碱性成纤维细胞因子对根尖牙乳头干细胞生物学作用及机制研究
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摘要
根尖牙乳头干细胞（Stem cells from the apical papilla，SCAP）是位于未发育完成恒牙根尖乳头的间充质干细胞(mesenchymal stem cells, MSCs)。SCAP是一类具有成牙潜能的干细胞，具有比牙髓干细胞（dental pulp stemcells，DPSCs)更强的群体倍增能力、增殖能力、端粒酶活性及成牙本质潜能。研究发现SCAP是根部成牙本质细胞的主要来源，对根部牙本质形成发挥重要作用。SCAP独特的功能和特点决定了其不仅仅是一种研究牙根发育时成牙本质细胞分化机制的体外细胞，而且是一种很好的牙组织工程种子细胞，可用于牙髓牙本质和生物学牙根的再生，具有良好且广阔的临床应用前景。
牙根的发育形成是由一系列上皮和间充质信号的相互作用调控的。碱性成纤维细胞因子（Basic fibroblast growth factor，bFGF）是纤维生长因子（fibroblast growth factors, FGFs）家族的成员之一，在牙组织形态发生中发挥重要作用，并有效促进牙根的延长和牙周组织的形成。在小鼠磨牙牙根形成的各个阶段，分化的成牙本质细胞均表达bFGF，提示bFGF参与了牙根形成的信号调控。bFGF是一种多效性生长因子，参与细胞的增殖，分化和迁移。同时也是维持人胚胎干（human embryonic stem，hES)细胞自我更新能力的重要因子。对成体干细胞，bFGF对不同分化阶段的细胞以及不同间充质干细胞的生物学效应表现并不相同。文献报道bFGF对牙髓干细胞的作用可表现上调或降低ALP的活性；促进或抑制其成牙本质细胞分化。SCAP是根部成牙本质细胞的来源，具有很强的成牙本质细胞分化潜能，而bFGF对SCAP的生物学作用如何，目前尚未见报道。
本研究采用体外培养的SCAP为实验模型，研究bFGF对SCAP增殖、分化等生物学效应，从而明确bFGF对SCAP增殖和分化的调控作用；进而明确bFGF对SCAP干细胞干性（stemness）的影响；明确在bFGF调控SCAP增殖分化中MAPKs信号通路是否发挥作用；这将为进一步研究SCAP的生物学特性、成牙本质细胞分化机制、牙髓牙本质再生及生物学牙根等组织工程应用提供实验基础，同时为将来SCAP应用于临床治疗提供新的思路和途径。
课题所取得的主要结果如下:
1．根尖牙乳头干细胞的分离、培养、鉴定及多向分化能力检测
通过酶消化法原代培养根尖牙乳头细胞，利用有限稀释培养法成功分离出根尖牙乳头干细胞克隆；采用成骨诱导、成脂诱导实验验证了根尖牙乳头干细胞具备多向分化能力；采用免疫组织化学染色检测了根尖牙乳头细胞中充质干细胞标志物STRO-1，波形丝蛋白和CD24的表达；流式细胞仪定量分析了根尖牙乳头干细胞的STRO-1阳性表达率。
2．碱性成纤维细胞因子提高根尖牙乳头干细胞干性
以体外培养的SCAP为靶细胞，研究了不同浓度bFGF对SCAP的促增殖作用：MTT比色法发现不同浓度bFGF可提高SCAP的细胞存活率，以5,10,20ng/ml浓度促增殖的效果显著；bFGF可加速SCAP细胞周期进程；bFGF可提高3,6,12代SCAP细胞克隆形成率；bFGF可明显降低SCAP的ALP活性，抑制体外矿化结节形成， bFGF对体外矿化诱导1，2，4w后的SCAP可不同程度降低成牙本质标记基因DSPP、ALP、OCN和BSPmRNA表达； bFGF抑制SCAP细胞向成骨方向分化；而以bFGF预培养1w后SCAP的成骨能力增加；bFGF增加成脂诱导3w SCAP中PPAR-γ2mRNA表达；bFGF可提高不同培养代数SCAP中STRO-1的阳性表达率及干细胞基因Nanog, Oct4, Sox2及Rex1的mRNA表达，从而提高SCAP的干细胞特性及未分化潜能。
3．碱性成纤维细胞因子对根尖牙乳头干细胞生物学作用机制的初步探讨
bFGF可激活MAPKs信号通路，促进SCAP中p38、ERK、JNK通路磷酸化蛋白的表达水平；在应用p38、ERK、JNK通路的抑制剂阻断各通路后，可部分逆转bFGF下调SCAP中DSPP、ALP、OCN和BSP表达的作用；bFGF分别联合JNK、ERK和p38通路抑制剂作用后，Nanog、Oct4和Sox2的表达在各实验组呈现了不同程度的下调；以JNK通路抑制剂（SP600125）和ERK通路抑制剂（U0126）组较为明显。实验结果显示MAPKs信号通路参与了bFGF对SCAP生物学作用的调控。
综上所述，本研究认为bFGF可以促进根尖牙乳头干细胞的增殖；调控细胞的分化能力从而增加SCAP的干细胞干性（stemness）及未分化潜能；MAPKs信号通路参与了bFGF对根尖牙乳头干细胞分化特性的调控。本实验结果为进一步研究根尖牙乳头干细胞的生物学特性及分化机制提供了研究基础；为进一步研究bFGF对根尖牙乳头干细胞的分化调控机制提供了研究基础；同时为牙髓牙本质再生及生物学牙根的组织工程研究提供了新的研究思路。
Introduction
Stem cells from the apical papilla (SCAP) are a population ofmesenchymal stem cells (MSCs) residing in the apical papilla of incompletelydeveloped teeth. SCAP are a distinct source of potent dental stem/progenitorcells and appear to have a greater dentinogenic potential than dental pulp stemcells (DPSCs). The distinction between dental pulp and apical papilla is thatapical papilla is the precursor tissue of the radicular pulp. Recently, SCAP havebeen shown to possess the ability to regenerate vascularized human dentalpulp-like tissues in emptied root canal space and produce new dentin-like tissueon existing dentinal walls. These pieces of evidence support the hypothesis thatSCAP may be the source of primary odontoblasts that are responsible for theformation of root dentin.
