本文關(guān)鍵詞：骨唾液酸蛋白糖基化修飾對成骨的影響　出處：《蘇州大學(xué)》2012年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 骨唾液酸蛋白 成骨細胞 糖基化修飾 凝集素 質(zhì)譜

【摘要】：目的 探討骨唾液酸蛋白(Bone Sialoprotein, BSP)糖基化修飾對成骨的影響及可能的作用機制。 方法 1.建立MC3T3-E1Subclone14成骨細胞培養(yǎng)模型,倒置顯微鏡觀察細胞形態(tài),RT-PCR和Western-Blot檢測成骨細胞相關(guān)因子骨鈣素、骨唾液酸蛋白等mRNA和蛋白水平的表達,堿性磷酸酶(Alkaline phosphatase, ALP)、von Kossa染色以鑒定成骨細胞和骨礦化的形成。 2.在成骨細胞培養(yǎng)模型中加入人重組骨唾液酸蛋白(Recombinant Human Bone Sialoprotein, rhBSP)及經(jīng)O-糖苷酶消化的去O-糖基化rhBSP,使用熒光標(biāo)記的具有特異識別O糖鏈結(jié)構(gòu)的凝集素VVL,VVA(Biotinylated Vicia Villosa Lectin, VVL,VVA)檢測細胞中O糖鏈,并通過流式細胞儀驗證；同時選擇在成骨細胞培養(yǎng)模型中高表達ppGalNAcT1和T4(polypeptide GalNAc transferases, ppGalNAcTs)為研究對象,利用RNA干擾技術(shù),分別構(gòu)建和篩選出高效的T1siRNA和T4siRNA表達載體轉(zhuǎn)染入MC3T3-E1Subclone14成骨細胞培養(yǎng)模型中,ALP、von Kossa染色及RT-PCR、Western-blot等成骨細胞生物學(xué)檢測手段觀察BSP O-糖基化修飾對成骨的影響。 3.在成骨細胞培養(yǎng)模型中加入不同濃度的α2,3神經(jīng)氨酸苷酶,使用熒光標(biāo)記的具有特異識別a2,3唾液酸結(jié)構(gòu)的凝集素MALII(Maackia amurensis leukoagglutinin Ⅱ,MALⅡ)檢測細胞表面的a2,3唾液酸糖鏈結(jié)構(gòu),并通過流式細胞儀驗證；Western-blot檢測BSP蛋白水平的變化；同時利用ALP、von Kossa染色及RT-PCR、Western-blot等成骨細胞生物學(xué)檢測手段觀察BSP相關(guān)的末端唾液酸化對成骨的影響。 4.在成骨細胞培養(yǎng)模型中加入rhBSP、肽N-糖苷酶F消化的去N-糖基化rhBSP及不同濃度的衣霉素(N-糖基化糖鏈合成抑制劑),使用熒光標(biāo)記的具有特異識別N糖鏈結(jié)構(gòu)的凝集素LEA(Lycopersicon esculentum agglutinin, LEA)檢測細胞中N糖鏈,并通過流式細胞儀驗證；同時利用ALP、von Kossa染色及RT-PCR, Western-blot等成骨細胞生物學(xué)檢測手段觀察BSP N-糖基化修飾對成骨的影響。 5.在MC3T3-E1Subclone14成骨細胞培養(yǎng)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檢測BSP蛋白水平的變化,同時利用凝集素標(biāo)記、流式細胞儀鑒定、質(zhì)譜分析等方法,探討B(tài)SP的糖鏈結(jié)構(gòu)。 結(jié)果 1.成功建立了MC3T3-E1Subclone14成骨細胞培養(yǎng)模型,證明MC3T3-E1Subclone14細胞培養(yǎng)至10d左右ALP染色呈強陽性反應(yīng),即成骨細胞大量形成,von Kossa染色至14d左右呈強陽性反應(yīng),說明成骨細胞分泌的骨基質(zhì)進入礦化期。 2.在成骨細胞培養(yǎng)模型中加入rhBSP及O-糖苷酶消化的去O-糖基化rhBSP, ALP、von Kossa染色結(jié)果顯示：BSP O-糖基化修飾影響了成骨細胞和骨礦化的形成；同時成功構(gòu)建和篩選出在MC3T3-E1Subclone14成骨細胞培養(yǎng)模型中高表達的高效siRNA表達載體ppGalNAcT1-1545和ppGalNAcT4-784,轉(zhuǎn)染入成骨細胞培養(yǎng)模型后有效抑制了成骨細胞分化和骨礦化的形成,證實BSP O-糖基化修飾影響成骨主要為ppGalNAcT1、T4兩個糖基轉(zhuǎn)移酶。 3.BSP相關(guān)的末端唾液酸化對骨礦化的影響主要以a2,3唾液酸為主,它可能通過影響維生素D受體VDR的表達,從而影響骨的礦化,但對成骨細胞的分化無明顯影響。 4.在成骨細胞培養(yǎng)模型中加入rhBSP、肽N-糖苷酶F消化的去N-糖基化rhBSP及不同濃度的衣霉素,ALP、von Kossa染色等結(jié)果顯示：BSPN-糖基化修飾及N-糖基化糖鏈合成抑制劑衣霉素對成骨無明顯影響。 5.肽N-糖苷酶F釋放糖蛋白中N-連接的糖鏈；質(zhì)譜檢查發(fā)現(xiàn),經(jīng)唾液酸酶處理后的BSP,其N-糖鏈末端的唾液酸脫落,1142(H5N4F1)、1366(H6N5F1)、1279(H6N5)、1504(H7N6)等出現(xiàn)在唾液酸酶處理后的圖譜中,此結(jié)果與Western-blot鑒定BSP的表達基本相符。同時流式細胞儀分析、Western-blot檢測進一步證實BSP是一個含N-糖鏈、O-糖鏈及末端唾液酸的糖基化蛋白。 綜上所述,我們成功建立了MC3T3-E1Subclone14成骨細胞培養(yǎng)模型；證實BSP O-糖基化修飾影響成骨主要涉及ppGalNAcT1、T4兩個糖基轉(zhuǎn)移酶；BSP相關(guān)的末端唾液酸化主要影響骨礦化的形成,并以α2,3唾液酸為主,但對成骨細胞的分化無明顯影響；BSP N-糖基化修飾及相關(guān)的N糖鏈對成骨無明顯影響；同時我們利用現(xiàn)有的質(zhì)譜技術(shù)初步分析了BSP的糖鏈結(jié)構(gòu),.進一步證實BSP是一個含N-糖鏈、O-糖鏈及末端唾液酸結(jié)構(gòu)的糖基化蛋白,且BSP糖基化修飾影響成骨主要以BSP O-糖基化修飾為主。
[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.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R363

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