骨唾液酸蛋白糖基化修饰对成骨的影响

发布时间：2018-01-01 02:08

本文关键词：骨唾液酸蛋白糖基化修饰对成骨的影响　出处：《苏州大学》2012年博士论文　论文类型：学位论文

【摘要】：目的探讨骨唾液酸蛋白(Bone Sialoprotein, BSP)糖基化修饰对成骨的影响及可能的作用机制。方法 1.建立MC3T3-E1Subclone14成骨细胞培养模型,倒置显微镜观察细胞形态,RT-PCR和Western-Blot检测成骨细胞相关因子骨钙素、骨唾液酸蛋白等mRNA和蛋白水平的表达,碱性磷酸酶(Alkaline phosphatase, ALP)、von Kossa染色以鉴定成骨细胞和骨矿化的形成。 2.在成骨细胞培养模型中加入人重组骨唾液酸蛋白(Recombinant Human Bone Sialoprotein, rhBSP)及经O-糖苷酶消化的去O-糖基化rhBSP,使用荧光标记的具有特异识别O糖链结构的凝集素VVL,VVA(Biotinylated Vicia Villosa Lectin, VVL,VVA)检测细胞中O糖链,并通过流式细胞仪验证；同时选择在成骨细胞培养模型中高表达ppGalNAcT1和T4(polypeptide GalNAc transferases, ppGalNAcTs)为研究对象,利用RNA干扰技术,分别构建和筛选出高效的T1siRNA和T4siRNA表达载体转染入MC3T3-E1Subclone14成骨细胞培养模型中,ALP、von Kossa染色及RT-PCR、Western-blot等成骨细胞生物学检测手段观察BSP O-糖基化修饰对成骨的影响。 3.在成骨细胞培养模型中加入不同浓度的α2,3神经氨酸苷酶,使用荧光标记的具有特异识别a2,3唾液酸结构的凝集素MALII(Maackia amurensis leukoagglutinin Ⅱ,MALⅡ)检测细胞表面的a2,3唾液酸糖链结构,并通过流式细胞仪验证；Western-blot检测BSP蛋白水平的变化；同时利用ALP、von Kossa染色及RT-PCR、Western-blot等成骨细胞生物学检测手段观察BSP相关的末端唾液酸化对成骨的影响。 4.在成骨细胞培养模型中加入rhBSP、肽N-糖苷酶F消化的去N-糖基化rhBSP及不同浓度的衣霉素(N-糖基化糖链合成抑制剂),使用荧光标记的具有特异识别N糖链结构的凝集素LEA(Lycopersicon esculentum agglutinin, LEA)检测细胞中N糖链,并通过流式细胞仪验证；同时利用ALP、von Kossa染色及RT-PCR, Western-blot等成骨细胞生物学检测手段观察BSP N-糖基化修饰对成骨的影响。 5.在MC3T3-E1Subclone14成骨细胞培养模型中,加入肽N-糖苷酶F、O-糖苷酶、唾液酸酶、β-半乳糖苷酶和氨基葡糖苷酶等,Western-blot检测BSP蛋白水平的变化,同时利用凝集素标记、流式细胞仪鉴定、质谱分析等方法,探讨BSP的糖链结构。结果 1.成功建立了MC3T3-E1Subclone14成骨细胞培养模型,证明MC3T3-E1Subclone14细胞培养至10d左右ALP染色呈强阳性反应,即成骨细胞大量形成,von Kossa染色至14d左右呈强阳性反应,说明成骨细胞分泌的骨基质进入矿化期。 2.在成骨细胞培养模型中加入rhBSP及O-糖苷酶消化的去O-糖基化rhBSP, ALP、von Kossa染色结果显示：BSP O-糖基化修饰影响了成骨细胞和骨矿化的形成；同时成功构建和筛选出在MC3T3-E1Subclone14成骨细胞培养模型中高表达的高效siRNA表达载体ppGalNAcT1-1545和ppGalNAcT4-784,转染入成骨细胞培养模型后有效抑制了成骨细胞分化和骨矿化的形成,证实BSP O-糖基化修饰影响成骨主要为ppGalNAcT1、T4两个糖基转移酶。 3.BSP相关的末端唾液酸化对骨矿化的影响主要以a2,3唾液酸为主,它可能通过影响维生素D受体VDR的表达,从而影响骨的矿化,但对成骨细胞的分化无明显影响。 4.在成骨细胞培养模型中加入rhBSP、肽N-糖苷酶F消化的去N-糖基化rhBSP及不同浓度的衣霉素,ALP、von Kossa染色等结果显示：BSPN-糖基化修饰及N-糖基化糖链合成抑制剂衣霉素对成骨无明显影响。 5.肽N-糖苷酶F释放糖蛋白中N-连接的糖链；质谱检查发现,经唾液酸酶处理后的BSP,其N-糖链末端的唾液酸脱落,1142(H5N4F1)、1366(H6N5F1)、1279(H6N5)、1504(H7N6)等出现在唾液酸酶处理后的图谱中,此结果与Western-blot鉴定BSP的表达基本相符。同时流式细胞仪分析、Western-blot检测进一步证实BSP是一个含N-糖链、O-糖链及末端唾液酸的糖基化蛋白。综上所述,我们成功建立了MC3T3-E1Subclone14成骨细胞培养模型；证实BSP O-糖基化修饰影响成骨主要涉及ppGalNAcT1、T4两个糖基转移酶；BSP相关的末端唾液酸化主要影响骨矿化的形成,并以α2,3唾液酸为主,但对成骨细胞的分化无明显影响；BSP N-糖基化修饰及相关的N糖链对成骨无明显影响；同时我们利用现有的质谱技术初步分析了BSP的糖链结构,.进一步证实BSP是一个含N-糖链、O-糖链及末端唾液酸结构的糖基化蛋白,且BSP糖基化修饰影响成骨主要以BSP O-糖基化修饰为主。