Tooth root formation is mediated through a series of epithelial-mesenchymal interactions regulated by several signaling pathways. Basic fibroblast growth factor (bFGF) is a member of FGFs, which plays an importantrole in morphogenesis and histogenesis of tooth and they effectively promote thetooth root elongation and periodontal tissue formation. More importantly, bFGFwas observed in differentiating odontoblasts at the apical end of the tooth and inthe furcation zone of the developing root at all the stages suggesting that bFGFmay participate in the signaling network associated with root development.bFGF is known an angiogenic and pleiotropic growth factor involved in theproliferation, differentiation and migration of numerous cell types. It is knownbFGF is a critically important factor to maintain self renewal ability of humanembryonic stem (hES) cells in cultures. For adult stem cells, the effects of bFGFon MSCs of different tissue origin are different. bFGF appears to exertdifferential effects on different cell types and the stage of differentiaton of cellsmay respond differently to bFGF as well. Some studies, however, have reportedthat bFGF can up-regulate or down-regulate the ALP activity, promote orsupress the differentiation potential of DPSCs. However, the effects of bFGF onSCAP have not yet been reported. Thus, it is important to investigate the effectsof bFGF on the biologic functions of SCAP in order to understand its regulatoryrole in this context.
The purpose of this study was to examine the effects of bFGF on thebehavior of SCAP in terms of cell proliferation, differentiation potential,stemness, and whether MAPK was involved in the differentiation of SCAP bybFGF.
Main results
1. Isolation of SCAP and characterization of multipotent differentiationability.
Human third molars with developing roots were collected due to orthodonticreasons. Procedures were performed according to the approval of theinstitutional review board and the informed consent of the patients. SCAP wereisolated, cultured and expanded as previously described. Cells were collectedand prepared for limiting dilution procedures to obtain single-colony-derivedstrains. Growth characteristics and multipotent differentiation of the cell wereassessed.
