Molecular fingerprinting of TGFbeta-treated embryonic maxillary mesenchymal cells.
Molecular fingerprinting of TGFbeta-treated embryonic maxillary mesenchymal cells.
复制标题
TGFbeta处理的胚胎上颌间充质细胞的分子指纹图谱。
DOI：
10.1034/j.1600-0544.2003.00264.x
复制
发表时间：
2003
影响因子：
3.1
通讯作者：
Greene,RM
中科院分区：
文献类型：
--
作者：
Pisano,MM;Mukhopadhyay,P;Greene,RM
转化生长因子-β（TGF β）家族代表一类信号分子，在正常胚胎发育中发挥核心作用，特别是在颅面区域的发育中。该家族的成员对继发腭的发育至关重要，在那里它们调节上颌骨和腭间充质细胞增殖和细胞外基质合成。该生长因子家族的功能是特别关键的，因为任何一个过程的扰动都会导致腭裂。虽然TGF β 1对胚胎颅面组织的细胞和表型作用已被广泛分类，但在上颌骨/腭发育中作为TGF β 1下游介质发挥作用的特定基因定义不清。基因表达阵列提供了在一项研究中对数百至数千个差异表达基因进行快速、同时评估的能力。由于TGF β作用的下游后遗症仅部分确定，因此利用互补DNA（cDNA）表达阵列技术（Clontech的TMMouse cDNA表达阵列）描绘来自TGF β处理的鼠胚胎上颌间充质细胞的原代培养物的差异表达基因的谱。用32P标记的cDNA探针与基于膜的cDNA阵列（1178个基因）进行杂交，所述cDNA探针由从TGF β处理或载体处理的胚胎上颌间充质细胞分离的RNA合成。将所得的磷光图像进行RT-PCR ImageTM分析，以确定对照和TGF β处理的上颌间充质细胞之间的基因表达差异。在1178个排列的基因中，552个（47%）表现出可检测的表达水平。22个基因的稳态水平上调，而其他8个基因的稳态水平下调，响应于TGF β的两倍或更大的因子。受影响的基因可以分为三个一般的功能类别：转录因子和一般的DNA结合蛋白;生长因子/信号分子;和细胞外基质和相关蛋白。通过与丰富的组成型表达的mRNA进行比较来评价每个基因的杂交程度：泛素、甘油醛-3-磷酸脱氢酶（GAPDH）、鸟氨酸脱羧酶（ODC）、细胞质β-肌动蛋白和40 S核糖体蛋白。在这些基因的表达水平中没有观察到响应于TGF β处理的可检测的变化。通过真实的实时定量聚合酶链反应验证基因表达谱分析结果。利用cDNA微阵列技术，使我们能够描绘一个初步的转录图谱的TGF β反应性在胚胎上颌间充质细胞。差异表达基因的概况提供了揭示性的见解，潜在的分子调控机制所采用的TGF β 1在编排颅面个体发育。
The transforming growth factor‐ß (TGFß) family represents a class of signaling molecules that plays a central role in normal embryonic development, specifically in development of the craniofacial region. Members of this family are vital to development of the secondary palate where they regulate maxillary and palate mesenchymal cell proliferation and extracellular matrix synthesis. The function of this growth factor family is particularly critical in that perturbation of either process results in a cleft of the palate. While the cellular and phenotypic effects of TGFß on embryonic craniofacial tissue have been extensively cataloged, the specific genes that function as downstream mediators of TGFß in maxillary/palatal development are poorly defined. Gene expression arrays offer the ability to conduct a rapid, simultaneous assessment of hundreds to thousands of differentially expressed genes in a single study. Inasmuch as the downstream sequelae of TGFß action are only partially defined, a complementary DNA (cDNA) expression array technology (Clontech's AtlasTMMouse cDNA Expression Arrays), was utilized to delineate a profile of differentially expressed genes from TGFß‐treated primary cultures of murine embryonic maxillary mesenchymal cells. Hybridization of a membrane‐based cDNA array (1178 genes) was performed with32P‐labeled cDNA probes synthesized from RNA isolated from either TGFß‐treated or vehicle‐treated embryonic maxillary mesenchymal cells. Resultant phosphorimages were subject to AtlasImageTManalysis in order to determine differences in gene expression between control and TGFß‐treated maxillary mesenchymal cells. Of the 1178 arrayed genes, 552 (47%) demonstrated detectable levels of expression. Steady state levels of 22 genes were up‐regulated, while those of 8 other genes were down‐regulated, by a factor of twofold or greater in response to TGFß. Affected genes could be grouped into three general functional categories: transcription factors and general DNA‐binding proteins; growth factors/signaling molecules; and extracellular matrix and related proteins. The extent of hybridization of each gene was evaluated by comparison with the abundant, constitutively expressed mRNAs: ubiquitin, glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), ornithine decarboxylase (ODC), cytoplasmic beta‐actin and 40S ribosomal protein. No detectable changes were observed in the expression levels of these genes in response to TGFß treatment. Gene expression profiling results were verified by Real‐Time quantitative polymerase chain reaction. Utilization of cDNA microarray technology has enabled us to delineate a preliminary transcriptional map of TGFß responsiveness in embryonic maxillary mesenchymal cells. The profile of differentially expressed genes offers revealing insights into potential molecular regulatory mechanisms employed by TGFß in orchestrating craniofacial ontogeny.