| 摘 要
【目的】 探讨染色体10q26上HTRA1启动子区域单核苷酸多态性(Single Nucleotide Polymorphism, SNP)与年龄相关性黄斑变性（Age-Related Macular Degeneration，AMD）的关联性以及HTRA1（High Temperature Requirement Factor A, HTRA）在人眼各层次中的表达。【方法】 1. 以犹他洲高加索人群为研究对象，采集各型AMD患者和对照组人群血样并提取DNA。 2. 以SNP rs11200638为中心选取染色体10q26上HTRA1基因内或HTRA1上游的9个标签SNP位点，通过单核苷酸引物延伸法对犹他洲高加索人群中的各型AMD患者和种族、年龄匹配的正常对照组人群进行基因分型，并用卡方检验检测其与AMD的关联性。 3. 从健康成年人的血样提取RNA，通过RT-PCR克隆HTRA1基因的编码区，在原核中进行HTRA1重组蛋白的表达和纯化，并用Western Blot检测HTRA1重组蛋白的抗原性。 4. 用兔抗人HTRA1多克隆抗体对AMD患者和正常人眼球冰冻切片进行免疫组化反应，检测HTRA1在人眼各层次中的表达。【结果】 1. SNP rs11200638与晚期AMD的2种类型，GA和湿性AMD，具有几乎一致的显著关联性，其与软性玻璃膜疣也具有一定的关联性，但相对晚期AMD较低。SNP rs10490924与各型AMD也具有显著的关联性，但不及rs11200638显著。 2. 以pET32a为载体构建的重组质粒，在BL21细菌中经IPTG诱导后，获得高效的HTRA1重组蛋白表达。通过Ni-NTA蛋白纯化柱纯化，获得高纯度并具有抗原性的重组HTRA1蛋白。 3. HTRA1在玻璃膜疣和新生血管组织上均有表达。【结论】 1. HTRA1启动因子内的rs11200638除了与湿性AMD有关联以外与GA和软性玻璃膜疣均有显著的关联性。HTRA1很可能是晚期AMD，包括GA和湿性AMD的主要危险因子，对软性玻璃膜疣的形成亦有一定的影响。 2. 由于His.Tag与Ni-NTA蛋白纯化柱的特异性结合，使我们获得了高纯度的HTRA1重组蛋白。通过Western Blot对纯化、透析后的重组蛋白抗原性检测，发现该蛋白具有很好的抗原性。因此，只需通过放大培养，即可获得大量高纯度的并具有天然抗原性的HTRA1重组蛋白，以用于动物模型的体内功能研究和抗体的制备。 3．HTRA1可能在玻璃膜疣和新生血管的形成中起到了重要的作用，玻璃膜疣和新生血管形成这2个阶段可能并非完全独立的，而是紧密联系在一起的。【关键词】 年龄相关性黄斑变性，HTRA1，SNP，rs11200638，克隆, 蛋白表达
ABSTRACT
【Objective】 To investigate the association of 9 single-nucleotide polymorphisms (SNP) around HTRA1 promoter region at Chromosome 10q26 with different phenotypes of Age-Related Macular Degeneration (AMD). Immunohistochemistry was used to determing HTRA1 expression in the human eye. 【Method】 1. All participants were recruited from a Caucasian cohort in Utah. DNA was extracted from their blood. 2. 9 SNPs at chromosome 10q26 centered around SNP rs 11200638 were genotyped in AMD cases and age and ethnicity matched normal controls. The chi-squared test for trend for the additive model over alleles was performed to assess evidence for association. 3. The total RNA was extracted from normal human blood and converted into first strand cDNA by SuperScriptTM III reverse transcriptase. The coding sequence of HTRA1 were cloned by PCR and were inserted into pET32a vector after enzyme digestion. The recombinant plasmid was transformed into BL21 after sequencing confirmation, and then induced by IPTG to get high expression of recombinant HTRA1 protein. The induced bacteria were lysed by lysis buffer. The recombinant protein was purified from the bacteria lysate by Ni-NTA protein purification columns. 4. Immunohistochemistry was used to determing HTRA1 expression in the human eye by using the rabbit anti-human HTRA1 polyclonal antibody. 【Result】 1. HTRA1 promoter region SNP rs11200638 contributes equally to both forms of advanced AMD (GA and wet AMD). It also confers risk to soft confluent drusen, although to a lesser extent. rs10490924 was found to have a significant association with different phenotypes of AMD, but inferior to rs11200638. 2. The HTRA1 coding sequence was cloned by two steps RT-PCR. After enzyme digestion and ligation, the coding sequence was inserted into pET32a vector. The BL21, transformed with recombinant plasmid, showed the high-level expression of HTRA1 protein after ITPG induction. The high purity recombinant protein was purified due to specific binding between His.Tag and Ni-NTA. The Western Blot showed that dialysised protein still had its native antigen. 3. The drusen and choroidal neovascularization were immunolabeled by HTRA1 antibody in AMD patients. 【Conclusion】1. rs11200638 in the promoter region of HTRA1 remains to be the most significantly associated variant for GA in the Utah population and is a major risk factor for GA and soft confluent drusen, in addition to wet AMD. It may play an inportant role in pathogenesis of advanced AMD and also confers risk to soft confluent drusen. 2. The high purity and large volume protein is the most important thing for protein structural and functional study. Those optimized Tags in pET32a vector help to produce soluble fusion protein and His.Tag make it easy to purify the fusion protein from bacteria lysate. The HTRA1 coding sequence was cloned by RT-PCR and inserted into pET32a vector. The recombinant plasmid shows high-level expression of fused HTRA1 protein after transformed into BL21 and IPTG induction. The high purity HTRA1 protein with native antigen was purified and was ready for further study. 3. HTRA1 may play an important role in neovascularization and drusen formation. The two processes, may not be completely independent, rather, they may converge. 【Key words】 Age-Related Macular Degeneration, HTRA1，SNP，rs11200638, clone, protein expression
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