體外製備全層皮膚等價物

In-vitro Preparation of Full Thickness Skin Equivalent

黃怡超(Yi-Chau Huang)

指導教授 ： 林峯輝

國立臺灣大學/工學院/醫學工程學研究所/博士(2005年)

https://doi.org/10.6342/NTU.2005.00684

摘要

摘要
由於纖維母細胞能適時適量的提供生長因子幫助多種細胞增生，本研究的首要目標，即是以纖維母細胞促進表皮角質幹細胞增生。以含有纖維母細胞的供養膠體及其配方液的最佳化組合來培養皮角質幹細胞，使角質幹細胞增生，不易產生分化，為組織工程皮膚提供大量且高品質的角質幹細胞。
供養膠體的最佳細胞密度由其生產之KGF與GM-CSF濃度來做選擇，最佳之細胞密度為1x105 cells/gel。可以分泌足夠量的生長因子在想要收集的培養液中，收集的培養液可以進ㄧ步與新鮮培養液配製成配方液。並比較配方液與供養膠體，對於促進角質幹細胞增生的貢獻，來挑選最佳的配方液與最佳的供養膠體為何。並以cytokeratin-10與TUNEL染色法，判斷角質細胞有沒有產生我們不想要的分化與凋亡。培養的結果顯示，角質幹細胞不利於在過滿盤的情況下增生，並且產生分化與凋亡。我們發現在繼代培養的實驗中，對於促進角質細胞增生及抑制角質細胞分化凋亡的功能來說，供養膠體的效果比配方液來的佳。角質幹細胞與最佳供養膠體共同培養於67%配方液中，可以維持角質幹細胞繼代培養的存活性與增生能力，並可以防止細胞分化與凋亡。藉著供養膠體與配方液的組合，我們提供了一個經濟而有效的方法，經由細胞間的即時溝通，不需要昂貴的添加物，即可大量的生產品質優良的角質幹細胞。另ㄧ方面，我們以改變培養液中鈣離子濃度的方式，調控角質幹細胞的增生與分化能力。將角質細胞培養於高、中、低鈣離子濃度之培養液中，分別為DMEM(1.2mM)、DK11(0.4mM)及K(0.03mM) 。測定角質細胞的MTT活性、增生能力及細胞群落形成的大小與形成效率。以定量PCR分析Caspase-14的表現量，判斷角質細胞分化的程度，並和增生能力作關聯性的分析。結果發現高鈣離子濃度會抑制角質細胞的增生、使細胞的存活性與群落形成效率降低。Caspase-14的表現增加，證明了較高的鈣離子濃度有促進分化的效果。整體而言，培養液中鈣離子的適度增加，提高了角質化的速度，角質的形成對於皮膚的保護功能有加分的作用。最後，我們將纖維母細胞接種於預先製備好的三維膠原蛋白支架中，培養成真皮等價物之後，以組合的配方液幫助角質幹細胞接種於其表面，再以增生培養方式建構表皮基底層。在不同的培養階段探討EGF的添加，對於表皮形成的影響。分析細胞存活性、基因表現、組織型態及免疫染色之間的關連性，綜合評估細胞-支架-訊息因子之間交互調控的作用機制。於氣-液介面下培養的結果發現，持續性的添加10ng/ml的EGF，會造成異常的分化結果，包括：較薄的表皮分化層、不佳的細胞-基質介面及不良的組織形態等等。但是適時的添加EGF可以得到良好的組織型態，這是由於表皮角質細胞與真皮纖維母細胞間，有即時溝通的結果，經由生長因子的paracrine與autocrine，或是回饋機制的調控，維持組織的正常狀態。實驗的結果說明，EGF可以參考細胞特定基因的表現狀況(例如：KGF與TGF-beta1)，作適度的添加。

