【作者】 马祖兴；

【导师】 胡宝成； 陈晓婷； 王宗华；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 细胞外基质磷酸糖蛋白/成骨细胞/骨细胞因子45 (Matrix Extracellular PhosphoglycoprotEin/Osteoblast/Osteocyte Factor 45, MEPE/OF45)自2000年被首次克隆以来,作为小型的N-端连接整合素的糖蛋白(Small Integrin -Binding Ligand, N-linked Glycoprotein, SIBLING)家族的一员,它的功能研究就一直局限在调节骨代谢和磷(体内)平衡方面。近年来,本实验室及其国外合作实验室的前期研究揭示了MEPE/OF45蛋白的新功能,即MEPE/OF45参与细胞DNA损伤应答;MEPE/OF45能够增强细胞对DNA损伤诱导剂(DNA Damage Inducers)的耐受性。本实验室在用基因表达谱芯片研究MEPE/OF45功能过程中还发现MEPE/OF45能够抑制细胞凋亡,并且MEPE/OF45的高表达伴随着SERPINB2[Serine(or cysteine) proteinase inhibitor, clade type-2]及SERPINB2相关蛋白RB(retinoblastoma)的高表达。因此,研究三者之间可能的调控关系对揭示MEPE/OF45的抗辐射及抗凋亡功能具有重要意义。在本研究中我们获得了以下的结果:(1)原核表达了SerpinB2基因、纯化了SERPINB2和MEPE/OF45的融合蛋白,并用纯化的蛋白免疫小鼠制备了抗SERPINB2和抗MEPE/OF45的多克隆抗体,用ELISA测定抗体的效价可达1:20000,Western印迹表明抗体的特异性较好;(2)构建了SerpinB2-siRNA载体,筛选得到2条SerpinB2-siRNA,可对293T细胞内源性SERPINB2的表达有抑制作用;(3)用瞬时转染过表达MEPE/OF45、microRNA抑制MEPE/OF45的表达及siRNA敲低SERPINB2的表达,研究了MEPE/OF45与SERPINB2和RB在蛋白水平的相互关系,发现MEPE/OF45、SERPINB2、RB蛋白表达水平具有一致性,有可能是MEPE/OF45影响凋亡的途径之一。该工作不仅有助于加深对细胞DNA损伤应答机制的认识,还可能为今后放射治疗的靶分子研究提供有意义的线索。更多还原

【Abstract】 Since MEPE/OF45 (Matrix Extracellular Phospho-glycoprotein/ Osteoblast/ Osteocyte Factor 45) was first cloned in 2000, however, its role as one member of SIBLING (Small Integrin-Binding Ligand N-linked Glycoprotein) family had been limited to regulation of bone metabolism and phosphate homeostasis.Recently, the previous research in our lab and our collaborative lab has shown a new function of MEPE/OF45. That is, MEPE/OF45 is involved in cellular DNA damage response and affects cellular resistance to DNA damage inducers. By using gene microarray to explore the function of MEPE/OF45, we found that MEPE/OF45 may inhibite apoptosis, and it is found that the cells with high expression of MEPE/OF45 show high expressions of SERPINB2 and RB. Therefore, it is very important to study the correlation of MEPE, SERPINB2 and RB in elucidation the redioresistance and inhibiting apoptosis of MEPE/OF45.Taken together, in this study we identified that (1) Serine (or cysteine) proteinase inhibitor B2(SerpinB2) gene was expressed in E.coli, SERPINB2 and MEPE/OF45 proteins were purified, and the polyclonal antibody against SERPINB2 and MEPE/OF45 were prepared in mice, the titer of these antibodies were determined by ELISA and were about 1:20000,. the specificity was identified by Western blot and SERPINB2 and MEPE/OF45 expressed in prokaryotic and eukaryotic cells can be recognized by these antiserum. (2) SerpinB2-siRNA vectors were constructed, and two SerpinB2-siRNAs which repress SerpinB2 expression in 293T cells were obtained, SerpinB2 expression was repressed by siRNAs. (3) By using overexpression of MEPE/OF45, inhibiting expression of MEPE/OF45 by miRNA and decreasing expression of SERPINB2 by siRNA, the correlation of MEPE/OF45, SERPINB2 and RB in protein levels were studied and the expression levels were accordant, it may be one of the pathways that MEPE/OF45 affectes apoptosis. Our data will not only increase our knowledge on DNA damage response but also give a clue to future research concerned on targets of radiotherapy.更多还原