Cellular effects of enamel matrix derivative are associated with different molecular weight fractions following separation by size-exclusion chromatography.
Cellular effects of enamel matrix derivative are associated with different molecular weight fractions following separation by size-exclusion chromatography.
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DOI：
10.1902/jop.2009.070420
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发表时间：
2009-04
影响因子：
4.3
通讯作者：
Cochran DL
中科院分区：
文献类型：
--
作者：
Johnson DL;Carnes D;Steffensen B;Cochran DL
牙釉质基质衍生物（EMD）已被证明可以促进软组织愈合和牙周组织的再生，但这种作用的机制尚不清楚。据推测，EMD中最丰富的蛋白——淀粉原蛋白是导致EMD效果的主要蛋白。本研究的目的是分离EMD，并将EMD的特异性细胞效应与不同分子量的分子量相关联。新鲜溶解的EMD经凝胶过滤分离，收集45份7ml馏分，脱盐，冻干，重悬。分析各组分对人微血管内皮细胞（HMVEC）增殖分化及骨祖细胞（C2C12）分化的影响。在骨形态发生蛋白（BMP）诱饵受体noggin预孵育前后，测定碱性磷酸酶活性（C2C12）作为成骨分化的标志物。血管生成（HMVEC）被评估为内皮细胞分化的标志。酶法测定使用聚丙烯酰胺凝胶与变性I型胶原共聚合，以确定每个部分的明胶溶解活性。用Sephadex G-100进行隔离层析后，EMD分离成三个主要的蛋白峰。峰1与柱空隙体积有关，峰3在盐体积附近洗脱。峰II在这两个峰之间洗脱。微血管细胞的增殖和血管生成活性与II峰和III峰均相关。碱性磷酸酶活性表明，骨祖细胞的分化是由峰I和峰II前缘的EMD成分诱导的。另外观察到，这种分化被预先用noggin处理的部分抑制，这表明这种活性是由BMP而不是淀粉原蛋白或其他未知蛋白诱导的。凝胶分离EMD峰1和峰2的早期部分检测到明胶溶解活性。EMD刺激的细胞活动与单一分子量的物种无关。noggin破坏C2C12碱性磷酸酶活性的事实表明，对骨祖细胞分化的影响是bmp样蛋白(s)的结果，而对增殖和血管生成的影响与峰II和峰III中存在的低分子量物种有关。最后，未加热的EMD显示明胶溶解活性，也可以在EMD成分的尺寸排除分离后检测到。这些活性的质量与报道的潜伏和活性MMP-20一致。
Enamel matrix derivative (EMD) has been shown to enhance both soft tissue healing and regeneration of the periodontium, but the mechanisms of this action are still unknown. It is assumed that amelogenin, the most abundant protein in EMD, is the protein primarily responsible for the effects of EMD. The purpose of this study was to fractionate EMD and associate specific cellular effects of EMD with different molecular weight fractions following size exclusion chromatography. Freshly dissolved EMD was fractionated by gel filtration and forty-five 7ml fractions collected, desalted, lyophilized, and resuspended. These fractions were analyzed for their effects on differentiation of osteoprogenitor cells (C2C12) and proliferation and differentiation of human microvascular endothelial cells (HMVEC). Alkaline phosphatase activity (C2C12) was measured as a marker for osteogenic differentiation before and after pre-incubation of the fractions with the bone morphogenetic protein (BMP) decoy receptor, noggin. Angiogenesis (HMVEC) was evaluated as a marker for endothelial cell differentiation. Enzymographic assays used polyacrylamide gels co-polymerized with denatured type I collagen to determine gelatinolytic activities in each fraction. EMD fractionated into three major protein peaks following size exclusion chromatography with Sephadex G-100. Peak I was associated with the column void volume, while peak III eluted near the salt volume. Peak II eluted between these two peaks. Proliferation and angiogenic activities were associated with both peak II and peak III for the microvascular cells. Differentiation of osteoprogenitor cells, indicated by alkaline phosphatase activity, was induced by EMD components present in peak I and the leading edge of peak II. The additional observation that this differentiation was inhibited by prior treatment of the fractions with noggin suggested the activity was induced by BMP rather than amelogenin or other unknown proteins. Gelatinolytic activities were detected in the early fractions of peaks 1 and 2 of gel fractionated EMD. The cellular activities stimulated by EMD are not associated with a single molecular weight species. The fact that noggin abolishes C2C12 alkaline phosphatase activity suggests that effects on osteoprogenitor cell differentiation are the result of a BMP-like protein(s), while effects on proliferation and angiogenesis are associated with lower molecular weight species present in peak II and peak III. Finally, unheated EMD displays gelatinolytic activities that are also detectable following size exclusion separation of EMD constituents. The masses of these activities were consistent with those reported for latent and active MMP-20.
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影响因子：
4.3
作者：
Schlueter, Scott R.;Carnes, David L., Jr.;Cochran, David L.
通讯作者：
Cochran, David L.
影响因子：
7.6
作者：
Iwata, T;Morotome, Y;Oida, S
通讯作者：
Oida, S
影响因子：
4.2
作者：
AOBA, T;FUKAE, M;MORENO, EC
通讯作者：
MORENO, EC
影响因子：
4.8
作者：
Malinda, KM;Nomizu, M;Ponce, ML
通讯作者：
Ponce, ML
影响因子：
4.8
作者：
Steffensen, B;Bigg, HF;Overall, CM
通讯作者：
Overall, CM