Hypertrophy is induced during the in vitro chondrogenic differentiation of human mesenchymal stem cells by bone morphogenetic protein-2 and bone morphogenetic protein-4 gene transfer.
Hypertrophy is induced during the in vitro chondrogenic differentiation of human mesenchymal stem cells by bone morphogenetic protein-2 and bone morphogenetic protein-4 gene transfer.
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DOI：
10.1186/ar2822
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发表时间：
2009
影响因子：
4.9
通讯作者：
Evans CH
中科院分区：
文献类型：
--
作者：
Steinert AF;Proffen B;Kunz M;Hendrich C;Ghivizzani SC;Nöth U;Rethwilm A;Eulert J;Evans CH
本研究比较了骨形态发生蛋白（BMP）-4和BMP-2基因转移作为软骨形成和肥大的人原代间充质干细胞（MSC）保持为颗粒培养物的代理。通过cre-lox组合构建携带编码人BMP-4的cDNA的腺病毒载体（Ad.BMP-4），并与先前产生的BMP-2（Ad.BMP-2）、绿色荧光蛋白（Ad.GFP）或萤火虫荧光素酶（Ad.Luc）的腺病毒载体进行比较。用5 × 102病毒颗粒/细胞的Ad.BMP-2或Ad.BMP-4感染人骨髓来源的MSC的培养物，接种成聚集体并在确定的无血清培养基中培养三周。未转导的细胞或用标记基因转导的培养物作为对照。通过ELISA测定BMP-2和BMP-4的表达，并通过组织学、免疫化学、生物化学和RT-PCR分析聚集体的软骨形成和肥大。培养基中BMP-2和BMP-4的水平最初为30至60 ng/mL，此后下降。BMP-4和BMP-2基因是原代MSC中软骨形成的等效诱导剂，如通过腔隙形成、蛋白聚糖和II型胶原的强染色、GAG合成水平增加以及与软骨细胞表型相关的mRNA表达所判断的。然而，BMP-4修饰的聚集体表现出较低的倾向，向肥大的进展，判断碱性磷酸酶，膜联蛋白5，免疫组化染色的X型胶原蛋白，和腔隙大小的表达。BMP-2和BMP-4在通过团块培养的原代人MSC引起软骨形成方面同样有效。然而，由BMP-2和BMP-4基因转移引发的软骨形成显示出相当多的肥大分化的证据，细胞在形态和功能上都类似于生长板软骨细胞。这表明在软骨修复中使用这些候选基因时要谨慎。
The present study compares bone morphogenetic protein (BMP)-4 and BMP-2 gene transfer as agents of chondrogenesis and hypertrophy in human primary mesenchymal stem cells (MSCs) maintained as pellet cultures. Adenoviral vectors carrying cDNA encoding human BMP-4 (Ad.BMP-4) were constructed by cre-lox combination and compared to previously generated adenoviral vectors for BMP-2 (Ad.BMP-2), green fluorescent protein (Ad.GFP), or firefly luciferase (Ad.Luc). Cultures of human bone-marrow derived MSCs were infected with 5 × 102 viral particles/cell of Ad.BMP-2, or Ad.BMP-4, seeded into aggregates and cultured for three weeks in a defined, serum-free medium. Untransduced cells or cultures transduced with marker genes served as controls. Expression of BMP-2 and BMP-4 was determined by ELISA, and aggregates were analyzed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy. Levels of BMP-2 and BMP-4 in the media were initially 30 to 60 ng/mL and declined thereafter. BMP-4 and BMP-2 genes were equipotent inducers of chondrogenesis in primary MSCs as judged by lacuna formation, strong staining for proteoglycans and collagen type II, increased levels of GAG synthesis, and expression of mRNAs associated with the chondrocyte phenotype. However, BMP-4 modified aggregates showed a lower tendency to progress towards hypertrophy, as judged by expression of alkaline phosphatase, annexin 5, immunohistochemical staining for type X collagen protein, and lacunar size. BMP-2 and BMP-4 were equally effective in provoking chondrogenesis by primary human MSCs in pellet culture. However, chondrogenesis triggered by BMP-2 and BMP-4 gene transfer showed considerable evidence of hypertrophic differentiation, with, the cells resembling growth plate chondrocytes both morphologically and functionally. This suggests caution when using these candidate genes in cartilage repair.
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通讯作者：
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