關鍵字

配方液 ； 供養膠體 ； 膠原蛋白 ； 角質細胞 ； 纖維母細胞 ； 皮膚等價物 ； 生長因子

並列摘要

Abstract
Fibroblasts produce a spectrum of necessary growth factors essential for growth and proliferation of a variety of cell types. First aim of this study was prepared the feeder gel with optimum fibroblast density and optimum conditioned medium that promoted keratinocyte proliferation without further differentiation for skin equivalent tissue engineering.
The optimum cell density in collagen feeder gel for optimum selected medium preparation will be determined by checking the level of keratinocyte growth factor (KGF) and granulocyte/macrophage colony-stimulating factor (GM-CSF) in conventional medium. The results showed that the cell density of 1x105 cells/gel in the feeder gel is better to produce optimum selected medium.
The conditioned medium is prepared by mixing the optimum selected medium and MCDB 153 medium together in different ratios for keratinocyte growth. The keratinocyte viability will be measured by MTT assay to determine the optimum conditioned medium. From the study, 67% conditioned medium was supposed the better medium for the keratinocyte proliferation. In this experiment, the optimum cell density in feeder gel to co-culture with keratinocyte is also determined as 1x105 cells/gel.
Cytokeratin-10 and TUNEL stain will be used to check the cell differentiation and apoptosis, respectively. The results suggest that keratinocyte should not be cultured in post-confluent condition due to toward undesired apoptosis and differentiation. The result of cell viability from passages to passages shows that the optimum feeder gel plays more important role to the keratinocyte proliferation than that of optimum conditioned medium. Keratinocytes cultured with optimum feeder gel in 67% conditioned medium could effectively promote proliferation, inhibit apoptosis and prevent from differentiation. The combination of conditioned media and feeder gel to culture keratinocyte without external supplements can provide an inexpensive way for keratinocyte proliferation and construct an environment for real-time communication between the two cells. The results conclude that keratinocyte cultivation in feeder gel with modified medium should be feasible in the production of high quality keratinocyte for skin equivalents preparation.
The second approach of preparing skin equivalents is regulation of proliferated and differentiated capacity. We investigated Ca2+ effects on the proliferation and differentiation using the primary keratinocytes model. Keratinocytes were incubated in DMEM (containing 1.2 mM Ca2+ concentration) or DK11 medium (containing 0.4 mM Ca2+ concentration) or K medium (containing 0.03 mM Ca2+ concentration). Cell viability was assessed with the MTT assay. Crystal violet assay was evaluated the proliferation rate and colony formation size of keratinocyte. Real-time PCR used to determine the terminal differentiated keratinocyte which expressed Caspase-14. Proliferation assays and real–time PCR were correlated with either proliferation or differentiation in cultured human skin epidermal keratinocytes.
High Ca2+ concentration was inhibited the cell viability and proliferation rate of keratinocyte. Ca2+ also increased caspases-14 expression, and inhibited cell viability, and cell colony forming efficiency. These results are consistent with Ca2+ induction of the keratinocyte differentiation. Thus, the overall Ca2+ actions connote protective functions for the epidermis that appear to include the triggering or acceleration of the differentiation.
At last, we designed a novel culture system included a self-designed 3-D collagen scaffold with different pore size and specific culture media for different culture stages. This skin equivalent culture model provides a new investigating system to study the role of extracellular matrix and growth factors including epidermal growth factor (EGF), keratinocyte growth factor (KGF), transforming growth factor beta 1 (TGF-beta1), in the cell-cell and cell-matrix interactions. Keratinocytes were seeded onto the dermal equivalent and incubated under submerged condition for 5 days then proceeding to air-liquid interface cultured either with or without EGF addition. In this study, EGF has a positive effect on the keratinocyte migration and proliferation in the submerged stage. However, when 10ng/ml of EGF was continual added in the air-lifted stage, a less organized and thin differentiated keratinocyte layers were found. Continual 10ng/ml of EGF addition in the air-lifted stage resulted in uneven cell-matrix interface, and disorganization of the suprabasal layers. On the contrary, in the air-lifted stage without excess EGF, the epithelium cells will stratify, differentiate, and form an epidermis completed with basal, spinous, granular, and cornified layers. The results showed that time scale modulation of EGF on keratinocyte cell behavior depend on the expression of paracrine or autocrine growth factors (e.g. KGF and TGF-beta1).

並列關鍵字

conditioned medium ； feeder gel ； collagen ； keratinocyte ； fibroblast ； skin equivalent ； growth factors

參考文獻

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