不同代次诱导多能干细胞增殖及牙周定向分化的能力

Comparative study of proliferative and periodontal differentiation propensity of induced pluripotent stem cells at different passages

目的:研究不同代次诱导多能干细胞(induced pluripotent stem cells,i PSCs)在增殖及向牙周定向分化能力上的区别,为今后深入研究和可能的临床应用提供理论基础。方法:复苏不同代次(P5、P10、P15、P20)人牙龈成纤维细胞来源的i PSCs,观察细胞形态并比较其增殖能力,进一步将各代次细胞形成拟胚体,于含生长分化因子-5(growth/differentiation factor-5,GDF-5)的培养基中诱导分化14 d,同时分别设置未诱导自发分化组为阴性对照,茜素红染色观察矿化结节并半定量分析钙盐沉积量,qRT-PCR及免疫荧光染色分别检测牙周相关基因及蛋白的表达,评价各代次间分化能力的差异。结果:不同代次的i PSCs均具有很强的体外增殖能力,经牙周定向诱导的细胞呈成纤维细胞样生长,茜素红染色可形成"矿化"结节,细胞钙盐沉积量较自发分化组显著增高且差异有统计学意义(P5:t=2.125,P=0.003;P10:t=2.246,P=0.021;P15:t=3.754,P=0.004;P20:t=3.933,P=0.002),但相同诱导条件下不同代次间钙盐沉积量差异无统计学意义(牙周定向诱导组:F=2.365,P=0.109;自发分化对照组:F=2.901,P=0.067),此外,相同代次细胞中诱导组牙周相关标记物的表达均高于对照组且差异有统计学意义(P<0.05),但不同代次间的蛋白表达差异无统计学意义(骨涎蛋白:F=0.9267,P=0.450;波形蛋白:F=0.9171,P=0.455;牙骨质蛋白1:F=2.129,P=0.137)。结论:不同代次不会影响i PSCs的增殖及分化能力,体外长期培养的i PSCs易于扩增,较高代次者经诱导仍可高效定向分化,适合作为牙周组织工程的种子细胞。

Objective： To compare the proliferative and periodontal specific differentiation abilities of induced pluripotent stem cells（ i PSCs） at different passages,and to investigate whether long term culturing would have a negative influence on their proliferation and specific differentiation capacity,thus providing a theoretical basis for further in-depth research on periodontal regeneration and the possible clinical applications of i PSCs. Methods： IPSCs derived from human gingival fibroblasts at passages 5,10,15 and 20 were recovered and cultured in vitro. Their morphology and proliferation rates were observed respectively. We further induced the i PSCs at different passages toward periodontal tissue under the treatment of growth/differentiation factor-5（ GDF-5） for 14 days through the EB routine,then compared the periodontal differentiation propensities between the different passages of i PSCs by detecting their calcified nodules formation by Alizarin red staining and assaying their relative periodontal tissue related marker expressions by qRT-PCR and immunofluorescence staining,including bone related markers： osteocalcin（ OCN）,bone sialoprotein（ BSP）; periodontal ligament related markers： periostin,vimentin; and cementum related markers： cementum attachment protein（ CAP）,cementum protein 1（ CEMP1）. The untreated spontaneous differentiation groups were set as negative controls respectively. Results： i PSCs at different passages all showed a high proliferative capacity when cultured in vitro and turned into a spindlelike shape similar to fibroblasts upon periodontal specific differentiation. All i PSCs formed typical calcified nodules upon GDF-5 induction by Alizarin red staining in comparison to their untreated controls. The relative calcium deposition at all passages had been significantly upgraded under the treatment of GDF-5（ P5： t = 2. 125,P = 0. 003; P10： t = 2. 246,P = 0. 021; P15： t = 3. 754,P = 0. 004; P20： t = 3. 933,P = 0. 002）,but no significant difference in their calcium deposition were detected within passages 5,10,15 and 20（ periodontal differentiation： F = 2. 365,P = 0. 109; spontaneously differentiation： F =2. 901,P = 0. 067）. Periodontal tissue related marker expressions of i PSCs at all passages had also been significantly upgraded under the treatment of GDF-5（ P 〈0. 05）,but still,no significant difference in their expression levels of periodontal tissue related proteins were detected within passages（ BSP： F =0. 926 7,P = 0. 450; vimentin： F = 0. 917 1,P = 0. 455; CEMP1： F = 2. 129,P = 0. 1367）. Conclusion： Our results preliminarily confirmed that long term culturing won＇t influence the proliferation capacity and periodontal specific differentiation propensity of i PSCs,as they can still proliferate and differentiate toward periodontal cells with high efficiency upon growth factor induction after continuous passaging.Therefore,i PSCs could be recognized as a promising cell source for future possible application in periodontal tissue regeneration.