成骨生长肽抗体的制备及其对NIH3T3细胞促增殖作用机理
发布时间：2018-07-23 13:52
【摘要】：目的:成骨生长肽(OGP)是从损伤后再生的骨髓培养基质中分离纯化所得的一种具有14个氨基酸的小分子多肽。OGP广泛存在于哺乳动物的血清中,进化上高度保守,因此具有很好的同源性。研究发现OGP及其C端5肽可以促进造骨和造血作用。对体外培养的MC3T3E1成骨细胞、NIH3T3成纤维细胞和成骨性Rosl7/2细胞等均有促增殖和分化作用。但目前,OGP的细胞作用机理还不明确。为此,本实验采用中科院生化所人工合成的sOGP,设计poly-OGP制备OGP抗体,观察sOGP对NIH3T3细胞的促增殖作用,并利用制备抗体的免疫分析法及其他技术探讨OGP及其C端5肽对NIH3T3细胞的增殖作用机理。 方法:实验第一部分 Merrifield固相合成法合成寡肽。用Boc系统进行OGP的合成。HPLC、质谱分析鉴定其分子的均一性和准确性。制备多聚分支肽(Poly-OGP)和KLH-OGP。利用Poly-OGP及KLH-OGP作为抗原免疫新西兰兔制备抗血清。以抗血清利用放射免疫法(RIA法)及ELISA法测定sOGP浓度。 实验第二部分 将NIH3T3细胞以细胞浓度为5×10~4/ml,接种入96孔培养板,使用10%NCS-1640培养液,其中含不同浓度的sOGP或其sOGP(10-14)。48小时后以MTT法观察细胞增殖情况。将细胞浓度为5×10~4/ml的细胞培养液中的sOGP或sOGP(10-14)的浓度调整到10~(-11)mol/l,48小时后以细胞计数法,细胞周期检测观察细胞增殖情况,以RT-PCR法及ELISA法观察Ⅰ型胶原的变化。 实验第三部分采用流式细胞FITC荧光染色法,观察在增殖浓度sOGP及sOGP(10-14)作用下的NIH3T3细胞膜表面OGP的变化。观察在OGP抗血清作用下,sOGP及sOGP(10-14)的促细胞增殖作用的变化。观察在百日咳毒素(PTX),一种Gi抑制剂的作用下,OGP及OGP(10-14)的促细胞增殖作用,细胞膜OGP免疫荧光染色及细胞周期的变化。 结果:合成OGP经HPLC鉴定分子高度均一,纯度为99.28%,经质谱分析sOGP分子量为1523.5,与理论分子量1523.75相符。Poly-OGP的分子量平均为40KD左右。Poly-OGP可免疫动物得到可以测定的抗血清。 MTT法,细胞周期法,细胞计数法可以观察不同浓度sOGP及sOGP(10-14)
[Abstract]:Objective: osteogenic growth peptide (OGP) is a small peptide with 14 amino acids, which is isolated and purified from the regenerated bone marrow culture matrix after injury. It is widely present in mammalian serum and is highly conserved in evolution. So it has good homology. It was found that OGP and its C-terminal 5 peptide could promote osteogenesis and hematopoiesis. The proliferation and differentiation of NIH3T3 fibroblasts and Rosl7/2 osteoblasts cultured in vitro were enhanced. But at present, the mechanism of OGP cell action is not clear. In this study, we designed poly-OGP to prepare OGP antibody, and observed the proliferation of NIH3T3 cells by sOGP, which was synthesized by the Institute of Biochemistry of Chinese Academy of Sciences (CAS), which was synthesized by the Institute of Biochemistry of Chinese Academy of Sciences (CAS). The mechanism of proliferation of NIH3T3 cells by OGP and C-terminal 5 peptide was studied by immunosorbent assay and other techniques. Methods: in the first part of the experiment, oligoseptides were synthesized by Merrifield solid state synthesis. OGP was synthesized by Boc system, and its homogeneity and accuracy were identified by mass spectrometry. To prepare branched polypeptide (Poly-OGP) and KLH-OGP. New Zealand rabbits were immunized with Poly-OGP and KLH-OGP as antigens to prepare antiserum. The concentration of sOGP was determined by radioimmunoassay (RIA) and ELISA method in antiserum. In the second part of the experiment, NIH3T3 cells were inoculated into 96-well culture plate at a cell concentration of 5 脳 10 ~ (4) / ml. The proliferation of NIH3T3 cells was observed by MTT method with different concentrations of sOGP or its sOGP (10-14) .48 hours later. The concentration of sOGP or sOGP (10-14) in 5 脳 10~4/ml cell culture medium was adjusted to 10 ~ (-11) mol 路L ~ (-1) for 48 hours. Cell cycle assay was used to detect cell proliferation, and the changes of type 鈪，
本文编号：2139618
[Abstract]:Objective: osteogenic growth peptide (OGP) is a small peptide with 14 amino acids, which is isolated and purified from the regenerated bone marrow culture matrix after injury. It is widely present in mammalian serum and is highly conserved in evolution. So it has good homology. It was found that OGP and its C-terminal 5 peptide could promote osteogenesis and hematopoiesis. The proliferation and differentiation of NIH3T3 fibroblasts and Rosl7/2 osteoblasts cultured in vitro were enhanced. But at present, the mechanism of OGP cell action is not clear. In this study, we designed poly-OGP to prepare OGP antibody, and observed the proliferation of NIH3T3 cells by sOGP, which was synthesized by the Institute of Biochemistry of Chinese Academy of Sciences (CAS), which was synthesized by the Institute of Biochemistry of Chinese Academy of Sciences (CAS). The mechanism of proliferation of NIH3T3 cells by OGP and C-terminal 5 peptide was studied by immunosorbent assay and other techniques. Methods: in the first part of the experiment, oligoseptides were synthesized by Merrifield solid state synthesis. OGP was synthesized by Boc system, and its homogeneity and accuracy were identified by mass spectrometry. To prepare branched polypeptide (Poly-OGP) and KLH-OGP. New Zealand rabbits were immunized with Poly-OGP and KLH-OGP as antigens to prepare antiserum. The concentration of sOGP was determined by radioimmunoassay (RIA) and ELISA method in antiserum. In the second part of the experiment, NIH3T3 cells were inoculated into 96-well culture plate at a cell concentration of 5 脳 10 ~ (4) / ml. The proliferation of NIH3T3 cells was observed by MTT method with different concentrations of sOGP or its sOGP (10-14) .48 hours later. The concentration of sOGP or sOGP (10-14) in 5 脳 10~4/ml cell culture medium was adjusted to 10 ~ (-11) mol 路L ~ (-1) for 48 hours. Cell cycle assay was used to detect cell proliferation, and the changes of type 鈪，
本文编号：2139618
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