游離輻射在毛囊生長期造成的萎縮反應與早發性再生

Abstract

放射線治療是現代癌症醫學的常用的治療手法之一，利用具穿透性的游離輻射對體內的腫瘤進行非入侵性的治療。可是這種治療手法會同時對附近的正常組織造成傷害，從中帶來許多副作用，其中落髮為廣為人知的副作用之一，落髮為病人帶來外觀上的影響，使他們心理上造成壓力。毛髮週期包括生長期，衰退期及休止期，過去對小鼠注射化療藥物後對毛髮影響的研究中，根據注射的劑量會得到萎縮性生長期及萎縮性衰退期兩個路徑，前者出現短暫修復，後者則無，可是萎縮性衰退期會經過一短暫休止期後會提早進入下一個生長期。本研究主要分為兩部分，一.探討高劑量游離輻射在毛囊生長期造成的傷害；二.在輻射傷害下毛囊在生長週期的影響。實驗使用出生後32天的C57BL/6母鼠，在毛囊處於生長期時，以8.5Gy銫-137伽瑪射線照射小鼠背部，觀察毛髮掉落情形及收集背部皮膚，以出生後40天，處於自然衰退期的小鼠作為對照組，以組織切片及免疫螢光染色進行分析。實驗結果顯示，照射游離輻射後，小鼠在第7天毛髮完全掉落，在組織切片下可以觀察到毛囊在第10天時型態接近休止期。利用γH2AX免疫螢光染色可以發現在照射游離輻射後6小時，毛囊表現嚴重的DNA斷裂，在TUNEL及Cleaved caspase-3的染色中可以發現在24小時內，毛囊出現大量細胞凋亡，同時表現BrdU的細胞數目相對正常生長期有顯著減少。另一方面，毛囊在受輻射後的48小時內出現同源重組(HR)及非同源性末端接合(NHEJ)來修復斷裂的DNA，表現的位置主要為毛囊球，在48小時後因傷害過大無法修復的情況下開始萎縮，隨後進入短暫的休止期。在第二部分中，實驗採用細胞分選儀收集對照組及輻射組休止期的不同細胞群，以即時聚合酶鏈式反應的方式觀察在休止期中不同細胞群分析其基因表現，結果顯示在照射游離輻射後，在毛囊inner bulge的部分，抑制進入生長期的信號降低，在bulge及次級毛胚可以發現輻射組的休止期比正常的休止期更具有進入生長期的條件，可是在輻射傷害導致的休止期，細胞的信號傳遞功能可能受到干擾，所以毛囊需要一個短暫的時間進行調整，才可以進入下一個生長週期。

Radiotherapy is one of the commonly used in cancer treatment. By use of ionizing radiation(IR) for non-invasive treatment of tumors in the body. However, this treatment will also cause damage to the nearby normal tissue, resulting in many side effects. IR induced alopecia is one of well-known side effects, it will affect the patient’s life quality. Hair cycle includes growth (anagen), regression (catagen) and quiescence (telogen). Mice were injected with chemotherapy drugs on the impact of hair research, it will induce dystrophic anagen or dystrophic catagen pathway; this has not a primary recovery, and that will. Otherwise, dystrophic catagen has a short telogen and early entry into next hair cycle. In this study divides into two parts: 1.The effect of IR on anagen hair follicle. 2.The effect of hair cycle after IR. In our experiment, C57BL/6 female mice were irradiated with gamma rays (8.5Gy) from radioactive isotope Cs-137 on the back skin when a hair follicle in the anagen stage, 40 days old B6 mice were used in the control group, the analysis was performed by H&E and immunofluorescence staining. The results showed that after irradiation of IR, the mice appeared alopecia at day 7 post irradiation. Histologically, the hair follicle structure was changed to telogen like phase after 10 days’ post irradiation. Immunofluorescence staining by γH2AX indicated serious DNA damage on the hair bulb at 6 hours post irradiation. In TUNEL and Cleaved caspase-3 staining can be found hair follicles appear a large number of apoptosis cells within 24 hours, the number of BrdU positive cells also decreased after IR treatment. On the other hand, homologous recombination (HR) and non-homologous terminal binding (NHEJ) repair marker Rad51 and DNAPkcs had occurred in the hair follicles after IR within 48 hours. Unfortunately, the repair would not success and hair follicles entered into dystrophic catagen. In part two, we will discuss why dystrophic telogen has a short resting time. We sorted Inner bulge, bulge and hair germ cells from normal telogen (P49, P59) and the radiation group(IRd10, d17) by FACSCalibur. qPCR data suggest that radiation group inner bulge inhibition signal was reduced. In bulge and secondary hair germ, we found radiation group cells were more active than normal telogen cells, moreover, we found IRd10 telogen cells signaling function may be disturbed, so the hair follicles need a short time to revise before they enter the next hair cycle.