Singh G D, Johnston J, Ma W, Lozanoff S
Department of Dental Surgery and Periodontology, Dundee Dental Hospital and School, University of Dundee, Scotland, UK.
Cleft Palate Craniofac J. 1998 Jan;35(1):65-76. doi: 10.1597/1545-1569_1998_035_0065_cpfifb_2.3.co_2.
This study tested the hypothesis that altered craniofacial morphology does not affect the expression of extracellular matrix (ECM) molecules such as fibronectin (FN), laminin (LN), type IV collagen, and tenascin-C (TN) but is associated with failure of palatal shelf elevation and fusion concomitant with cleft palate formation.
To test this hypothesis, a comparative immunohistological analysis of FN, LN, type IV collagen, and TN was undertaken on brachyrrhine (Br/Br) mice and normal (+/+) fetuses during secondary palate formation. Normal and Br/Br fetuses were collected at gestational days E13 and E14 (representing prefusion stages) and E15 and E18 (representing postfusion stages). Cryostat palatal sections (8 microm) were postfixed in methanol, washed, and stained with primary antibody. All sections were washed and coated with secondary antibody (swine-anti-rabbit IgG) and mounted with citifluor.
Immunohistological analysis showed that LN and type IV collagen were located near the presumptive medial epithelial seam (MES) or edge (MEE) in +/+ or Br/Br fetuses, respectively. Fibronectin showed a homogeneous distribution at all stages in both groups of mice. In contrast, TN became localized below the presumptive MES or MEE in both groups of mice at E14. In +/+ animals at E15, TN dissipated and became confined to the oral basement membrane by E18. At E15 and E18 in cleft Br/Br mutants, TN stained beneath the MEE.
Although the distributions of ECM molecules are similar during normal and cleft palatogenesis, differences in TN expression are associated with cleft palate formation.
本研究验证了以下假设，即颅面形态改变不会影响细胞外基质（ECM）分子如纤连蛋白（FN）、层粘连蛋白（LN）、IV型胶原蛋白和腱生蛋白-C（TN）的表达，但与腭裂形成时腭突抬高和融合失败有关。
为验证该假设，在次级腭形成过程中，对短鼻（Br/Br）小鼠和正常（+/+）胎儿进行了FN、LN、IV型胶原蛋白和TN的比较免疫组织学分析。在妊娠第E13和E14天（代表融合前阶段）以及E15和E18天（代表融合后阶段）收集正常和Br/Br胎儿。将恒冷箱腭部切片（8微米）用甲醇后固定、洗涤，并用一抗染色。所有切片洗涤后用二抗（猪抗兔IgG）包被，并用citifluor封固。
免疫组织学分析表明，LN和IV型胶原蛋白分别位于+/+或Br/Br胎儿的假定内侧上皮缝（MES）或边缘（MEE）附近。纤连蛋白在两组小鼠的所有阶段均呈均匀分布。相比之下 在E14时，两组小鼠的TN均定位于假定MES或MEE下方。在E15时的+/+动物中，TN消散，并在E18时局限于口腔基底膜。在E15和E18时，腭裂Br/Br突变体中，TN在MEE下方染色。
尽管在正常和腭裂发生过程中ECM分子的分布相似，但TN表达的差异与腭裂形成有关。
