摘要：眼輪部幹細胞與其「利基細胞」(Niche cells)之間的交互作用，是目前有關幹細胞研究的熱門議題。眼輪部上皮幹細胞的「利基細胞」據推論很可能是與眼輪部幹細胞緊密接觸的間質細胞「mesenchymal cell」。但是目前常用在體外培養眼輪部幹細胞過程中所使用的酵素，例如Dispase 和trypsin-EDTA，均會破壞上皮幹細胞與其周圍細胞之間的物理性接觸；因此可能使得培養之細胞失去了維持幹細胞特色的環境。如何能在培養眼輪部幹細胞的過程中，盡量維持幹細胞與「利基細胞」間的緊密接觸，充分利用「利基細胞」對幹細胞的影響來增進體外培養之上皮細胞層之幹細胞培養效能，乃為一個有趣且重要的課題。這在臨床上治療眼輪部受損病患，或在基礎研究上探討眼輪部幹細胞的特質上都有很大的意義。本實驗室過去在角膜上皮細胞和眼輪部幹細胞的課題上有深入的研究，本計劃即是利用過去實驗室的基礎，分別在「眼輪部上皮細胞」和「口腔黏膜上皮細胞」的幹細胞培養上，深入探討「利基細胞」的保存對於體外培養這些不同上皮幹細胞的影響。計畫分三年進行，第一年著重利用Collagenase A (膠原蛋白脢)取代傳統的Dispase和Trypsin-EDTA，用以分離「紐西蘭白兔」之眼輪部上皮細胞層與其下之間質層(Stroma)；而用此方法取得之細胞團塊(cell cluster method)理論上將維持幹細胞與利基細胞之間的接觸，且此團塊將放置於羊膜上繼續培養。而用傳統方法(Dispase 和Trypsin-EDTA)處理之分散輪部上皮細胞(cell suspension method)，和用組織塊直接培養之上皮細胞層 (explant method)，將當成對照組，比較培養之上皮細胞層的生長速度、細胞型態、利基細胞的存在與表現、colony forming ability、幹細胞標記(ABCG2、P63)和分化細胞(K3、K4、K12、K13)之特質表現(利用西方點墨法和RTPCR)等的異同。實驗中同時比較有或沒有3T3 細胞株的共同培養下，是否導致不同的培養結果。同時也比較口腔黏膜上皮細胞的培養是否有類似表現。第二年計畫，則用trypsin/EDTA 和protein tyrosine phosphatase (PTP)的抑制劑 (sodium orthovanadate)打破幹細胞與利基細胞之間的接觸(cell-cell contact)，用同樣於第一年實驗中的方法，應證這些方法的培養成效是否因為細胞和細胞之間的接觸受到破壞而產生變化。此外，也將注意sodiumorthvanadate 的使用是否產生不良的藥物毒性。同樣的比較也將用在口腔黏膜上皮細胞的培養上面，用以應證同樣的效果出現在不同的上皮細胞上。第三年計畫，則將這些不同方法培養的上皮細胞層移植在眼輪部受損的兔子眼球上。移植之後，將在第一、第二、第四週利用活體共軛焦顯微鏡(in vivo confocal microscopy)和壓印式細胞學檢查(impression cytology)定期追蹤細胞之變化；另外，也將在犧牲動物之後，利用免疫染色法、colony forming ability、幹細胞標記(ABCG2、P63)和分化細胞(K3、K4、K12、K13)之特質表現(利用西方點墨法和RTPCR)來比較這些不同方法所培養的細胞層是否有不同的臨床療效。本實驗初步的假說是利用Collagenase A 取代傳統酵素作用方法，將更快速地培養細胞、讓細胞維持較類似幹細胞的特色、不需要3T3 細胞的共同培養、且有更好的移植效果。如果本計劃的假說得到應證，將可更安全有效地在細胞治療上嘉惠病患。
Abstract: The interaction between limbal stem cell and its niche cells is a hot and impartantresearch topic at present. The niche cells of limbal stem cells are supposed to bemesenchymal cell. However, the commonly used enzyme during the process of limbalepithelial cellular culture, such as dispase and trypsin-EDTA may potentially disrupt theinteraction of limbal stem cells and niche cells. Such reaction may decrease the positiveinfluence of niche cells on maintaining the stemness of limbal stem cells, and affect thecultivated cell sheets. It is thus interesting and important to find out new methods ofmaintaining cell-cell contact of niche cells and stem cells during the process of limbal stemcell sheets especially in dealing with patients with limbal insufficiency. Our lab hasadequate background ability and publication in the research of limbal stem cells and cornealepithelial cells. The purpose of this project is to based on the previous background andfocus on the interaction of niche cells and limabl stem cells in tissue engineering and celltherapy.The first year of the project is to use collagenase A to replace traditional dispase andtrypsin-EDTA, and isolate intact limabl epithelial cell sheets for culturing (cell clustermethod). Cell suspension method (dispase treatment followed by trypsin/EDTA) and tissueexplant method will be control. We will compare the cell growth rate, cell morphology, theexistence of niche cells, colony forming ability, the expression level of differentiationmarkers (K3, K4, K12, K13) by IHC, RTPCR and Western blot analysis among thesegroups. The need of 3T3 co-culture will also be evaluated. We will also evaluate the effecton oral mucosal epithelial cells. In the second year, we will focus on using trypsin/EDTA orprotein tyrosine phosphatase (PTP) generalized inhibitor “sodium orthovanadate” to disruptthe cell-cell interaction between niche cells and limbal stem cells. We will evaluate whetherdisruption of cell-cell interaction affect the culture result. The cell growth rate, cellmorphology, the existence of niche cells, colony forming ability, the expression level ofdifferentiation markers by IHC, RTPCR and Western blot analysis will be evaluated. Wewill also evaluate the effect on oral mucosal epithelial cells. In the third year, we willtransplant the cell sheets from different methods onto the rabbit corneas with mechanicallimbal injury. At postoperative 1, 2 and 4 weeks, in vivo confocal microscopy andimpression cytology will be examined. At postoperative 4 weeks, IHC and colony formingability will be evaluated. We will also compare the different surgical outcomes.The project hypothesized that using collagenase A to replace transitional methodsduring cull culture may rapidly get adequate cell numbers for transplantation, maintainingthe cultured cells in stem cell like status, decrease the dependence of 3T3 coculture and getbetter surgical results. If the hypothesis is correct, such culturing method may benefit thepatients with limbal damage.
