【作者】 祝海宝；

【导师】 郭定宗；

【作者基本信息】 华中农业大学 ， 临床兽医， 2009， 博士

【摘要】 成骨生长肽（Osteogenic growth peptide,OGP）是一种高效促成骨及造血刺激因子。普遍存在于哺乳动物的血液中,对促进骨形成、松质骨密度及骨折的愈合具有重要的意义。有研究发现,OGP通过自分泌及旁分泌途径,促进人及啮齿目动物的成骨细胞及骨髓基质干细胞分化、碱性磷酸酶（Alkaline phosphatase,ALP）活性及基质矿化。本试验从以下五个方面探讨了OGP对奶牛骨髓基质干细胞及成骨细胞作用及调控机制,并对OGP转基因细胞微囊生物学特性进行研究。1.奶牛骨钙素抗体制备以奶牛骨组织提取的RNA为模板,利用RT-PCR技术扩增出奶牛骨钙素（Bonecalcium protein,BGP）全长cDNA,然后将扩增产物重组到PMD-18T载体中,测定了全基因的核苷酸序列。序列分析表明,奶牛骨钙素全长cDNA为303bp,编码100个氨基酸,与GenBank中的X53699的序列完全相同。通过重叠延伸PCR技术连接单链DNA片段人工定点同义突变,将奶牛骨钙素成熟蛋白基因中的大肠杆菌稀有密码子同义突变成大肠杆菌常用密码子并亚克隆到PET-32a表达载体,转化到宿主菌BL₂₁（DE₃）,经IPTG诱导后,成功表达出奶牛骨钙素融合蛋白,并成功制备奶牛骨钙素多抗血清,为后续试验提供了抗体。2.OGP对奶牛骨髓基质干细胞增殖、分化及矿化的影响及其调控机制研究本试验研究了OGP对奶牛体外培养的骨髓基质干细胞增殖及分化特性的作用。结果发现,成骨生长肽能促进奶牛骨髓基质干细胞的增殖,并能促进奶牛骨髓基质干细胞ALP活性及矿化的形成,另外能刺激BGP mRNA的表达。OGP能刺激奶牛骨髓基质干细胞内皮一氧化氮合成酶（Endothelial nitric oxide synthases,eNOS）的表达,当eNOS表达受到抑制时,OGP对奶牛骨髓基质干细胞的促分化活性受到抑制。由此认为,OGP能刺激奶牛骨髓基质干细胞的增殖及向成骨细胞分化,并且是通过eNOS介导。3.OGP对奶牛成骨细胞增殖、分化及矿化的影响及其调控机制研究本试验研究了外源性OGP对体外培养的奶牛成骨细胞增殖、分化及矿化的影响。组织块移行法分离奶牛成骨细胞,通过形态观察及碱性磷酸酶活性鉴定。MTT法检测不同浓度OGP对成骨细胞生长的作用。细胞质碱性磷酸酶活性及骨钙素水平分别通过比色法检测及放射免疫法测定。硒素红染色观察矿化结节。结果显示,成骨生长肽能明显促进奶牛成骨细胞的增殖,最大效应浓度为10^-9M;并且能促进成骨细胞ALP活性、骨钙素及矿化结节的形成;当抑制osterix基因表达,成骨生长肽促进奶牛成骨细胞ALP活性及骨钙素分泌相应受到抑制。由此揭示,OGP对奶牛成骨细胞增殖及分化具有促进,并且是通过上调osterix介导。4.体内氧自由基对移植细胞微囊活性的影响为了揭示细胞微囊移植过程中宿主体内氧自由基的动态变化特征。将细胞微囊、空微囊以及生理盐水在移植小鼠体内后1、4、7天,检测血清自由基,MDA及SOD水平。过氧化氢及MDA水平在空微囊移植后瞬时升高,后又降低到正常水平,但是细胞微囊移植组,移植后持续升高。NO及SOD水平在空微囊及细胞微囊移植组均升高,但是生理盐水注射组无明显变化。细胞微囊在移植7天后回收,通过AO/EB染色发现,细胞具有良好的活性。由此认为,NO是微囊特异性的刺激形成因子,氧自由基在微囊移植早期迅速升高,但是由于体内抗氧化物酶升高从而能有效清除。5.OGP转基因细胞微囊体内及体外增殖及分泌特性研究为OGP转基因细胞微囊在体内及体外的增殖及分泌特征。本试验检测了OGP转基因细胞微囊在体外培养条件下的增殖及OGP分泌活性,并将微囊细胞移植入骨质疏松疾病模型体内,检测微囊内细胞增殖及OGP分泌活性,并对骨质疏松疾病模型体内氧自由基及抗氧化物酶的活性进行了检测。结果显示,OGP转基因细胞微囊在体外培养条件下具有良好的增殖及分泌活性,而在骨质疏松疾病模型体内细胞大量死亡,宿主动物血清OGP含量无明显变化。骨质疏松模型血清氧自由基含量明显升高,抗氧化物酶活性降低,当注射抗氧化剂后,能明显降低血清自由基含量。由此认为,OGP转基因细胞微囊具备治疗移植的潜力,在骨质疏松疾病模型体内移植失败,主要原因是,骨质疏松模型体内氧自由基含量高,导致微囊内细胞坏死或凋亡。更多还原

