内蒙古绒山羊皮肤毛囊毛乳头细胞系的建立及其基因表达谱型的研究
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摘要
毛乳头控制毛囊的新生和毛囊的大小,是毛囊生长和毛囊周期的控制中枢。内蒙古绒山羊体表皮肤具有两种大小和质地差异显著的毛囊,初级毛囊(产粗毛)和次级毛囊(产绒)。绒山羊初级毛囊与次级毛囊大小上的显著差异使其成为研究毛囊形态发生发育机制的优良模型。而次级毛囊比初级毛囊更为明确的毛囊年生长周期及每个周期的易于划分等特点也为研究毛囊周期机制提供了良好的实验材料。本研究通过Ⅱ型胶原酶消化结合机械分离的方法,共建立了三种毛乳头细胞系,分别为毛囊生长期初级毛囊毛乳头细胞、生长期次级毛囊毛乳头细胞和休止期次级毛囊毛乳头细胞。我们对三种毛乳头细胞的形态和生长情况进行了检测,发现体外培养条件下所有三种毛乳头细胞均呈三角形或多角形,均高表达毛乳头细胞特异性标记分子α-平滑肌肌动蛋白。不同的是,只有毛囊生长期初级毛囊毛乳头细胞和生长期次级毛囊毛乳头细胞能够在体外培养条件下形成细胞凝集块,休止期次级毛囊毛乳头细胞在任何代次均不能形成。随后的细胞增殖能力检测发现休止期次级毛囊毛乳头细胞较另外两种毛乳头细胞增殖能力弱。高通量转录组测序技术在检测基因表达水平的方面具有高效、全面和精确的特点。以山羊基因组全序列测序结果为研究背景,利用高通量转录组测序的方法我们对三种毛乳头细胞基因表达谱型进行了分析。基因表达差异性分析显示,毛囊生长期的初级毛囊和次级毛囊的毛乳头细胞基因表达的差异主要表现在血管新生、细胞外基质-受体相互作用,以及Wnt/β-catenin/Lefl KEGG信号通路方面,预示着这些基因在毛囊的形态发生发育过程中可能起到关键作用；毛囊生长期次级毛囊毛乳头细胞和休止期次级毛囊毛乳头细胞差异表达的基因主要富集于细胞周期、细胞黏附分子、细胞因子-细胞因子受体相互作用,以及p53KEGG信号通路上,并揭示在次级毛囊休止期中期毛乳头细胞在促进次级毛囊的退行和绒纤维生长的停止的同时,能够抑制自身细胞凋亡并为完整功能毛乳头的重建或恢复活性做准备。所有这些发现将为进一步研究出生后的毛囊形态发生发育和毛囊周期性生长提供信息。
Dermal papilla is considered the control center of hair follicle neogenesis, size, growth and hair cycle. Inner Mongolia Cashmere goats(Copra hircus) have a double coat comprising the primary (producing guard hair) and secondary hair follicles (producing cashmere), which have dramatically different sizes and textures. That makes the Cashmere goats become a potent model for hair follicle morphogenesis research. The secondary hair follicles have a circannual growth cycle, with each growth phase can be easily distinguished. That provides us good materials for researches on the hair cycle. In the present study by using microdissection combined with collagenase II digestion, we established three dermal papilla cell lines with respect to the anagen primary hair follicles, anagen secondary hair follicles, and telogen secondary hair follicles. We had clarified the similarities and differences in their morphology and growth characteristics, and found that all the three types of dermal papilla cells exhibited a triangular or polygon shape, and were all positive for the a-smooth muscle actin, which is a specific marker for cultured dermal papilla cells. However, only those dermal papilla cells derived from the anagen primary hair follicles or the anagen secondary hair follicles could form cell aggregates. The telogen secondary hair follicle-derived dermal papilla cells could not form cell aggregates at any passage. Subsequent cell proliferation ability analyses showed that the telogen secondary hair follicle-derived dermal papilla cells had lower proliferation rate than the other two cell types. There are great advantages in measuring gene expression levels of the high-throughput transcriptome sequencing technology, by which we identified gene expression patterns of the three dermal papilla cell lines on the research background of complete goat genome sequence. Gene expression difference analyses revealed that the majority of the differentially expressed genes between the anagen primary hair follicle dermal papilla cells and the anagen secondary hair follicle dermal papilla cells are involved in vascularization, ECM-receptor interaction and Wnt/(3-catenin/Lefl signaling pathways from the Kyoto Encyclopedia of Gene and Genomes (KEGG) database, suggesting their critical roles in hair follicle morphogenesis. Enrichment analyses revealed that differently expressed genes between the anagen secondary hair follicle dermal papilla cells and the telogen secondary hair follicle dermal papilla cells were significantly enriched in cell cycle, cell adhesion molecules, cytokine-cytokine receptor interaction and p53signaling pathway from the KEGG database, and suggested that in the middle of telogen the secondary hair follicle dermal papilla cells seemed on the one hand to promote the degeneration of the secondary hair follicles and cessation of cashmere growth, while on the other hand to resist self-apoptosis and prepare for the regeneration or revivification of fully functional dermal papillae. All these findings will provide us information for further researches on postnatal hair follicle morphogenesis and hair growth cycles.
