The Investigation of Hair Follicle Regeneration Utilizing Human Interfollicular Dermal and Epidermal Stem Cells

哺乳动物毛囊中的毛乳头细胞（Dermal papilla cells，DPC）具有诱导毛囊发育的能力。通过扩增毛乳头细胞（DPC）并将其移植到秃发皮肤中，从理论上讲是可能的，本研究采用基因芯片技术和RT-PCR技术，对人头皮毛乳头细胞和真皮成纤维细胞进行比较，以阐明组织工程毛囊中诱导毛发的遗传因素。在微阵列分析中，我们发现TGF基因与真皮成纤维细胞相比显著上调，并且它是参与毛发再生的候选基因之一。RT-PCR分析显示，与真皮成纤维细胞相比，培养的毛乳头细胞中TGF-β mRNA显著且持续上调。此外，我们发现角质形成细胞分泌的可溶性因子显著上调TGF β-2 mRNA.Immunohistochemistry（IHC）的人头皮皮肤和毛囊的生物标志物，以前报告为特异性IFKC进行。还通过FACS分析和免疫染色研究了培养的角质形成细胞中生物标志物的表达。免疫组化结果显示，K15蛋白表达于隆突区。CD 34主要表达于下区，包括隆突亚区和外根鞘（ORS）。CD 271（p75神经营养因子受体）在K15阴性的下ORS中表达。所有这些都只表达在最外层粘附到基底membranes.We研究了两个在体内模型的重现性，包括“室模型”，移植培养的人DPC到裸鼠的硅胶室，和“三明治模型”，植入培养的人DPC与大鼠唯一的皮肤下的裸鼠的皮肤。“三明治模型”比“室模型”和其他体内模型更有用，用于测试人DPC在再现性方面的诱导活力。

Dermal papilla cells (DPCs) in the mammalian hair follicles have been shown to have an ability to induce and develop hair follicles. It is theoretically possible to regenerate human hair by expanding DPCs and transplanting them in the bald skin.We performed a microarray analysis and RT-PCR comparing human dermal papilla cells and dermal fibroblasts both obtained from adult scalp skin, in order to elucidate the genetic factors responsible for hair induction in tissue engineered hair follicles. In the microarray analysis we found that TGFs gene was significantly upregulated compared to dermal fibroblasts and that it was one of the candidate genes involving in hair regeneration. RT-PCR analysis revealed significant and sustained upregulation of TGFs mRNA in cultured dermal papilla cells compared to dermal fibroblasts. Furthermore, we found soluble factors secreted by keratinocytes significantly upregulated TGF beta-2 mRNA.Immunohistochemistry (IHC) of human scalp skin and hair follicles for the biomarkers previously reported as specific for IFKC was performed. The expression of the biomarkers in cultured keratinocytes was also investigated by FACS analysis and immunostaining. Keratin 15 (K15) was expressed in the bulge area in IHC. CD34 was expressed in the lower area, which includes the sub-bulge area and outer root sheath (ORS). CD271 (p75 neurotrophin receptor) was expressed in the lower ORS that was negative for K15. All of them were expressed only in the outermost layer adhering to the basement membrane.We examined reproducibility of two of in vivo models for trichogenesis previously reported, including 'chamber models', grafting cultured human DPCs into silicone chambers on nude mice, and 'sandwich models', implanting cultured human DPCs with rat sole skin under the skin of nude mice. 'Sandwich models' were more useful than 'chamber models' and other in vivo models for testing the inductive viability of human DPCs in terms of reproducibility.