2. Biological effects of bFGF on SCAP.
Different concentration of bFGF all increased the proliferation of SCAP. Theconcentration of5,1020ng/ml were significantly increased SCAP proliferation..bFGF proceeded into the G2/S phase of SCAP. The numbers of CFU-Fformation of SCAP at passages3,6and12were increased with bFGF. Incontrast, bFGF reduced the ALP activity and mineral nodule formation. bFGFdown-regulated the expression of ALP, BSP, DSPP and OCN especially afterSCAP cultured in osteo/dentinogenic medium for2week. SCAP pretreated withbFGF for1week can increase the ability of calcium deposition. bFGFup-regulated the expression of PPAR-γ2after3weeks of adipogenic induced.Regardless of the passage numbers, bFGF increased the percentage of STRO-1+cells significantly and increased the expression levels of pluripotent gene (Oct4,Nanog, Sox2and Rex1) expression levels. These results indicated bFGF canincrease the stemness of SCAP.
3. The mechanism of biological effects of bFGF on SCAP differentiationand remain stemness.
After SCAP treated with bFGF for different time, the phosphorylation ofJNK, P38and ERK were determined by Western blot assay. bFGF can inducedphos-JNK, phos-ERK and phos-p38expression.
The inhibitors U0126, SP600125or SB203580can partly withdraw thesuppression of bFGF on the expression of DSPP, BSP and OCN in SCAP, whichwere cultured in mineralization differentiation media for1week. After treatedwith SP600125，U0126and SB203580respectively, SCAP were cultured instandard medium with or without the presence of bFGF for6days. Theinhibitors only could be partially withdraw the effect of bFGF, Nanog, Oct4andSox2decreased but Rex1not. ERK and JNKpathway play an important role inthe effect of bFGF to SCAP.Conclussions
In summary, our results suggest that bFGF enhanced the stemness of SCAPevidenced by the increased proliferation, CFU-F forming capacity, andexpression of STRO-1, Nanog, Sox2, Oct4and Rex1, while bFGF suppressedALPase activity, calcified nodule formation and osteo/dentinogenicdifferentiation. MAPKs pathway take part in the effects of bFGF on the biologicfunctions of SCAP. Therefore, in this system, bFGF appears to function as afactor to maintain SCAP stemness, by enhancing self-renewal ability andpreventing cell differentiation. This will facilitate the long-term usage of thesecells for various tissue regeneration applications including pulp/dentinregeneration, bioroot formation and even for neural tissue regeneration.
