生長期與休止期毛囊以高低劑量游離輻射照射後之活體細胞動態

Abstract

隨著癌症患者的數量持續增多，放射治療的需求也逐漸升高。然而，這種治療方式就如同一把雙面劍，它能有效地清除癌細胞，但對於鄰近的正常組織卻可能產生不可逆的傷害。最為大眾所熟知的副作用，即是病人在接受治療後經常會產生落髮的情況。這種現象背後的原因是因為放療的主要原理在於摧毀快速分裂的細胞，而癌細胞正是這種細胞的典型例子。然而，多數人類毛囊(hair follicles)處於生長期（anagen），在此期間毛囊內的基質細胞（matrix cells）也會快速分裂，進而產生毛髮。因此，當毛囊受到放射性傷害時，其內部的基質細胞也會同時受損，進一步影響到毛髮的生長。更重要的是，毛囊是一種精緻的微型器官，其內部各部位都在結構與生理功能上相互緊密連結，任何一處的損傷都可能導致整體毛囊的損害，進而引發永久性的落髮。有趣的是，處於生長期的毛囊對於不同劑量的放射線所造成的傷害會有不同的反應，例如低劑量可能會引發失養性生長期（dystrophic anagen），而高劑量則可能引發失養性衰退期（dystrophic catagen）。然而，關於這兩種反應中的細胞動態和毛囊再生機制，目前還有許多細節尚待釐清。 過去的研究多是透過組織切片的技術來探討毛囊的相關課題，但這種技術卻有著一些限制，例如在過程中的任何步驟都有可能傷害到樣本中，而使毛囊呈現的並非是其在生物體中真正的狀態；難以追蹤毛囊於生物體中的真正的動態變化，並且切片採樣時間點之間的連續變化也無法得知；因為切面的角度的不同而可能會產生視覺上的錯覺。然而，隨著雙光子顯微鏡(Two-Photon Microscopy, TPM)的發展，我們已經可以對於毛囊進行即時的活體動態觀察。因此，我們利用雙光子顯微鏡開發了一個可以穩定模擬人類毛囊狀態的動物模型，並以此模型與雙光子顯微鏡來追蹤不同時期的毛囊在受到不同劑量的游離輻射(ionizing radiation)傷害後的反應。透過對於毛囊的每日追蹤(daily tracing)和縮時攝影(time-lapse)，我們得以揭示許多過去研究中難以發現或確認的現象。 本研究揭示了許多先前在組織切片無法確定獲觀察到的受損的毛囊結構，特別是在高劑量的游離輻射傷害下，例如在衰退期(catagen)中毛囊的上皮鏈(epithelial strand)會有斷裂的現象，以及真皮乳頭(dermal papilla)受到傷害後的各種反應等，並將我所觀察到現象進行了詳細的分類和定義。我們認為這些現象背後都有其各自生理上的意義，雖然目前仍無法闡釋其背後機制，但若我們能夠對這些現象有更清晰的理解，或許就能更有效地應對臨床上因放療而引起的落髮問題。因此，清楚地定義與分類可以幫助我們更快地聚焦問題，對於未來解決放療所引起的落髮有著積極的意義。

The demand for radiotherapy (RT) is gradually rising due to the continual increase in the number of cancer patients. The RT can be likened to a double-edged sword, as it has the potential to successfully eradicate cancer cells, but it also carries the risk of causing irreversible harm to adjacent healthy tissues. The most observed adverse effect is alopecia, which is frequently experienced by patients following treatment. The underlying cause of this phenomenon can be attributed to the fundamental principle of RT, which is designed to eradicate rapidly proliferating cells, with cancer cells serving as a prime illustration. Most human hair follicles (HFs) are primarily in the anagen, characterized by rapid division of matrix cells within the HFs, resulting in the production of hair shafts. Therefore, the damage caused by RT not only affects the HFs, but also has an impact on the internal matrix cells, thereby further influencing the process of hair growth. More significantly, HF is a kind of intricate miniorgan, with each component intricately interconnected in terms of both structural and physiological functionality. Hence, any form of damage has the potential to cause a significant impairment in HF function, leading to persistent hair loss. Interestingly, HFs in the anagen phase exhibit distinct responses when exposed to different doses of ionizing radiation (IR). For example, a low dose IR may trigger a dystrophic anagen, while a high dose IR could induce a dystrophic catagen. However, there are still numerous aspects about the cell dynamics and mechanisms of HF regeneration in these two reactions that require further elucidation. Earlier studies have employed tissue sectioning methods to investigate research about HFs. However, this technique is associated with certain limitations. For instance, any step in the process has the potential to damage the sample, thereby compromising the accurate representation of HFs in the organism. Additionally, it is challenging to monitor the true dynamic changes of HFs within the organism, and it is not feasible to ascertain the continuous changes that transpire between the sampling time points. Furthermore, visual distortions may arise due to variations in the angles of the slicing planes. With the advent of Two-photon microscopy (TPM), the real-time observation of in vivo cell dynamics in HFs has become possible. Therefore, we developed a stable in vivo animal model to simulate the state of human HFs. This model, along with TPM, was utilized to track the responses of HFs at different phases when subjected to varying doses of IR. Through the implementation of daily tracing and timelapse imaging techniques on HFs, numerous previously elusive or unverified phenomena have been successfully identified and confirmed. This study presents a comprehensive analysis of HF structures that have been found to be damaged, providing new insights that were previously unconfirmed or unobservable in tissue sections. Notably, this study highlights the impact of high doses of IR damage on these structures. For instance, the epithelial strand (ES) within the catagen of HFs may be disconnected, or dermal papilla (DP) reacts differently to IR damage. The phenomena were subsequently characterized and defined. We believe that each of these phenomena holds physiological significance, and although the underlying mechanisms remain elusive, a more comprehensive comprehension of these phenomena could potentially enhance the management of clinical radiotherapy-induced alopecia (RIA). Therefore, the establishment of clear definitions and classifications can facilitate a more efficient identification of the problem, thereby playing a crucial role in the resolution of RIA in the future.