不同代次诱导多能干细胞增殖及牙周定向分化的能力

发布时间：2018-06-11 23:26

本文选题：多能干细胞 + 细胞代次　；参考：《北京大学学报(医学版)》2017年01期

【摘要】：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(P0.05),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。
[Abstract]:Objective: to study the difference of proliferation and periodontal directional differentiation between different generations of induced pluripotent stem cells in order to provide a theoretical basis for further research and possible clinical application. Methods: I PSCs derived from human gingival fibroblasts were resuscitated from different generations of P5, P10, P15 and P20. The morphology of the cells was observed and their proliferative abilities were compared. The differentiation was induced in the medium containing growth factor -5 (GDF-5) for 14 days, and the group without spontaneous differentiation was set as negative control. Alizarin red staining was used to observe the mineralized nodules and semi-quantitative analysis of calcium salt deposition. The expression of periodontal related genes and proteins were detected by RT-PCR and immunofluorescence staining respectively, and the differentiation ability of different generations was evaluated. Results: I PSCs of different generations had strong proliferative ability in vitro. The cells induced by periodontal orientation grew like fibroblasts, and alizarin red staining could form "mineralized" nodules. Cell calcium deposition was significantly higher than that of spontaneous differentiation group (P 5: t 2. 125% P0. 003) P10t1 2. 246U P0. 021 + P15: t 3. 754 + P0. 004 + P20: t 3. 933 P0. 002, but there was no significant difference in calcium deposition between different generations under the same inducement condition (periodontal orientation induction group: F 2. 365U P0. 109; spontaneous control group: F 2. 901P 0.067, P 0. 067), but under the same inducement condition, there was no significant difference in the amount of calcium deposit between different generations (P = 2. 365P0. 109); the spontaneous control group: F2. 901P0. 067, in addition, there was no significant difference in the amount of calcium deposition between different generations under the same inducement. In the same generation, the expression of periodontal markers in the induction group was higher than that in the control group, and the difference was statistically significant (P 0.05), but there was no significant difference in protein expression among different generations (bone sialoprotein: F0. 9267 P0. 450; vimentin 1: F0. 9171, P0. 455; cementum protein: 1: F2. 129, P0. 137). Conclusion: the ability of proliferation and differentiation of I PSCs is not affected by different generations. The long term cultured I PSCs are easy to expand, and the higher generation can still be induced to differentiate effectively, which is suitable for periodontal tissue engineering seed cells.
【作者单位】：北京大学口腔医学院·口腔医院牙周科口腔数字化医疗技术和材料国家工程实验室口腔数字医学北京市重点实验室;
【基金】：国家自然科学基金(81470739)资助~~
【分类号】：R781

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