牙根的发育形成是由一系列上皮和间充质信号的相互作用调控的。碱性成纤维细胞因子（Basic fibroblast growth factor，bFGF）是纤维生长因子（fibroblast growth factors, FGFs）家族的成员之一，在牙组织形态发生中发挥重要作用，并有效促进牙根的延长和牙周组织的形成。在小鼠磨牙牙根形成的各个阶段，分化的成牙本质细胞均表达bFGF，提示bFGF参与了牙根形成的信号调控。bFGF是一种多效性生长因子，参与细胞的增殖，分化和迁移。同时也是维持人胚胎干（human embryonic stem，hES)细胞自我更新能力的重要因子。对成体干细胞，bFGF对不同分化阶段的细胞以及不同间充质干细胞的生物学效应表现并不相同。文献报道bFGF对牙髓干细胞的作用可表现上调或降低ALP的活性；促进或抑制其成牙本质细胞分化。SCAP是根部成牙本质细胞的来源，具有很强的成牙本质细胞分化潜能，而bFGF对SCAP的生物学作用如何，目前尚未见报道。
本研究采用体外培养的SCAP为实验模型，研究bFGF对SCAP增殖、分化等生物学效应，从而明确bFGF对SCAP增殖和分化的调控作用；进而明确bFGF对SCAP干细胞干性（stemness）的影响；明确在bFGF调控SCAP增殖分化中MAPKs信号通路是否发挥作用；这将为进一步研究SCAP的生物学特性、成牙本质细胞分化机制、牙髓牙本质再生及生物学牙根等组织工程应用提供实验基础，同时为将来SCAP应用于临床治疗提供新的思路和途径。
课题所取得的主要结果如下:
1．根尖牙乳头干细胞的分离、培养、鉴定及多向分化能力检测
通过酶消化法原代培养根尖牙乳头细胞，利用有限稀释培养法成功分离出根尖牙乳头干细胞克隆；采用成骨诱导、成脂诱导实验验证了根尖牙乳头干细胞具备多向分化能力；采用免疫组织化学染色检测了根尖牙乳头细胞中充质干细胞标志物STRO-1，波形丝蛋白和CD24的表达；流式细胞仪定量分析了根尖牙乳头干细胞的STRO-1阳性表达率。
2．碱性成纤维细胞因子提高根尖牙乳头干细胞干性
以体外培养的SCAP为靶细胞，研究了不同浓度bFGF对SCAP的促增殖作用：MTT比色法发现不同浓度bFGF可提高SCAP的细胞存活率，以5,10,20ng/ml浓度促增殖的效果显著；bFGF可加速SCAP细胞周期进程；bFGF可提高3,6,12代SCAP细胞克隆形成率；bFGF可明显降低SCAP的ALP活性，抑制体外矿化结节形成， bFGF对体外矿化诱导1，2，4w后的SCAP可不同程度降低成牙本质标记基因DSPP、ALP、OCN和BSPmRNA表达； bFGF抑制SCAP细胞向成骨方向分化；而以bFGF预培养1w后SCAP的成骨能力增加；bFGF增加成脂诱导3w SCAP中PPAR-γ2mRNA表达；bFGF可提高不同培养代数SCAP中STRO-1的阳性表达率及干细胞基因Nanog, Oct4, Sox2及Rex1的mRNA表达，从而提高SCAP的干细胞特性及未分化潜能。
3．碱性成纤维细胞因子对根尖牙乳头干细胞生物学作用机制的初步探讨
bFGF可激活MAPKs信号通路，促进SCAP中p38、ERK、JNK通路磷酸化蛋白的表达水平；在应用p38、ERK、JNK通路的抑制剂阻断各通路后，可部分逆转bFGF下调SCAP中DSPP、ALP、OCN和BSP表达的作用；bFGF分别联合JNK、ERK和p38通路抑制剂作用后，Nanog、Oct4和Sox2的表达在各实验组呈现了不同程度的下调；以JNK通路抑制剂（SP600125）和ERK通路抑制剂（U0126）组较为明显。实验结果显示MAPKs信号通路参与了bFGF对SCAP生物学作用的调控。
综上所述，本研究认为bFGF可以促进根尖牙乳头干细胞的增殖；调控细胞的分化能力从而增加SCAP的干细胞干性（stemness）及未分化潜能；MAPKs信号通路参与了bFGF对根尖牙乳头干细胞分化特性的调控。本实验结果为进一步研究根尖牙乳头干细胞的生物学特性及分化机制提供了研究基础；为进一步研究bFGF对根尖牙乳头干细胞的分化调控机制提供了研究基础；同时为牙髓牙本质再生及生物学牙根的组织工程研究提供了新的研究思路。
Introduction
Stem cells from the apical papilla (SCAP) are a population ofmesenchymal stem cells (MSCs) residing in the apical papilla of incompletelydeveloped teeth. SCAP are a distinct source of potent dental stem/progenitorcells and appear to have a greater dentinogenic potential than dental pulp stemcells (DPSCs). The distinction between dental pulp and apical papilla is thatapical papilla is the precursor tissue of the radicular pulp. Recently, SCAP havebeen shown to possess the ability to regenerate vascularized human dentalpulp-like tissues in emptied root canal space and produce new dentin-like tissueon existing dentinal walls. These pieces of evidence support the hypothesis thatSCAP may be the source of primary odontoblasts that are responsible for theformation of root dentin.
