【摘要】：目的:成骨生長肽(OGP)是從損傷后再生的骨髓培養(yǎng)基質(zhì)中分離純化所得的一種具有14個氨基酸的小分子多肽。OGP廣泛存在于哺乳動物的血清中,進(jìn)化上高度保守,因此具有很好的同源性。研究發(fā)現(xiàn)OGP及其C端5肽可以促進(jìn)造骨和造血作用。對體外培養(yǎng)的MC3T3E1成骨細(xì)胞、NIH3T3成纖維細(xì)胞和成骨性Rosl7/2細(xì)胞等均有促增殖和分化作用。但目前,OGP的細(xì)胞作用機(jī)理還不明確。為此,本實(shí)驗采用中科院生化所人工合成的sOGP,設(shè)計poly-OGP制備OGP抗體,觀察sOGP對NIH3T3細(xì)胞的促增殖作用,并利用制備抗體的免疫分析法及其他技術(shù)探討OGP及其C端5肽對NIH3T3細(xì)胞的增殖作用機(jī)理。 方法:實(shí)驗第一部分 Merrifield固相合成法合成寡肽。用Boc系統(tǒng)進(jìn)行OGP的合成。HPLC、質(zhì)譜分析鑒定其分子的均一性和準(zhǔn)確性。制備多聚分支肽(Poly-OGP)和KLH-OGP。利用Poly-OGP及KLH-OGP作為抗原免疫新西蘭兔制備抗血清。以抗血清利用放射免疫法(RIA法)及ELISA法測定sOGP濃度。 實(shí)驗第二部分 將NIH3T3細(xì)胞以細(xì)胞濃度為5×10~4/ml,接種入96孔培養(yǎng)板,使用10%NCS-1640培養(yǎng)液,其中含不同濃度的sOGP或其sOGP(10-14)。48小時后以MTT法觀察細(xì)胞增殖情況。將細(xì)胞濃度為5×10~4/ml的細(xì)胞培養(yǎng)液中的sOGP或sOGP(10-14)的濃度調(diào)整到10~(-11)mol/l,48小時后以細(xì)胞計數(shù)法,細(xì)胞周期檢測觀察細(xì)胞增殖情況,以RT-PCR法及ELISA法觀察Ⅰ型膠原的變化。 實(shí)驗第三部分采用流式細(xì)胞FITC熒光染色法,觀察在增殖濃度sOGP及sOGP(10-14)作用下的NIH3T3細(xì)胞膜表面OGP的變化。觀察在OGP抗血清作用下,sOGP及sOGP(10-14)的促細(xì)胞增殖作用的變化。觀察在百日咳毒素(PTX),一種Gi抑制劑的作用下,OGP及OGP(10-14)的促細(xì)胞增殖作用,細(xì)胞膜OGP免疫熒光染色及細(xì)胞周期的變化。 結(jié)果:合成OGP經(jīng)HPLC鑒定分子高度均一,純度為99.28%,經(jīng)質(zhì)譜分析sOGP分子量為1523.5,與理論分子量1523.75相符。Poly-OGP的分子量平均為40KD左右。Poly-OGP可免疫動物得到可以測定的抗血清。 MTT法,細(xì)胞周期法,細(xì)胞計數(shù)法可以觀察不同濃度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，

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