无机三氧化聚合物调控根尖牙乳头干细胞分化的机制研究

发布时间：2018-05-03 06:09

  本文选题：无机三氧化聚合物 + 根尖牙乳头干细胞　； 参考：《南京医科大学》2012年硕士论文

【摘要】：生物活性材料无机三氧化聚合物（mineral trioxide aggregate, MTA）现已广泛应用于根尖诱导成形术的临床治疗，并取得了较好的效果，但其对牙根发育及再生过程中的重要种子细胞——根尖牙乳头干细胞（stem cells from apicalpapilla, SCAPs）增殖分化能力的影响，以及其中的作用机制尚不清楚。本文将分三个部分就这一问题进行探讨分析： 第一部分MTA条件培养基的制备和诱导浓度筛选 研究目的：制备MTA条件培养基，筛选最佳诱导浓度，观察其对SCAPs增殖能力的影响。 研究方法：制备MTA条件培养基，碱性磷酸酶（alkaline phosphatase, ALP）活性检测实验筛选诱导SCAPs矿化的最佳浓度，通过茜素红染色实验及10%氯代十六烷基吡啶（cetylpyridinium chloride, CPC）溶液溶解钙离子定量实验佐证。另外，采用四甲基偶氮噻唑盐（methyl-thiazolyl-tetrazolium, MTT）法绘制SCAPs生长曲线，计算细胞群体倍增时间（population doubling time, PDT），以及流式细胞术（flow cytometry, FCM）检测细胞周期分布，计算细胞的增殖指数（proliferation index, PI），检测MTA条件培养基对SCAPs的增殖能力的影响。 实验结果： ALP活性检测结果显示，MTA条件培养基诱导3d时，2mg/mL组的SCAPs细胞ALP活性最高（P 0.01）。采用该浓度MTA诱导14d时，茜素红染色结果显示，诱导组矿化结节形成能力明显高于未诱导组（P 0.01）。PDT和细胞周期分析后的PI结果均显示，两组SCAPs的增殖能力无明显差异（P0.05）。 结论：2mg/mL MTA不影响SCAP细胞的增殖能力，但对SCAPs的矿化能力有明显的促进作用。 第二部分MTA对SCAPs分化以及形态发生能力的影响 研究目的：进一步明确MTA对SCAPs牙向/骨向分化以及形态发生能力的影响。 研究方法：提取MTA诱导3d和7d时的细胞总RNA和总蛋白，通过实时定量逆转录聚合酶链式反应实验（real-time reverse transcription polymerase chainreaction, real-time RT-PCR）检测牙向/骨向分化相关标志基因（ALP, DSPP,RUNX2, OCN等）的表达情况，蛋白免疫印迹实验（Western blot）检测牙向/骨向分化相关标志蛋白（DSP, RUNX2, OCN等）的表达情况。另外，将细胞团复合根管支架材料，体内移植后，观测诱导后SCAPs的形态发生能力。 实验结果：Real-time RT-PCR和Western blot结果显示，MTA诱导组OCN、RUNX2、DSP等基因/蛋白表达水平明显高于未诱导组，提示MTA可增强SCAPs的牙向/骨向分化能力。体内移植实验发现，与未诱导组相比，MTA诱导后的SCAPs可以形成类牙本质牙髓复合体结构，并且表达较高水平的DSP、RUNX2、OCN等矿化相关蛋白。 结论：MTA明显促进SCAPs的牙/骨向分化、牙本质牙髓复合体形成能力。 第三部分MTA调控SCAPs分化的NF-κB通路机制 研究目的：探讨核因子κB（nuclear factor kappa B, NF-κB）通路在MTA诱导的SCAPs分化过程中的调控作用。 研究方法：选取MTA条件培养基刺激0h、0.25h、0.5h、3h、6h和12h，分别提取各时间点的SCAPs胞浆蛋白和胞核蛋白，Western blot检测通路相关蛋白（NF-κB P65、IκB-α、磷酸化NF-κB P65、磷酸化IκB-α）的表达，检测NF-κB通路的激活情况。另外，制作细胞爬片，将上述六个时间点的SCAPs固定后，对NF-κB P65进行免疫荧光染色，观察P65的核转位情况，，间接反映NF-κB通路的活性状态。 实验结果：NF-κB通路关键蛋白的Western blot和荧光染色检测结果表明，P65核转位明显。表达结果显示：P65逐渐由胞浆转入核内；p-P65在0.25h表达已显著升高，并在0.5h时继续上升，但随后开始下降；IκB-α先略有下降，然后恢复；p-IκB-α在0.25h表达即到最高，0.5h时虽开始下降但仍为高表达。提示NF-κB通路被激活。NF-κB P65的荧光染色检测亦表明，P65转入细胞核内启动相关转录程序。 结论：MTA诱导SCAPs的牙/骨向分化和形态发生，可能是通过激活NF-κB通路来实现的。
[Abstract]:Mineral trioxide aggregate (MTA), a bioactive material, has been widely used in the clinical treatment of apex inducement and has achieved good results. However, it is an important seed cell in the process of root development and regeneration (stem cells from apicalpapilla, SCAPs) in root development and regeneration. The influence of chemical capacity and the mechanism of action are not yet clear. This article will discuss and analyze the problem in three parts.
Part one preparation of MTA conditioned medium and screening of induced concentration
Objective: to prepare MTA conditioned medium and screen the best induction concentration and observe its effect on SCAPs proliferation.
