Quantitative analysis of ceramide metabolites in Ca2+ and vitamin C treated keratinocytes : Ca2+와 vitamin C로 분화된 표피 각질형성세포에서 Ceramide 대사체 함량 분석

다국어 초록 (Multilingual Abstract)

Ceramides which are the major constituent of sphingolipids in epidermis known to play diverse roles in the outermost layers of skin such as water holding capacity and physical barrier property. Moreover they can be hydrolyzed into free sphingoid bases such as C18 Sphingosine(SO), C18 Sphinganine(SA) or further metabolized to C18 So-1-Phosphate(S1P), C18 Sa-1-Phosphate(SA1P) in keratinocytes. The significance of the ceramides metabolites has been emerged; altered levels of SO, SA in skin disorders and roles of S1P, SA1P as signaling lipids have been reported. However, the overall metabolism of sphingoid bases and their phosphates during keratinocytes differentiation has not been discussed in great details. Thus, in this study we analyzed these ceramide metabolites in the process of keratinocyte differentiation. 3 distinct keratinocyte differentiation stage were prepared with 0.07 mM Ca2+ (proliferation stage), 1.2 mM Ca2+ (early differentiation stage) in serum free culture medium, and serum containing medium with Vit C (50 ㎕/㎖) (late differentiation stage). And also serum containing medium was used to elucidate whether Vit C per se increases sphingoid bases and their phosphates. Sphingoid bases and their phosphates after alkaline phosphatase reaction were also determined by HPLC system. Compared to 0.07 mM Ca2+, levels of SO, SA, S1P and SA1P were not altered in 1.2mM Ca2+ treatment. However, in keratinocytes cultured under Vit C containing serum medium, levels of SO, SA, S1P and SA1P were dramatically higher than those in 0.07 and 1.2mM Ca2+ treated cells, but compared to serum containing medium, Vit C did not enhance their production significantly. Taken together, we demonstrate that the late differentiation process of keratinocytes induced by Vit C and serum was accompanied with dramatic increase of sphingoid bases and their phosphates, but Vit C per se had no effects on their production.

국문 초록 (Abstract)

표피 내에는 칼슘의 기울기가 존재하여, 낮은 칼슘농도(0.07mM)에서는 각질형성세포는 증식하는 반면에 고 칼슘의 농도(1.2mM)는 세포의 분화를 유도하게 된다. 그리고 비타민 C(50㎍ per ㎖)와 10%의 Serum, 고 칼슘(1.2mM)의 농도가 유지가 되면 세포의 분화는 더 진행이 된다고 알려져 있다. 이러한 세포배양방법을 이용하여 세포 분화에 따른 세포 형태학적, 많은 생화학적 변화가 밝혀져 있지만, 세라마이드 대사체 연구는 아직 밝혀진 바가 없으며, 또한, 사전연구에서 비타민 C는 스핑고지질의 합성에 관련한 효소 및 지질함량을 증가시킨다고 보고되었지만 세라마이드 대사체에 관련한 변화를 아직 밝혀지지 않았다. 이에 본 연구에서는 칼슘과 비타민 C를 이용하여 각질형성세포의 분화를 유도한 후 세라마이드 대사체인 C18 Sphingosine(SO)과 C18 Sphinganine(SA), 그리고 그들의 인산화 형태인 Sphingo-sine 1 Phosphate(S1P)와 Sphinanine 1 Phosphate(SA1P)를 HPLC를 이용하여 함량분석을 통해 각질형성세포에 따른 이들의 변화를 관찰하였고, 비타민 C가 이들의 함량을 증가시키는지 알아보았다. SO, SA, S1P, 그리고 SA1P 모두 후기 분화가 진행되었을 때, 크게 증가하지만, 칼슘의 변화에 따른 변화는 동반되지 않았다. 그래서 이들 대사체들은 표피의 후기 분화에 필수적이라고 생각한다. 반면, 후기분화를 촉진한다고 보고되었던 비타민 C 자체는 SO, SA, S1P, 그리고 SA1P 생성에 아무런 영향은 주지 않았다.

목차 (Table of Contents)

• Chapter Ⅰ.Literature review 1
• 1.The Skin 2
• 2.Keratinocytes
• 2.1.Keratinocyte differentiation 4
• 2.2.Sphingolipid synthesis in epidermis 7
• 2.3.Sphingoid bases 10
• 2.4.Sphingoid base 1 phosphates 12
• 3.Nutrients regulating keratinocyte differentiation
• 3.1.Calcium 16
• 3.2.Vitamin C 19
• 4.Sphingolipids in food 22
• References for literature review 24
• Chapter Ⅱ.Quantitative analysis of ceramide metabolites in Ca2+ and vitamin C treated keratinocytes 31
• Abstract 32
• Introduction 34
• Materials and methods 36
• Results 41
• Discussion 53
• Summury 59
• References 60
• 한글초록 65