Dermal papilla is considered the control center of hair follicle neogenesis, size, growth and hair cycle. Inner Mongolia Cashmere goats(Copra hircus) have a double coat comprising the primary (producing guard hair) and secondary hair follicles (producing cashmere), which have dramatically different sizes and textures. That makes the Cashmere goats become a potent model for hair follicle morphogenesis research. The secondary hair follicles have a circannual growth cycle, with each growth phase can be easily distinguished. That provides us good materials for researches on the hair cycle. In the present study by using microdissection combined with collagenase II digestion, we established three dermal papilla cell lines with respect to the anagen primary hair follicles, anagen secondary hair follicles, and telogen secondary hair follicles. We had clarified the similarities and differences in their morphology and growth characteristics, and found that all the three types of dermal papilla cells exhibited a triangular or polygon shape, and were all positive for the a-smooth muscle actin, which is a specific marker for cultured dermal papilla cells. However, only those dermal papilla cells derived from the anagen primary hair follicles or the anagen secondary hair follicles could form cell aggregates. The telogen secondary hair follicle-derived dermal papilla cells could not form cell aggregates at any passage. Subsequent cell proliferation ability analyses showed that the telogen secondary hair follicle-derived dermal papilla cells had lower proliferation rate than the other two cell types. There are great advantages in measuring gene expression levels of the high-throughput transcriptome sequencing technology, by which we identified gene expression patterns of the three dermal papilla cell lines on the research background of complete goat genome sequence. Gene expression difference analyses revealed that the majority of the differentially expressed genes between the anagen primary hair follicle dermal papilla cells and the anagen secondary hair follicle dermal papilla cells are involved in vascularization, ECM-receptor interaction and Wnt/(3-catenin/Lefl signaling pathways from the Kyoto Encyclopedia of Gene and Genomes (KEGG) database, suggesting their critical roles in hair follicle morphogenesis. Enrichment analyses revealed that differently expressed genes between the anagen secondary hair follicle dermal papilla cells and the telogen secondary hair follicle dermal papilla cells were significantly enriched in cell cycle, cell adhesion molecules, cytokine-cytokine receptor interaction and p53signaling pathway from the KEGG database, and suggested that in the middle of telogen the secondary hair follicle dermal papilla cells seemed on the one hand to promote the degeneration of the secondary hair follicles and cessation of cashmere growth, while on the other hand to resist self-apoptosis and prepare for the regeneration or revivification of fully functional dermal papillae. All these findings will provide us information for further researches on postnatal hair follicle morphogenesis and hair growth cycles.
引文
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