[Abstract]:objective
To investigate the effect of glycosylation of Bone Sialoprotein (BSP) on osteogenesis and its possible mechanism of action.
Method
1. establishment of MC3T3-E1Subclone14 osteoblast cell culture model, cell morphology was observed by inverted microscope, RT-PCR and Western-Blot detection of osteoblast related factor expression of osteocalcin, bone sialoprotein, mRNA and protein levels of alkaline phosphatase (Alkaline, phosphatase, ALP, von) Kossa staining to identify the osteoblastic bone formation and bone mineralization.
2. in the osteoblast culture after the recombinant human bone sialoprotein model (Recombinant Human Bone Sialoprotein, rhBSP and O-) by glycosidase digestion to O- glycosylation of rhBSP, using fluorescence labeled with VVL lectin specific recognition of O sugar chain structure, VVA (Biotinylated Vicia Villosa Lectin, VVL, VVA) detection O sugar chain in the cell, and verified by flow cytometry; at the same time in osteoblast culture of ppGalNAcT1 and high expression of T4 model (polypeptide GalNAc transferases, ppGalNAcTs) as the research object, using RNA interference technology, and the screening of efficient T1siRNA and T4siRNA expression vector was transfected into MC3T3-E1Subclone14 osteoblast cell culture model. ALP were constructed, von Kossa and RT-PCR Western-blot staining, osteoblast biology detection means to observe the BSP glycosylation of O- into osteoblasts.
3. in the osteoblast culture with different concentration in the model of alpha 2,3 neuraminidase, using fluorescence labeled with MALII lectin specific recognition a2,3 sialic acid structure (Maackia amurensis leukoagglutinin II, MAL II) detection of a2,3 sialic acid sugar chain structure on the surface of cells was verified by flow cytometry; changes of Western-blot to detect the protein level of BSP; at the same time using ALP, von and RT-PCR Kossa staining, Western-blot osteoblast biology detection means to observe the BSP related terminal sialic acid on bone formation.
