探討氧化壓力造成毛囊間質乳突細胞老化影響表皮-真皮交互作用之機轉

Abstract

毛囊由表皮與真皮兩部分所建構，其中真皮由毛囊真皮乳突細胞所組成。毛囊真皮乳突細胞參與毛囊的發育、生長、週期的調控、型態與器官大小。根據文獻顯示真皮乳突細胞可以教育未分化的表皮細胞走向毛囊發育；也可在成體皮下誘導毛囊異位再生。透過毛囊發生學，了解真皮乳突細胞是來自未分化的間質層纖維母細胞，真皮乳突細胞與纖維母細胞都來自相同的間質細胞並共有大部分的基因叢。然而，被移除的真皮乳突細胞卻可以從附近的纖維母細胞補充而再生成為新的真皮乳突細胞。許多文獻指出經由輻射或化學法治療後引起的掉髮是因為毛囊表皮細胞受到損傷，卻較少文獻探討對毛囊間質細胞的影響。我們發現當纖維母細胞特化成毛囊真皮乳突細胞後，較能夠對抗環境中的壓力所造成的傷害。在氧化壓力傷害下，真皮乳突細胞有較好的存活率，我們發現是由於氧化壓力會使纖維母細胞產生較多的活化態caspase 3及gamma-H2A.X，毛囊真皮乳突細胞具有較高的抗細胞凋亡bcl-2表現量。在高劑量的輻射傷害後，毛囊真皮乳突細胞仍可進行細胞分裂，而纖維母細胞卻不能。因此我們認為毛囊真皮乳突細胞對於外界壓力具有較好的抵抗性。 化療造成的永久性禿髮會有毛髮變細的現象，文獻指出來自雄性禿的毛囊真皮乳突細胞有較佳的細胞分裂能力，並具有老化特徵的證據顯示，但相關機制尚未被釐清，其困難處是由於這些細胞無法在體外大量培養。我們研究發現經由氧化壓力傷害後，存活下來的毛囊真皮乳突細胞具有早熟性老化的現象：細胞分裂能力降低、老化標的因子p16及p21表現量上升。這些老化的毛囊真皮乳突細胞保留它特有的基因表現，並非轉變回纖維母細胞。老化的毛囊真皮乳突細胞無法接收來自角質細胞培養液的刺激生長，說明了表皮對真皮層的訊號傳遞受到損害。這些老化的毛囊真皮乳突細胞中，其誘導毛囊再生的基因(akp與versican)量下降、自我聚集能力變差，並且失去誘導毛囊新生的能力。另外也發現老化的毛囊真皮乳突細胞無法刺激毛囊角質細胞生長與失去維持毛囊幹細胞特性的能力，並無法教育角質細胞往毛囊分化。我們認為間質對表皮訊號傳遞以及幹細胞活化的能力受到損害。在微陣列基因與細胞激素晶片的分析結果顯示：老化細胞分泌大量的發炎因子(interleukin-4,5,6)，角質細胞培養在IL-6培養液中其生長能力與細胞聚落形成能力皆變差，我們認為老化的毛囊真皮乳突細胞是因為分泌interleukin 6而抑制角質細胞生長與幹細胞活化。 毛囊真皮乳突細胞對毛囊器官大小有很重要的調控。我們研究顯示毛囊真皮乳突細胞數量與毛囊器官的大小粗細有統計上的正相關。因此我們目標是藉由組織工程的方法去控制毛囊再生後的大小。我們先前的研究顯示生醫材料的親疏水性可以調控毛囊真皮乳突細胞的貼附與細胞聚集形成微組織球的能力，毛囊真皮乳細胞可以經由不同的播種數量，在聚乙烯醇的表面形成各種大小的微組織球體。此方法的優點是可快速取得並經由注射也保有其完整球體結構，並且保有其特化基因表現。我們發現較大的微組織可提高毛囊誘導效率，卻無法經由適當的微組織控制毛囊再生後器官的大小。 我們的研究顯示可以利用氧化壓力刺激而快速得到大量的老化毛囊真皮乳突細胞，並釐清其中老化真皮細胞在表皮-真皮交互作用後的影響機轉，可做為其他器官之表皮-真皮的研究模式，探討因氧化壓力或是年齡增長所導致的器官老化衰退現象並應用在臨床治療上的相關疾病預防。

