LYRM1基因过表达对P19细胞分化、增殖和凋亡的影响

【作者】 陈福坤；

【导师】 钱玲梅；

【作者基本信息】 南京医科大学 ， 内科学， 2010， 硕士

【摘要】 先天性心脏病（CHD）一直以来都是婴儿死亡的一个主要病因。大多数学者认为CHD主要与遗传和环境有关,其中遗传因素的作用起着主要作用。虽然目前已发现多个与心肌细胞发育、先天性心脏病发生相关的基因,但其确切机制并不十分清楚。因此,从分子水平上开展先天性心脏病的病因研究,发现新的靶基因十分必要。LYRM1（LYR motif containing 1）是一个新发现的基因,一篇有关其与肥胖关系的文献报道表明其可以促进前体脂肪细胞的增殖和抑制前体脂肪细胞的凋亡,多组织表达谱提示该基因中等丰度表达于人类心脏组织,且与线粒体功能密切相关。由于在脊椎动物的器官发育中,心脏与脂肪组织均起源中胚层、细胞增殖和细胞凋亡是心脏形态建成的两个重要进程、线粒体是心脏组织重要的能量来源,而目前有关其与先天性心脏病的关系尚未见报道,因此本文拟以该基因为研究对象,初步阐明其生物学特性及其对心肌细胞株P19细胞功能的影响。生物信息学分析提示,LYRM1基因全长1589 bp（NM₀20424）,开放阅读框302～670 bp,编码122个氨基酸（NP₀65157）;定位于染色体16p11.2区域,有4个外显子和3个内含子。LYRM1基因编码的蛋白是一亲水性的可溶蛋白,未发现信号肽序列,可能为非分泌蛋白;不存在跨膜结构域,应不属于膜蛋白。亚细胞定位分析提示,LYRM1基因定位于胞核的可能性较大;蛋白结构域分析发现,LYRM1基因编码蛋白存在一LYR结构域,提示可能参与线粒体功能及能量代谢;蛋白序列比对提示,LYRM1基因编码蛋白相对保守。P19细胞是从C3H/He雄性小鼠畸胎瘤中分离得到的,是一种能够在体外培养的胚胎干细胞,二甲基亚砜（DMSO）可将其诱导分化为心肌样细胞。进一步构建LYRM1基因稳定过表达P19细胞株,观察LYRM1基因过表达（overexpression）对P19细胞分化、增殖及凋亡的影响。通过倒置显微镜观察过表达LYRM1的P19细胞和含空载体的P19细胞向心肌细胞分化过程,LYRM1基因对P19细胞分化无明显影响;MTT比色法测定及细胞周期分析结果显示,LYRM1基因具有促进P19细胞增殖的作用;磷脂酰丝氨酸外翻分析及Caspase 3活性检测结果提示,LYRM1基因对P19细胞凋亡具有抑制作用。总之,本研究初步探讨了LYRM1基因的生物学特性及其对P19细胞功能的影响,为先天性心脏病的研究提供新的线索。更多还原

【Abstract】 Congenital heart disease (CHD) is always the leading cause of death in the first year of life. CHD is considered having something to do with environmental and genetic factors, and environmental factor plays the most important role. Although some genes were found in conection with CHD and the growth of myocytes, but the specific mechanism is unclear. Thus, identification and characterization of novel genes and proteins associated with CHD remain an important issue.LYRM1 gene is a new gene. A research has shown that it can promote proliferation and inhibit apoptosis of preadiprocytes. Many organizations expression profiles indicate that LYRM1 gene expressed in human heart tissue with medium abundance. And it was bound up with the mitochondrial function. During the development of vertebrate organs, both heart and adipose tissue result from mesoblast. And proliferation and inhibition of the cells are the two important processes of heart morphogenesis. Mitochondria are the important energy sources of heart tissue. And now the relation between LYRM1 and CHD is not clear. Therefore, this gene was selected for further analysis in this study.The LYRM1 is an mRNA of 1589 basepairs (bps) with a 369 bp open reading frame (ORF) which encodes a 122 amino acids (aas) protein with a molecular mass of 13.27 kDa. NCBI Map Viewer analysis revealed that the LYRM1 gene is located on chromosome 16 and is composed of 4 exons. Amino acid sequence analysis revealed that LYRM1 was not a membrane-spanning protein and was an unsecreted protein. LYRM1 localizes in the nucleus and has a LYR domain. A sequence comparison of mouse and rat LYRM1 orthologous genes suggests that LYRM1 represents a novel gene highly conserved across primates and rodents.P19 cell was derived from dysembryoma of C3H/He male mice. It is a kind of embryonic stem cell, which can be cultured in vitro. And it can be induced to be myocyte.We examined the effect of LYRM1 on cell differentiation, proliferation, and apoptosis in vitro by establishing a stable P19 cell line overexpressing LYRM1, and found that: (i) LYRM1 does not affect the differentiation of P19 cells; (ii) the result of the MTT assay indicated that LYRM1 can promote proliferation of P19 cell and cell cycle analysis showed that a non-significant increase in the percentage of cells in S-phase; and (iii) LYRM1 can prevent apoptosis induced by serum deprivation with the analysis of annexin V-FITC and caspase-3 activity. In summary, our data demonstrate that by increasing cell proliferation and lowering apoptotic rate, LYRM1 has the potential to affect the development of CHD.In conclusion, we characterized several original features and the relationship with P19 cells of LYRM1 gene. Which can provide new clue to the researchs of CHD.更多还原