【Abstract】 Osteogenic growth peptide (OGP) is highly effect to promote bone and blood-stimulating factor. It could promote bone formation, cancellous bone mineral density and fracture healing. Some studies have been found that OGP, through autocrine and paracrine way, could stimulat human and rodents osteoblasts and bone marrow stromal stem cell differentiation, alkaline phosphatase activity and matrix mineralization. In this study, the following five aspects of OGP on the cow bone marrow stromal cells and osteoblastic cells and its mechanism and characteristics of microcapsulated OGP genetically modified cells were studied.1. Preparation of dairy osteocalcin antibodyBovine osteocalcin cDNA gene was amplified from bone of bovine by RT-PCR. PCR product was cloned into PMD-18T plasmid. Sequencing analysis showed that bovine osteocalcin cDNA gene was 303 bp and encoded 100 amino acids, which completely matched with X53699 in GenBank. Through connecting three artificial single strands DNA which contain fixed-point mutated sites by Gene splicing by overlap extension PCR, we changed some rare codons to that Escherichia coli frequently used in mature bovine osteocalcin gene, then recombined the connected production into PET-32a plasmid and transformed into the competent expressive cells of E.coli BL21(DE3). Recombinant E.coli BL21 was induced by IPTG and osteocalcin fusion protein was successfully expressed, and successfully to expressed bovine BGP anti-body.2. Osteogenic actions of the osteogenic growth peptide on bovine marrow mesenchymal stromal cells in cultureThe OGP regulated marrow mesenchymal stem cells which derived from human and rodent proliferation and differentiation into osteoblasts. Whether OGP directly regulates the bovine marrow mesenchymal stem cells differentiating into osteoblasts remains unknown. In this study, we evaluated the effect of OGP on the growth and differentiation of bovine marrow mesenchymal stem cells in culture. Our results showed that OGP promoted differentiation of the bovine stem cells. OGP increased alkaline phosphatase (ALP) activity and mineralized nodule formation, and stimulated osteoblast-specific mRNA expression of Osteocalcin (BGP). On the other hand, OGP dose-dependently stimulated the expression of endothelial nitric oxide synthases. These results show for the first time a direct osteogenic effect of OGP on bovine marrow stromal cells in culture, which could be mediated by the induction of endothelial nitric oxide synthases.3. The stimulating effect of OGP on the proliferation, differentiation, and mineralization of osteoblastic cells from Holstein cattleThis study investigated the effect of exogenous osteogenic growth peptide on proliferation, differentiation and mineralization of osteoblastic cells from Chinese Holstein cattle. The osteoblastic cells were isolated and cultured, then identified through morphological observation and alkaline phosphatase staining methods. The effect of the different concentrations of osteogenic growth peptide on cell growth was assessed by MTT assay. Cytoplasmic alkaline phosphatase activity and osteocalcin levels were measured by colorimetric assay and radioimmunoassay, respectively.calcium nodules were observed using alizarin red S stain. The results showed that osteogenic growth peptide can significantly promote osteoblasts proliferation, and also stimulate the alkaline phosphatase activity and osteocalcin secretion.the max effect of concentration is 10-9M. when osterix expression was inhibited, the promotion of osteogenic growth peptide to alkaline phosphatase activity and osteocalcin secretion was inhibited accordingly. This revealed that osteogenic growth peptide can promote dairy osteoblast proliferation and differentiation and is mediated by up-regulating osterix.4. Responses of free radicals to subcutaneous implantation of alginate—chitosan-alginate (ACA) microcapsules in miceOur objective was to characterize the kinetics of free radicals when responding to encapsulated cell implantations in vivo. Cell viability and serum free radical, malondialdehyde and superoxide dismutase levels were evaluated at 1, 4 and 7 days after either saline injection or the implantation of empty microcapsules or encapsulated cells. Hydrogen peroxide and malondialdehyde levels showed an initial rise in the recipients of the empty microcapsules, before decreasing to the basal level. However, in rats receiving the encapsulated cells, the levels were higher at the end of study. Nitric oxide and superoxide dismutase increased after the implantation of microcapsules with or without the BHK-21 cells, but did not alter in response to the saline injection. The viability of encapsulated cells was high in vivo and some microcapsules had broken by day 7 post-implantation. These results suggest that nitric oxide played a role in the specific response to microcapsules. Free radicals increased rapidly immediately following microcapsule transplantation, but they caused only slight cellular damage before the microencapsulated cells were exposed.5. Release of OGP from microencapsulated engineered cells and proliferation characteristics in vivo and in vitroFor the study of engineered cells microcapsules in vivo and in vitro characteristics of the proliferation and secretion. Secretory of OGP and engineered cells proliferation in microcapsule cultured in vitro and in vivo was tested. Serum antioxidant enzyme activities, lipid peroxidation and nitric oxide levels in rabbit with glucocorticoid-induced osteoporosis were also assessed. The results demonstrated that OGP engineered cells microcapsules have good secretion and cell proliferation. But when transplanted into Osteoporosis model, a large number of cells in microcapsule dead. Serum free radicals in Osteoporosis model were significantly higher, but antioxidant activity decreased. The injection after anti-oxidants can significantly reduce the serum concentration of free radicals. Thus that, OGP microencapsulated genetically modified cells have the potential for the treatment of transplantation, in vivo model of osteoporosis disease transplant failure, mainly due to osteoporosis in vivo model of high levels of oxygen free radicals, leading to Microcystis necrosis or apoptosis within the cell.更多还原