Tooth root formation is mediated through a series of epithelial-mesenchymal interactions regulated by several signaling pathways. Basic fibroblast growth factor (bFGF) is a member of FGFs, which plays an importantrole in morphogenesis and histogenesis of tooth and they effectively promote thetooth root elongation and periodontal tissue formation. More importantly, bFGFwas observed in differentiating odontoblasts at the apical end of the tooth and inthe furcation zone of the developing root at all the stages suggesting that bFGFmay participate in the signaling network associated with root development.bFGF is known an angiogenic and pleiotropic growth factor involved in theproliferation, differentiation and migration of numerous cell types. It is knownbFGF is a critically important factor to maintain self renewal ability of humanembryonic stem (hES) cells in cultures. For adult stem cells, the effects of bFGFon MSCs of different tissue origin are different. bFGF appears to exertdifferential effects on different cell types and the stage of differentiaton of cellsmay respond differently to bFGF as well. Some studies, however, have reportedthat bFGF can up-regulate or down-regulate the ALP activity, promote orsupress the differentiation potential of DPSCs. However, the effects of bFGF onSCAP have not yet been reported. Thus, it is important to investigate the effectsof bFGF on the biologic functions of SCAP in order to understand its regulatoryrole in this context.
The purpose of this study was to examine the effects of bFGF on thebehavior of SCAP in terms of cell proliferation, differentiation potential,stemness, and whether MAPK was involved in the differentiation of SCAP bybFGF.
Main results
1. Isolation of SCAP and characterization of multipotent differentiationability.
Human third molars with developing roots were collected due to orthodonticreasons. Procedures were performed according to the approval of theinstitutional review board and the informed consent of the patients. SCAP wereisolated, cultured and expanded as previously described. Cells were collectedand prepared for limiting dilution procedures to obtain single-colony-derivedstrains. Growth characteristics and multipotent differentiation of the cell wereassessed.
2. Biological effects of bFGF on SCAP.
Different concentration of bFGF all increased the proliferation of SCAP. Theconcentration of5,1020ng/ml were significantly increased SCAP proliferation..bFGF proceeded into the G2/S phase of SCAP. The numbers of CFU-Fformation of SCAP at passages3,6and12were increased with bFGF. Incontrast, bFGF reduced the ALP activity and mineral nodule formation. bFGFdown-regulated the expression of ALP, BSP, DSPP and OCN especially afterSCAP cultured in osteo/dentinogenic medium for2week. SCAP pretreated withbFGF for1week can increase the ability of calcium deposition. bFGFup-regulated the expression of PPAR-γ2after3weeks of adipogenic induced.Regardless of the passage numbers, bFGF increased the percentage of STRO-1+cells significantly and increased the expression levels of pluripotent gene (Oct4,Nanog, Sox2and Rex1) expression levels. These results indicated bFGF canincrease the stemness of SCAP.
3. The mechanism of biological effects of bFGF on SCAP differentiationand remain stemness.
After SCAP treated with bFGF for different time, the phosphorylation ofJNK, P38and ERK were determined by Western blot assay. bFGF can inducedphos-JNK, phos-ERK and phos-p38expression.
The inhibitors U0126, SP600125or SB203580can partly withdraw thesuppression of bFGF on the expression of DSPP, BSP and OCN in SCAP, whichwere cultured in mineralization differentiation media for1week. After treatedwith SP600125，U0126and SB203580respectively, SCAP were cultured instandard medium with or without the presence of bFGF for6days. Theinhibitors only could be partially withdraw the effect of bFGF, Nanog, Oct4andSox2decreased but Rex1not. ERK and JNKpathway play an important role inthe effect of bFGF to SCAP.Conclussions
In summary, our results suggest that bFGF enhanced the stemness of SCAPevidenced by the increased proliferation, CFU-F forming capacity, andexpression of STRO-1, Nanog, Sox2, Oct4and Rex1, while bFGF suppressedALPase activity, calcified nodule formation and osteo/dentinogenicdifferentiation. MAPKs pathway take part in the effects of bFGF on the biologicfunctions of SCAP. Therefore, in this system, bFGF appears to function as afactor to maintain SCAP stemness, by enhancing self-renewal ability andpreventing cell differentiation. This will facilitate the long-term usage of thesecells for various tissue regeneration applications including pulp/dentinregeneration, bioroot formation and even for neural tissue regeneration.
引文
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