組織蛋白去乙醯化抑制劑(Valporic Acid, VPA)對人類牙乳突細胞骨分化及基因表現之影響

Abstract

細胞核內的DNA與組織蛋白(Histone)結合形成染色質(Chromatin)，外基因調控(epigenetic modification)即是指針對染色質而非DNA序列的基因表現調控。組織蛋白為DNA摺疊時所依賴的線軸，組織蛋白乙醯化(Acetylation)是外基因調控過程中的重要步驟，組織蛋白去乙醯化抑制劑(Histone deacetylation inhibitor)具有抑制組織蛋白去乙醯化酵素(Histone deacetylase)的功能，進而影響基因表現及細胞生長分化。近期細胞生物學研究已發現組織蛋白去乙醯化抑制劑可誘導癌細胞凋亡，也可促進正常細胞(肝細胞、骨細胞等)分化，因此在幹細胞誘導及組織工程應用上，組織蛋白去乙醯化抑制劑可能具有值得探討的潛力。牙乳突細胞群在牙齒自然發育過程中扮演重要的角色，可在適當誘導下表現多重分化的潛能，而許多基礎研究也證實培養自根尖牙乳突的前驅性細胞群，具有造骨細胞表型特徵。 本研究的目的在探討一種組織蛋白去乙醯化抑制劑(Valproic Acid, VPA)對於人類初代培養的根尖牙乳突細胞，在正常培養液或促進細胞基質鈣化培養液(含有L-ascorbic acid 2-phosphate (0.05mM)、sodium β-glycerophosphate (10 mM) 、 dexamethasone (10 -7 M)中，經不同的培養時間(12小時至15天)，其成骨分化指標基因的表現及細胞外基質礦化的影響。本研究結果發現人類根尖牙乳突細胞的鹼性磷酸酶活性表現隨培養時間漸增長而上升，顯示其具有似成骨細胞分化的基本特性；且在促進基質鈣化培養液中，根尖牙乳突細胞可明顯表現細胞外基質鈣化，並隨著培養時間的增長而增加。而VPA的刺激有早期提升ALP (Alkaline phospatase)、OC (Osteocalcin) 、COL-1 (Collagen type-I)和Cbfa-1 (Core-binding factor-1)基因表現的效應，而此效應於正常培養液和促進基質鈣化培養液中皆然。以即時定量聚合酶連鎖反應(Real-time PCR)發現ALP、OC和Cbfa-1的mRNA表現在VPA刺激一天後即為對照組的2-4倍，比單純誘導組提升10倍。但單純以VPA作誘導只能強化基因表現，並無細胞外基質鈣化。此外，隨著培養時間增加，未給予VPA的組別會漸漸趕上添加VPA組別的基因表現及基質礦化程度。研究結論: VPA可促進人類根尖牙乳突細胞的成骨基因表現、鹼性磷酸脢活性，提早表現細胞外基質礦化；但隨著培養時間增長，細胞外基質礦化的表現量與單純誘導組比較並未顯著升高。

Histones are responsible for the first level of DNA packing in eukaryotic chromatin.The organization of chromatin serves a dual purpose, one function is to pack the DNA into a compact form inside the nucleus of a cell, and the other is regulatory. Epigenetic modifications of chromatin play key roles in chromatin structure and the regulation of gene expression. Histone acetylation and deacetylation play a direct role in the regulation of gene transcription. Recently, histone deacetylase inhibitors (HDI) were shown to inhibit histone deacetylases ( HDACs), thus cause growth arrest and induce differentiation of transformed cells in culture and inhibit tumor growth in vivo. Normal cells also undergo cell cycle arrest and differentiation in the presence of HDI which means HDI had potential for applying on stem cells induction and tissue engineering. The dental papilla contributes to tooth formation and eventually converts to pulp tissue, so it had multipotent progenitor characteristic in different induction environment. There is evidence that the dental papilla cells were as potent in osteo/ dentinogenic differentiation and showed the osteoblastic phenotype. The purpose of this study was to examine the effect of Valporic acid, an histone deacetylase inhibitor, on the expression of osteogenic differentiation markers in the human dental papilla cells. The cells were cultured in standard medium or induction medium with osteogenic supplements including L-ascorbic acid 2-phosphate (0.05mM), sodium β- glycerophosphate (10 mM) and dexamethasone (10 -7 M). Our results demonstrated the progressive increase of ALP activity and extracellular matix mineralization in human dental papilla cells when cultured in induction medium. Theses results implied the osteoblastic phenotype of human dental papilla cells. The mRNA expression of ALP, OC, Cbfa-1 and COL-I was upregulated by VPA (1mM) in standard medium as well as induction medium, especially in short-term culture. Although the VPA in standard medium could upregulate the expression of osteoblastic marker genes, no significant extracellular matrix mineralization was noted. As to long-term culture, the upregulatory effect of VPA on osteoblastic marker genes progressively diminished in both standard and induction medium. The results of this study suggest that VPA can accelerate osteogenic differentiation of dental papilla cells. The expression of ALP activity and extracellular matrix mineralization in VPA-treated group was noted earlier than those in control group. However, in the long-term culture, the difference in the matrix mineralization did not reach statistically significant level.