【作者】 罗赛群；

【导师】 胡维新；

【作者基本信息】 中南大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 多发性骨髓瘤是一种后生发中心浆细胞异常增生所致的造血系统恶性肿瘤,瘤细胞具有极强的遗传不稳定性,导致染色体数量和结构的异常。1号染色体异常是多发性骨髓瘤患者最常见的染色体异常之一,表现为1p缺失和1q扩增。我们采用表达克隆法,从人浆细胞白血病细胞株ARH-77(一种多发性骨髓瘤细胞株)中克隆到一个新的与多发性骨髓瘤恶性相关的基因,命名为hMMTAG2(Genbank登录号:AY137773)。该基因定位于人类染色体1q42.13,cDNA全长1300bp,由8个外显子组成,编码分子量约为29kD的蛋白质。前期研究表明,将hMMTAG2基因导入NIH3T3细胞,发现该基因能促使NIH3T3细胞转化;同时将该基因导入COS-7细胞,其表达产物定位于细胞核。本课题拟在前期研究基础上,进一步阐明hMMTAG2基因的功能,揭示其与多发性骨髓瘤发生、发展的关系。构建pLXSN/TAG2载体,采用磷酸钙共沉淀法将hMMTAG2基因导入包装细胞PA317,建立稳定表达的细胞株PA317/TAG2。收集含有病毒颗粒的细胞培养上清(条件培养基),经离心、过滤后,感染NIH3T3细胞,经G418筛选,共获得6个稳定表达的NIH3T3/TAG2细胞克隆。以NIH3T3/pLXSN和NIH3T3分别作为空载体和空细胞对照,采用细胞生长计数、MTT法分析,发现hMMTAG2基因能促进细胞生长、增殖;同时,采用流式细胞分析,发现hMMTAG2基因能促进细胞从G1向S期转换;软琼脂集落培养分析表明,hMMTAG2基因能促进细胞克隆性生长;将NIH3T3/TAG2细胞经皮下注射至BALB/c裸鼠,能致裸鼠体内形成肿瘤;生物信息学分析表明,MMTAG2蛋白具有转录因子的基本结构;定量RT-PCR及WesternBlot分析显示NIH3T3/TAG2细胞中c-myc基因表达增高。这些结果提示,hMMTAG2基因过表达能促进MM细胞生长、增殖,促使细胞转化,促进肿瘤形成。hMMTAG2能上调c-myc的表达,提示MMTAG2可能作为核转录因子,通过c-myc依赖的途径而发挥转录调控作用,进一步调控下游靶基因的表达而导致MM的发生。更多还原

【Abstract】 Multiple myeloma (MM) is a hematological malignancy characterized by the uncontrolled proliferation of monoclonal plasma cells in post-germinal center. Multiple myeloma cells posses extreme genetic instability, induce number and structure abnormalities in MM cells. Chromosome 1 aberrations are very common in most hematologic malignancies and constitute the most common structural aberration in MM, characterized as chromosome 1p delete and 1q gain/amplification.We have identified a novel multiple myeloma tumor-associated gene from ARH-77 cell line cDNA gene pool which named as hMMTAG2 by expression cloning (Genbank accession Number: AY137773). The hMMTAG2 gene is located at chromosome 1 q42.13. The full length of hMMTAG2 cDNA is 1300bp, which contain 8 exons and encode a 29kD protein. The studies showed that hMMTAG2 can induce NIH3T3 cells transformation. The encoded products of hMMTAG2 are located at cell nuclus.We have constructed the pLXSN/TAG2 retroviral recombinant, and then transfected the package cell PA317, established the stable expressed cell line PA317/TAG2. The supernatant from the cell line PA317/TAG2 culture was harvested to infect NIH3T3 cells. 6 cell clones of stable expression hMMTAG2 gene were acquired by G418 selection. Cell growth curve analysis, MTT analysis, flow cytometry (FCM) and soft agar clone forming method were adopted to examine the biology activity of the transfected cells. The results showed that the hMMTAG2 could induce cell growth and cell proliferation, drive cell through G1 phase and induce S phase entry. Then, the NIH3T3/TAG2 cells were injected i.h. into BALB/c nude mice to understand whether they could form the implanted tumor. The results showed that hMMTAG2 has the ability of tumorgenesis. Bioinformatics analysis showed that MMTAG2 has a similar structure with transcrtipt factor. Meanwhile, we have found that the expression of c-myc mRNA and protein were upregulated in NIH3T3/TAG2 cells, whereas not in NIH3T3 cells.These data indicate that overexpression of hMMTAG2 induce cell proliferation and tumor formation. The overexpression of hMMTAG2 can upregulate the expression level of c-myc. The evidence suggest that MMTAG2 may play a role in inducing transcription and regulation as a nuclear transcript factor through c-myc dependent pathway.更多还原