Isolation and Characterization of Side Population Cells from Neonatal Murine Epidermis

過去研究指出，於5 µg/mL Hoechst 33342 染色 90分鐘條件下，收取表皮邊際細胞之效率甚低，約為0.3% 至 1.4%。於此條件下，收取之表皮邊際細胞於細胞週期具有慢細胞週期之特性，並表現表皮幹細胞之特定表面標幟，如: Sca-I, α6-integrin 與 β1-integrin 等。近年來研究指出，此表皮邊際細胞亦具有於體內與體外之分化特性。因此，本試驗以提高分離表皮邊際細胞之效率，並針對其特性及體外分化潛力做進一步探討。研究首先以螢光小鼠之胎鼠表皮細胞為細胞來源，針對染劑濃度與染色時間，找出最有利於分離邊際細胞之條件，並有效提高分離之效率，以減少染劑毒性對細胞所產生之死亡。結果顯示，若染劑濃度為3.5μg/mL，染色90分鐘，其收取之邊際細胞比例 (4.94 ± 0.6%) 顯著優於其他不同濃度之組別。而於染色時間試驗中，邊際細胞之比例會隨著染色時間之增加而有趨勢性的上升；且相較於30, 45, 60, 75, 120分鐘之組別，染色處理90分鐘，可獲得最佳之邊際細胞比例及細胞活力。因此，綜合染劑濃度與時間對細胞分離效率與活力之影響，Hoechst 33342 濃度為3.5μg/mL，染色90分鐘，呈現最優之細胞效率及細胞活力。驗二主要之目的為將分離出之表皮邊際細胞，分析其細胞週期、表面標幟之表現及體外分化之誘導。結果顯示，邊際細胞之S/G2/M 期百分比 (9.68% ± 1.87%) 明顯低於非邊際細胞 (19.43% ± 2.40%) (P<0.05)；表示所分離出之表皮邊際細胞與表皮幹細胞於體內之特性一致，均表現慢細胞週期之特性。表皮邊際細胞於α6 integrin high , β1 integrin high, CD71 dim 與Sca-1之表現，均與表皮幹細胞有相同之表現。確認表皮邊際細胞於細胞週期與表面標幟表現幹細胞之特性後，本試驗首次將表皮邊際細胞體外誘導分化至脂肪細胞，結果顯示，經過體外誘導12天後，細胞可染上Oil Red O，表示表皮邊際細胞成功於體外分化至脂肪細胞，進一步確認表皮邊際細胞之幹細胞分化潛力。合上述，本試驗初步建立分離表皮邊際細胞之最佳條件。試比較四組不同染劑濃度與六種不同染色時間點，依照邊際細胞比例與死亡率之結果提示，染色條件為3.5μg/mL、90分鐘，係最有利且最有效率之收取邊際細胞條件。而分析其細胞週期、表面標幟與體外脂肪細胞之轉分化後，證明均表現幹細胞之特性 : 慢細胞週期、表現α6 integrin high , β1 integrin high, CD71 dim 與Sca-1，與成功於體外轉分化至中胚層脂肪細胞。因此，表皮邊際細胞之潛力極具探討價值，未來將可以皮膚做為成體幹細胞來源，於再生醫學領域有所應用。Previous researchers had published the results which the harvested rate of epidermal SP cells are low, distributed among 0.3% to 1.4% when stained with the best condition: 5 µg/mL Hoechst 33342 for 90 min, for the isolation of SP cells. In addition, the cell cycle stage of SP cells are analyzed and demonstrated that they exhibit lower percentage of G2/M phase cell than non-SP cells. Furthermore, they express some markers of epidermal stem cell as Sca-I, α6-integrin and β1-integrin etc. Logically assume that they are epidermal stem/progenitor cells and belong to deep quiescent cells in vivo. Concern with diversified efficiency and dead cell rate of isolated SP cells, to develop more suitable conditions of isolating SP cells from murine epidermis is of great worth. Thus, the main objective of this study is to improve the efficiency of isolating SP cells derived from neonatal murine epidermis and clarify their particular characterizations.or further research, to decrease the damage of staining procedure is a dramatic topic. Therefore, the first experiment is to improve the harvested rate of SP cells and attempt to decrease the propidium iodide (PI) positive rate after the harmful staining procedure. Epidermal cells were isolated from neonatal mice skin (Day 1-3) and stained with different concentrations of dye and stop reaction in specific time points. The result reveals that the SP cells harvested rate is 4.94 ± 0.6% in 3.5 µg/mL treated group, which is more stable than other groups. In treated time assay, the SP cell rate gradually increased following with time elapsing till 90 min