4. in the osteoblast culture into rhBSP model, N- peptide N-glycosidase F digestion to N- glycosylation of rhBSP and different concentrations of tunicamycin (N- synthesis inhibitor glycosylation), using fluorescence labeled with LEA lectin glycan structure specific recognition of N (Lycopersicon esculentum agglutinin, LEA) detection N sugar chain in the cell, and verified by flow cytometry; at the same time, using ALP, von and RT-PCR Kossa staining, Western-blot osteoblast biological detection methods to observe BSP N- glycosylation on osteogenic effect.
5. in MC3T3-E1Subclone14 osteoblast cell culture model, adding peptide N- glucosidase F, O- glycosidase, sialidase, beta galactosidase and Glucosaminidase, Western-blot to detect changes in BSP protein levels, while using lectin staining, flow cytometry, mass spectrum analysis method of sugar the chain structure of BSP.
Result
1. successfully established MC3T3-E1Subclone14 culture model of osteoblast, MC3T3-E1Subclone14 cell culture to about 10d ALP staining showed strong positive reaction, which is formed into a large number of bone cells, von Kossa staining showed strong positive reaction to 14d, which indicates that the bone stromal osteoblasts secreted into the mineralization period.
2. in the osteoblast culture to ALP glycosylation of O- rhBSP, joining rhBSP and O- glycosidase digestion model in von, Kossa staining showed: BSP O- glycosylation affects osteoblastic bone formation and bone mineralization; at the same time, successfully constructed and screened by osteoblasts cultured in MC3T3-E1Subclone14, the high expression of siRNA in the model the expression vector ppGalNAcT1-1545 and ppGalNAcT4-784, were transfected into osteoblasts culture model can effectively inhibit osteoblast differentiation and the formation of bone mineralization, confirmed that BSP O- glycosylation influence osteogenesis were mainly ppGalNAcT1, T4 two glycosyltransferases.
The effect of 3.BSP related terminal sialidification on bone mineralization is mainly a2,3 sialic acid. It may affect the mineralization of bone by affecting the expression of vitamin D receptor VDR, but it has no significant effect on the differentiation of osteoblasts.
4., in the osteoblast culture model, rhBSP, peptide N- glycosidase F digestion, N- glycosylation rhBSP and different concentrations of yamomycin, ALP and von Kossa staining showed that BSPN- glycosylation and N- glycosylation inhibitor glycomycin inhibitor had no significant effect on osteogenesis.
5. peptide N- glucosidase F release sugar chain connection N- glycoprotein; mass spectrometry examination found that the sialidase treated BSP, sialic acid N- sugar chain at the end of the fall, 1142 (H5N4F1), 1366 (H6N5F1), 1279 (H6N5), 1504 (H7N6) appeared in sialidase treatment after the spectrum, the expression of this result and the identification of Western-blot BSP basically. At the same time, flow cytometry analysis, Western-blot assay further confirmed that BSP is a N- containing sugar chain, O- sugar chain and terminal sialic acid glycosylated protein.
In summary, we have successfully established a MC3T3-E1Subclone14 culture model of osteoblast; confirmed that BSP O- glycosylation influence osteogenesis mainly related to ppGalNAcT1, T4 two glycosyltransferase; BSP related terminal sialic acid mainly affects the formation of bone mineralization, and the alpha 2,3 sialic acid, but had no significant effect on osteoblast differentiation cells; BSP N- glycosylation and related N sugar chain has no obvious effect on osteogenesis; at the same time we use the existing mass spectrometry analyses of sugar chain structure, BSP. Further confirmed that BSP is a N- containing sugar chain, glycosylated protein O- sugar chain and terminal sialic acid structure. And the BSP glycosylation effect of osteogenic mainly BSP O- glycosylation.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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