EMBRYONIC MOUSE PRE-METATARSAL DEVELOPMENT IN ORGAN-CULTURE
EMBRYONIC MOUSE PRE-METATARSAL DEVELOPMENT IN ORGAN-CULTURE
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DOI：
10.1002/jez.1402650309
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发表时间：
1993-03-01
影响因子：
--
通讯作者：
SPOONER, BS
中科院分区：
文献类型：
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作者：
KLEMENT, BJ;SPOONER, BS
胚胎小鼠前跖骨在妊娠13天从胚胎中取出，并在确定的无血清培养基中培养15天。通过组织学分析，我们观察到培养的跖骨前组织经历了与体内正常生长的跖骨前组织相似的发育特征。最初的间质凝聚区经过分化和形态发生形成由软骨组织组成的不同的棒。这一分化步骤的标志是Il型胶原蛋白的合成。代谢标记，胃蛋白酶消化，SDS-PAGE和放射自显影被用来证明这种蛋白，当软骨组织存在于培养物中。在额外的培养时间后，软骨棒的特定区域发生终末软骨细胞分化和形态发生，形成肥大的软骨细胞带。这个分化步骤的一个标志是碱性磷酸酶的合成。在整个培养过程中，我们测量了这种酶的活性，发现在终软骨细胞分化时，这种酶的活性显著增加。软骨细胞肥大的另一个特征是细胞周围的基质钙化。用茜素红染色观察培养前跖骨的钙化基质。通过在培养基中添加ITS，我们观察到终软骨细胞在较短的培养时间内发生分化。在培养基中添加血清也能加速末梢分化，并且随着培养时间的延长，在杆状细胞的中心区域形成类骨基质。
Embryonic mouse pre-metatarsals were removed from embryos at 13 days of gestation and cultured in a defined, serum-free medium for up to 15 days. By histological analysis, we observe that the cultured pre-metatarsal tissue undergoes a similar developmental profile as premetatarsals growing normally in vivo. The initial mesenchyme condensation regions undergo differentiation and morphogenesis to form distinct rods made up of cartilage tissue. A marker of this differentiation step is the synthesis of type Il collagen. Metabolic labelling, pepsin digestion, SDS-PAGE, and autoradiography were used to demonstrate this protein when cartilage tissue is present in the cultures. After additional culture time, terminal chondrocyte differentiation and morphogenesis take place in specific regions of the cartilage rods to form bands of hypertrophied chondrocytes. One marker of this differentiation step is the synthesis of the enzyme alkaline phosphatase. We have measured the activity of this enzyme throughout the culture period and see a substantial increase at the time of terminal chondrocyte differentiation. Another feature of hypertrophied chondrocytes is that the matrix around the cells becomes calcified. Calcified matrix in our cultured pre-metatarsals was visualized by staining with alizarin red. By supplementing the defined culture medium with ITS, we observed that terminal chondrocyte differentiation took place in a shorter culture time. Supplementation of the medium with serum results in a similar acceleration of terminal differentiation, and, with additional culture time, an osteoid-like matrix forms around the central region of the rods.