variants of ASGPR H1, designated H1a and H1b, are extensively expressed in both human liver tissues and in human hepatoma cell lines HepG2 and Huh7.2. The protein encoded by the H1b variant is a secreted form of H1.3. sASGPR is a hetero-oligomeric complex of the secreted form of H1 and H2 and is able to bind a lactose-agarose substrate.4. RNA aptamer H1-A25 that can specifically bind to human hepatic ASGPR with high affinity was successfully selected by using SELEX.5. Aptamer H1-A25 can specifically bind to human hapatocytes.Significances of the study1. Our works provide the first evidence of the existence of splice variant H1b of human hepatic ASGPR H1, and prove that the protein encoded by the variant H1b is a secreted form of H1 which participates in the composing of sASGPR complex. The study improved the understanding of sASGPR, and laid a foundation for further comprehensive study of the function of ASGPR.2. This is the first reported RNA aptamer which could bind to a human hepatic specific receptor. And this newly isolated aptamer could be modified to deliver imaging, diagnostic, and therapeutic agents targeted at hepatic parenchymal cells.更多还原