【作者】 李新云；

【导师】 李奎；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2007， 博士

【摘要】 免疫系统是机体中一个极为重要的防御系统，其功能不正常，机体的生命就会受到威胁。免疫系统中起核心作用的是免疫细胞，均起源于骨髓中的造血干细胞。因此，造血干细胞的正常分化对维持机体免疫系统的功能是至关重要的。TMEM66（transmembrane protein 66）基因可能与免疫细胞的分化密切相关。本研究拟从分子水平、细胞水平及个体水平探讨TMEM66基因的功能。主要研究工作和结果如下：1 TMEM66基因功能预测TMEM66基因是一个新的功能基因，目前还没有该基因的专门研究报道，其功能也不清楚。本研究拟通过对TMEM66基本生物学信息进行收集整理，并对TMEM66的结构和功能进行初步预测，为研究该基因的功能奠定一定的基础。结果显示：TMEM66是一个三跨膜蛋白，含有一个DUF1183功能域；人TMEM66基因在分化成熟的免疫细胞如B淋巴细胞、T淋巴细胞、NK细胞、单核细胞、树突细胞中高表达，在脑、脊髓、淋巴结和血液中也高表达，而在白血病细胞（HL60、Molt4、K562）及恶性淋巴瘤细胞系（Raji、Daudi）等未分化成熟的免疫细胞中低表达；在癌变组织中该基因表达下调。以上研究结果表明：TMEM66基因可能与免疫细胞的分化相关，低表达TMEM66基因与癌症发生相关。2 TMEM66基因分子水平研究根据生物信息学分析的结果，推测TMEM66基因是一个与细胞分化相关的新基因。本研究拟对猪TMEM66基因进行克隆、定位、多态分析，并对猪和人TMEM66基因的表达规律进行研究，为阐明该基因的功能提供分子生物学依据。结果显示：扩增得到猪TMEM66基因片段长度为1886bp，包含一个完整ORF编码337氨基酸，与人TMEM66蛋白（NP₀57211）同源性达84％；猪TMEM66基因定位于猪15号染色体q臂与微卫星SW1673紧密连锁；在猪TMEM66基因CDs区发现了一个A／C多态，基因频率检测发现只在巴马猪群中存在等位基因A且A等位基因频率远远高于C等位基因，其它群体中都只有等位基因C，没有A等位基因；猪TMEM66在肺、脾、淋巴结、脑组织中高表达，而在骨骼肌、心肌和脂肪中低表达；人TMEM66在癌变组织或细胞中比其对应的正常组织表达量下降；TMEM66基因随着胚胎的生长发育呈现上调表达趋势；ATRA诱导U937分化过程中，TMEM66基因呈现上调趋势，48h表达到达高峰，以后呈现下调趋势；以上研究结果表明：TMEM66基因可能与细胞分化相关。3 TMEM66基因细胞水平研究TMEM66基因是一个膜蛋白，其亚细胞定位及表达水平与功能密切相关。本研究对人TMEM66基因进行了亚细胞定位；并构建慢病毒过表达载体，研究过表达该基因对细胞周期的影响，进一步从细胞水平探讨TMEM66基因的功能。结果显示：TMEM66分布于细胞核膜上。本研究中发现人TMEM66还存在其它两个转录本。第二转录本比TMEM66缺少了第一跨膜域及部分第一膜内域；第三转录本缺少了第一膜内域及第一膜内域。推测它们可能与TMEM66的功能调节相关。对TMEM66第三个转录本进行亚细胞定位，结果表明第三个转录本分布于细胞质及细胞核膜；对TMEM66互作蛋白IFIT5进行亚细胞定位分析，结果发现IFIT5分布于细胞质中；通过慢病毒介导TMEM66在U937细胞中过表达研究，发现过表达TMEM66导致细胞凋亡增加。经ATRA诱导不同时间点检测细胞周期，结果发现TMEM66过表达的U937细胞G2期比率比对照组高，凋亡比率也比对照组高。以上研究结果表明：TMEM66基因是一个核膜蛋白，过表达TMEM66基因导致细胞凋亡增加，在诱导细胞分化过程中，过表达TMEM66可导致细胞G2期抑制并促使G2期抑制的细胞发生凋亡。4 TMEM66基因突变体转基因小鼠构建为了从个体水平探讨TMEM66基因的功能，本研究采用显微注射法制作TMEM66突变体转基因小鼠，为进一步研究该基因的功能奠定基础。结果显示：出生的55只小鼠中，通过PCR法和Southern杂交检测，有6只为阳性鼠，传代研究发现3只阳性鼠可以稳定传代，外源基因片段以串联重复的方式整合到小鼠的基因组中，RT—PCR检测发现转入的突变基因在多个组织中转录。以上研究结果表明：通过显微注射法得到了3只可以传代的转基因阳性鼠，为以后从个体水平研究该基因的功能奠定了基础。更多还原