Hair follicle (HF) is composed of epithelium and mesenchyme. The dermal papilla (DP) is a component of HF mesenchyme and it is involved in the development, growth, cycle regulation, shape, and organ of hair. References showed DP can educate the undifferentiated epithelia cells going to follicular fate and induce ectopic hair neogenesis in adult skin. During HF morphogenesis, DP cells are from fibroblasts (FB), uncharacterized mesenchyme. DP and FB are derived from the same original mesencyme and shared the most genes. However, amputated DP could be regenerated from local fibroblast. There are many studies that have shown that radiotherapy and chemotherapy-induced hair dystrophy and even hair loss is associated with follicular epithelial damage. However, there is little literature on the damage of follicular mesenchyme. To address this question, we found that fibroblasts become more resistant to environmental stress when they differentiate into DP cells. DP cells had higher survival than FB under oxidative stress according to induce cleaved caspase 3, induce cell death, and gamma-H2A.X in FB. DP cells had higher bcl-2, anti-apoptotic factor, expression than FB. DP after radiation can still proliferate but FB can’t. However, DP cells have greater resistant ability to stress than fibroblasts. Permanent chemotherapy-induced alopecia had diffuse thinning hair. Previous studies have showed that balding DP cells from androgenetic alopecia (AGA) have smaller cell size and limited proliferation compared to non-balding DP cells. Many evidences show that premature senescence is a characteristic of balding DP cells. The exhaustive pathological mechanism is yet unclear. However, the problem is, it difficult to expand numerous balding or aged DP cells in culture. We found the survival DP cells tend to become senescence after oxidative stress. Aged DP cells became enlarged and flatness, having the limited proliferating ability and increasing senescence-associated beta galactosidase activity, p16 and p21. However, the oxidative stress induce senescence wasn’t happened in FB cells. These senescent DP cells conserved the DP specific genes. We suggested that the epithelium to mesenchyme signaling was impaired according to the senescent DP cells are irreversibly by keratinocyte conditioned medium. The HF inductive genes, akp and versican, were decreased in senescent DP cells. The senescent DP cells had less AKP activity and impaired self-aggregated ability. Importantly, they lost the HF induction ability. We also found that the senescent DP cells unable to stimulate HF keratinocytes (KC) growth and support clonal growth and stemness of HF keratinocytes. The senescent DP cells exhibited uredced ability to induce follicular differentiation. We suggested that the mesenchyme to epithelium signaling and stem cell activation were impaired. Base on screen of microarray and cytokine array, we found that the interleukins were up-regulated in senescent DP medium, especially in interleukin 6 (IL-6). The senescent DP cells inhibit of KC clonal growth and stem cell activation by induced IL-6 secretion. The DP plays a crucial role of controlling HF organ size. In our observation, we found that the DP cell number is correlated with HF thickness and is statistically significant. We purpose to control the size of regenerated hair follicles through tissue engineering. According to our previous results, the adhesivity and aggregation ability of DP cells can be controlled by different substratum hydrophobicity. We found that the DP cells could format DP spheroids at different sizes when cultured in polyvinyl alcohol (PVA)-coating plate by different cell densities. The advantages are rapid and to ensure that they are injectable. Characters of spheroidal microtissues obtained on PVA-coated surface. We found that larger DP spheroids showed higher HF induction efficiency but size of preformed DP spheroids did not affect the thickness of regenerated hair. In our studies, we set up a model of rapid and easy senescent DP cells acquisition to elucidate the role of senescent mesenchyme in the interaction of epithelium and mesenchyme for other organs. This model is a beneficial strategy to explore the pathological mechanism of organ atrophy as a result of oxidative stress or aging.