【作者】 卢学春；

【导师】 楼方定； 于力；

【作者基本信息】 中国人民解放军军医进修学院 ， 血液学， 2003， 博士

【摘要】 LRP16基因是于力等通过甲基化敏感性限制性路标基因组扫描技术比较初诊和复发急性髓系白血病细胞DNA甲基化修饰差异后，利用cDNA末端快速扩增技术于1999年12月从健康人外周血单个核细胞中克隆到的一条人类新基因。该基因cDNA序列全长共1225bp，定位于第11号染色体长臂11区。LRP16基因至少编码两种形式的翻译产物，其中短型编码蛋白翻译产物是一种核蛋白，长型编码蛋白亚细胞定位不明。 为探讨LRP16基因表达调控机制，研究LRP16基因功能以及LRP16基因在白血病中的表达情况，本研究首先对LRP16基因启动子分子进行了克隆和亚克隆，构建了启动子荧光素酶真核表达质粒，并对各个亚克隆片段启动子活性进行了检测，在此基础上对不同活性启动子片段可能存在的顺式结构进行了计算机辅助系统识别分析；以LRP16长型编码ORF为目的片段，利用神经网络系统对编码蛋白的一级结构、二级结构、蛋白进化树以及蛋白结构域进行了信息学分析，并构建了LRP16真核表达质粒，观察了LRP16过度表达对白血病细胞系K562增殖及细胞周期的影响；通过EST数据库、SAGE谱分析、GeneNote以及2DPAGE数据库对LRP16基因的表达谱进行了预测，在上述工作基础上，利用RT-PCR的方法对LRP16基因在白血病细胞系以及原代白血病细胞中的表达情况进行了检测。 结果表明：①LRP16基因启动子具有典型Ⅱ型真核启动子特点，有TATA盒和GC盒，其核心调控区域位于-600bp以内；在-2000bp至-1000bp范围内可能存在抑制基因表达的顺式元件；LRP16基因启动子既具有通用解放军总医院军医进修学院 第一部分 博士论文顺式作用元件也存在多种与包括细胞周期、造血调控、细胞分化、肿瘤发生以及应激反应有关的特异）烦式作用元件；②状P16基因可能具有3种不同的 ORF。其中长型编码蛋白平均分子量为为 3 5 5 0 5．11 Da，理论叶 值为9．58，富含甘氨酸门．4％）和亮氨酸门1．1％），其二级结构为混合型，a螺旋占36．000，p折叠占13．85y，其他结构占50．15y。有N－糖基化、蛋白激酶 C磷酸化位点、酪蛋白激酶 11磷酸化位点、酪氨酸激酶磷酸化位点、卜肉豆荔化位点和酚胺化位点等6种已知的修饰位点，分布于肽链上的18个不同位置；与组蛋白大HZAI的C末端具有高度同 源性，在进化上非常保守，从远古细菌到高等生物等物种均存在LRPlb 基因的表达；LRP16基因的过度表达对白血病细胞系 K5u i邮胞的增殖有 促进作用，这种增殖作用部分是通过促进细胞由GO期进A GI期及S期 来买现的；①LRP16”基因在包括骨髓、脐带血、脾脏、肝脏等多种生理状 态下的器官、组织和细胞中均有表达；但在包括脑脊液和血浆在内的体 液以及肝癌细胞分泌蛋白中没有表达，提示 LRP16基因编码蛋白不是分 泌型蛋白；④LRP基因编码蛋白与白血病关系密切，可能是一个与白血 病复发相关的基因。更多还原

【Abstract】 LRP16 is a novel gene which was cloned from human mononuclear cells by Yuli in 1999 using restriction length genomic scanning(RLGS) and the cDNA was isolated using the rapid amplification of cDNA end (RACE) technique (GeneBank Accession No. AF202922). The full length of LRP16 gene transcriptional copy has 1225 base pair of nucleotides and contains at least two open reading frames. The short coding protein is a nuclear protein, while the cell sub-location of the longer one is not clear.In order to explore the possible regulation mechanism of LRP16 gene expression, study the novel gene’s function and its expression pattern in leukemia cells, the present study first cloned promoter and sub-clone promoter molecules, then constructed sub-LRP16 gene promoter-pGL3-Basic vectors. After that, Luciferase activities of all sub-clone promoter sequences were analyzed. At last, the possible cis-acting elements were analyzed by computer-aid system. Computer-aid neurological system was used to predict the primary and the second structure of LRP16 coding protein, as well as its putative evolutionary tree. In the experiment of laboratory part, we first construct a eukaryotic expression vector containing the long type ORF of LRP16 gene and its expression was detected in K562 cells. The effects of LRP16 over expression on cell proliferation and its cell cycle were observed as well.The LRP16 gene expression pattern was analyzed by the scanning of EST database, SAGE database, GeneNote database and 2DPAGE database. Based on these prediction results, RT-PCR method was used to detect the LRP16 expression pattern in leukemia cell lines and primary leukemia cells from patients.Results showed: ㎜RP16 promoter is a typical class II eukaryotic promoter with a TATA box and several GC boxes, which has a core positive regulation sequence from -600bp to -200bp and a few putative negative czs-acting elements locating from -2000bp to -lOOObp. LRP16 gene promoter has both general and special cis-acting elements relating to cell cycle, hematopoiesis, cell proliferation, carcinogenesis and acute reacting process. (DLRP16 may have three types of ORFs, the molecular weight of the longest ORF coding protein is 35505.1 ID and the theoretical pi value is 9.58. LRP16 coding protein is rich in Glycine (11.4%) and Leucine(ll.l%)> which has a complex secondary siruciur6 witrr 36.00% of a-helix, 13.85% of 8-sheet and 50.15% of other secondary structures. Long type LRP16 coding protein has one N-glycosylation site, six protein kinase C phosphorylation sites, five casein kinase II phosphorylation sites, two tyrosine kinase phosphorylation sites, three N-myristoylation sites and one amidation site locating in 18 different positions. The coding protein has a similarity domain with C terminal sequence of macroH2Al. LRP16 coding protein has a very conserved evolutionary domain in it sequence, which is expressed in nearly all kinds of species. The over expression of LRP16 long type ORF accelerates the proliferation of leukemia cell line K562 and this is partly due to shorten the time from GO phase to Gl phase and S phase. ㎜RP16 gene expressed nearly in all organs, tissues and cells, including bone marrow, spinal cord blood, spleen, liver, lungs, brain and heart et al, while it has no expression in human cerebrospinal fluid, human plasma and HepG2 secreted Proteins. All these showed that LRP16 coding protein is not a secreted protein. 甃RP16 gene coding protein may be a leukemia related protein, which may has somewhat function relation to leukemia drug resistance and relapse.更多还原