and significant difference (P<0.05) was obtained comparing with 30, 45 and 60 min treated group, but has not significant difference with 120 min one. In the respect of live cell rate assay, the dead cell rate is trending down when concentration of dye is increasing. The extremely significant difference was observed between 3.5 µg/mL and 5 µg/mL treated groups (P<0.01). Furthermore, the 90 min treated group exhibits lower dead cell rate than 120 min one. Obviously, the appropriate condition that 3.5 µg/mL Hoechst 33342 treat primary cells for 90min was used for following trials.he second experiment is to analyze cell cycle stage of epidermal SP cells and non-SP cells, and verify whether epidermal SP cells possess the characteristics of stem/progenitor cells. The result shows that S and G2/M phases of SP cells (9.68% ± 1.87%) are significantly lower than epidermal non-SP cells (19.43% ± 2.40%) (P<0.05). Besides, surface marker analysis confirmed the sorted epidermal SP cells express higher levels α6-integrin and β1-integrin which are epidermal stem cell markers than non-SP cells. Furthermore, Sca-1, a progenitor cell marker, is expressed in SP cells, exclusive of non-SP cells. In addition, those cells are no contamination with blood and bone marrow derived cells based on the results show that the SP cells are negative expressions of CD45 and CD34. After examine cell cycle and cell surface marker, we use epidermal SP cells to trans-differentiate into adipocyte in vitro. The result shows that epidermal SP cells gradually change their morphology in differentiation medium between 12 days. We use Oil Red O to stain the cells after culture for 12 days, partial cells had give rise into adipocyte and expressed red after staining.aken together, a more efficient method for the isolation of epidermal SP cells have been established and verify harvested SP cells possess some characteristics resembling to epidermal stem cells whether in cell cycle, surface markers expression and adipocyte differentiation. These results suggest that the established conditions of isolating epidermal SP cells that can express stem cell characteristic.頁次試委員會審定書 II 謝 III 錄 IV 次 VI 次 VIII 要 IX文摘要 XI壹章 緒論 1貳章 文獻檢討 4.1 幹細胞 (stem cells) 4.2皮膚生理 7.2.1皮膚之基本構造 7.3邊際細胞 (side population cells) 22.3.1 邊際細胞之發展 22.3.2邊際細胞分離之原理 23.3.3自不同組織中取得邊際細胞之應用 27.4 表皮邊際細胞 (epidermal side population cells) 29.4.1表皮邊際細胞之研究進展 29参章 試驗研究 34驗一新生小鼠表皮分離邊際細胞之條件優化、前言 34、材料與方法 35.1 實驗動物 35.2 新生小鼠表皮細胞之分離 35.3小鼠表皮邊際細胞之篩選 36、結果與討論 41.1 Hoechst濃度對分離效果之影響 41.2 染色時間對分離效果之影響 44.3 Hoechst濃度與不同染色時間對於邊際細胞存活率之影響 46、結論 49驗二生小鼠表皮邊際細胞之特性探討 50、前言 50、材料與方法 51.1 藉由反轉錄聚合酶連鎖反應 (RT-PCR) 分析比較表皮邊際細胞與非邊際細胞 (non-side population, NSP) 對於BCRP1標幟膜蛋白之表現 51.2 細胞週期之分析 53.3 表面抗原分析 54.4 體外脂肪細胞之轉分化 55、結果與討論 58.1 表皮邊際細胞與非邊際細胞中BCRP1之表現形象 58.2 表皮邊際細胞之細胞週期 60.3 表皮邊際細胞之表面標幟 62.4 體外脂肪細胞之轉分化 64、結論 66肆章 總結 67伍章 參考文獻 68者小傳 7