目的 构建C57BL/6J小鼠腭突体外培养模型,研究血小板衍化生长因子受体α(PDGFR-α)在小鼠腭突体外培养不同时间段的表达变化.方法 应用悬浮培养法分别培养腭突24、36、48 h,体视显微镜及苏木素-伊红(HE)染色观察腭突发育情况.免疫组织化学检测腭突体外培养不同时间段组织中PDGFR-α的表达定位, Western blot检测腭突体外培养不同时间段组织中PDGFR-α的时空表达变化.结果 体视显微镜和HE染色结果均显示,体外培养24 h,双侧腭突向中线方向靠近生长,间隙变窄;36 h,镜下观察到双侧腭突接触,HE切片观察到腭中嵴上皮缝的形成;48 h,镜下观察到双侧腭突融合,HE切片观察到腭中嵴上皮缝消失.PDGFR-α在腭突体外悬浮培养24 h时表达最高,36及48 h后逐渐降低.结论 本实验改进的腭突体外悬浮培养方法模拟了腭突的体内发育环境,为应用体外腭突培养模型研究腭裂复杂的发病机制奠定了基础.笔者采用的体外悬浮培养方法证明腭突发育相关基因PDGFR-α在体外培养腭突的表达变化与其体内表达是一致的.%Objective This experiment builded in C57BL/6J mouse models of palatal process culture in vitro,to study the expression of platelet-derived growth factor receptor α (PDGFR-α) during different times. Methods Experiment cultured palates with the method of suspension culture for 24, 36, 48 h, palatal development was observed by stereomicroscope and hematoxylin-eosin (HE) staining. PDGFR-α expression location in different period was detected by immunohistochemical assays. The expression of PDGFR-α was examined by Western blot. Results Stereomicroscope and HE staining results showed that in vitro cultivation of 24 h,palatal process the space between bilateral palatal process reduced,bilateral palatal process contact and the crest epithelial seam could be seen in stereomicroscope and HE in 36 h, bilateral palatal process fusion and the crest epithelial seam disappearance could be seen in stereomicroscope and HE in 48 h. The expression level of PDGFR-α cultured palates for 24 h reached its peak and gradually reduced in 36 and 48 h. Conclusion The changes of PDGFR-α expression in cultivation of palatal process in vitro and in vivo had the same trend.In this study,improved method of suspension culture of palatal process in vitro simulated the development of the palatal process in vivo.The study had made a foundation for subsequent cleft palate of complex mechanismsd.
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