【Abstract】 The immune system is the essential part of the body to protect itself to counter detrimental agents from outside. The malfunction of the system sometimes caused lethal problems of normal life. The immune cells play a central role in the system; they derived from hematopoietic progenitor cell in bone marrow. So, the development and differentiation of hematopoietic progenitor cell is vital importance for the normal functioning of immune activities. TMEM66 (transmembrane protein 66) is a protein involved in the differentiation of the immune cells. Present studies focus in the gene function both in vitro and in vivo. The main results are as follows:1 The predicted function of TMEM66 geneUntil now, there is no report in the scientific literature regarding the function of TEME66 gene. At first, we colleted some related information about this gene employ some bioinformatics methods to deduce the possible biological function. This will be better for future study. The TEME66 protein is a tri-transmembrane protein which contains a DUF1183 domain. In human, the gene expression of TEME66 is high in differentiated immune cells such as B lymphocytes, T lymphocytes, monocytes and dendritic cells. It also showed high expression in brain, spinal cord, lymph note and blood. However, it showed low expression level in leukemic cell (HL60, Molt4 and K562), malignant lymphoma cells (Raji, Daudi) and other undifferentiated immune cells. It is down-regulated in carcinomatous tissues too. These results indicated that TEME66 is involved in the differentiation of immune cells and its aberrant low expression is associated with cancer.2 Molecular characterization of TEME66 geneTEME66 is a new gene which in relate to cell differentiation in our bioinformatics predictions. Present study cloned, mapped the porcine TEME66 gene, then identified several polymorphism sites in pig populations and detected its expression pattern in both human and pig, provided basic information for further research of this gene. Our PCR results showed the pig TMEM66 gene contains 1886bp including an integrity ORF and encoding a protein with 337 animo acids which shared 84% homologous with its human counterpart. The pig TMEM66 gene located in the q arm of porcine chromosome 15 and closely linked with marker SW1673. An A/C SNP is identified in the pig TMEM66 locus. The genotyping results showed the A allele is only presented in Bama pigs and it is the dominant allele in this population. However, there is only C allele in other pigs investigated. Pig TEME66 gene expression is abundant in lung, spleen, lymph note and brain, but in low level in skeletal muscle, heart and fat. The expression of human TEME66 gene is down-regulated in carcinomatous tissues or cells compared with normal tissues. The TMEM66 gene is up-regulated through the embryo development. After ATRA induction in U937 cells, the expression of TEME66 gene is increased, reached the plateau around 48 hours and then decreased. These results indicated that TEME66 gene involved in cell differentiation. In this study, there were two other transcriptions of human TMEM66 gene founded. They may participate to the regulation of the function of TMEM66 gene.3 TEME66 function in cellsTEME66 protein is a membrane protein; the subcellular localization of TEME66 protein could implicate its function. This study detected the subcellular localization of human TEME66 protein. We also studyed the overexpression of TMEM66 gene using lentivirus vector. The results indicated that TEME66 protein located in the nuclear membrane. The third transcript distributed across the cytoplasm and also nuclear membrane. The IFIT5 which was interacted with TMEM66 localized to cytoplasm. After overexpression of TEME66 by lentivirus infection in U937 cells, the apoptosis intensified. Then, ATRA induction was introduced in and the G2 period of U937 cells with overexpression of TEME66 is higher than that of control cells without TEME66 overexpression. The apoptosis percent is also higher in the overexression cell lines. These results indicated that TEME66 protein mainly localized to the nuclear membrane. During differentiation, TEME66 could cause the G2 period prolongation and induce the apoptosis of targeted G2 period cells.4 The TEME66 mutation transgenic mouseTo investigate the TEME66 function in vivo, we produced the TEME66 mutation transgenic mice by conventional micro injection method. After PCR and southern blot verification, we found 6 positive individuals among the 55 neonatal pups. Three positive mice could stably transfer the transgene to next generation through passage. The transgene is incorporated into the host genome in a serial multi-copy manner. RT-PCR analysis indicated that the transgene was expressed in many tissues. The results showed at least 3 mice embraced the TEME66 mutation transgene which could be stably passaged; provide a valuable model for the further study of TEME66 function.更多还原