Research methods: preparation of MTA conditioned medium, alkaline phosphatase (alkaline phosphatase, ALP) activity detection test to screen the best concentration of SCAPs mineralization, by alizarin red staining experiment and 10% Chloroalkyl sixteen alkyl pyridine (cetylpyridinium chloride, CPC) solution calcium ion quantitative test evidence. In addition, the use of four methyl azothiazide. The growth curve of SCAPs was plotted by methyl-thiazolyl-tetrazolium (MTT), and the cell multiplication time (population doubling time, PDT) was calculated and the cell cycle distribution was detected by flow cytometry (flow cytometry, FCM). The proliferation index (proliferation index) was calculated and the proliferation ability of the conditioned medium was detected. Influence.
The results of ALP activity detection showed that the ALP activity of SCAPs cells in 2mg/mL group was the highest (P 0.01) when MTA conditioned medium induced 3D. The results of alizarin red staining showed that the formation ability of the mineralized nodules in the induced group was significantly higher than that of the uninduced group (P 0.01).PDT and the PI results after cell cycle analysis were all displayed, two There was no significant difference in the proliferation ability of group SCAPs (P0.05).
Conclusion: 2mg/mL MTA does not affect the proliferation of SCAP cells, but has a significant effect on SCAPs mineralization.
The second part is the effect of MTA on SCAPs differentiation and morphogenesis.
Objective: to further clarify the effect of MTA on SCAPs's odontogenic / osteogenic differentiation and morphogenesis.
Methods: to extract the total RNA and total protein of 3D and 7d induced by MTA, and to detect the expression of tooth / bone differentiation related genes by real-time quantitative reverse transcriptase polymerase chain reaction (real-time reverse transcription polymerase chainreaction, real-time RT-PCR), protein immuno printing The trace test (Western blot) was used to detect the expression of DSP, RUNX2, OCN and so on. In addition, after transplantation, the morphogenetic ability of SCAPs was observed after transplantation in vivo.
The results of Real-time RT-PCR and Western blot showed that the expression level of OCN, RUNX2, DSP and other genes and proteins in MTA induced group was significantly higher than that in the uninduced group, suggesting that MTA could enhance the tooth / bone differentiation ability of SCAPs. In vivo transplantation experiment found that SCAPs after MTA inducement could form the dentinal pulp complex knot of the dentin like group compared with the uninduced group. It also expressed higher levels of mineralization related proteins such as DSP, RUNX2 and OCN.
Conclusion: MTA can significantly promote the SCAPs / odontoblast differentiation and dentin pulp complex formation ability.
The third part is the mechanism of NF- kappa B pathway regulated by MTA in SCAPs differentiation.
Objective: To investigate the regulatory role of B nuclear factor kappa B (NF- B) pathway in MTA induced SCAPs differentiation.
Methods: MTA conditioned medium was selected to stimulate 0h, 0.25h, 0.5h, 3h, 6h and 12h, respectively, to extract SCAPs cytoplasmic protein and nucleoprotein at each time point, and Western blot to detect the expression of pathway related proteins. After fixation with SCAPs at six time points, immunofluorescence staining of NF- kappa B P65 was performed to observe the nuclear translocation of P65 and indirectly reflect the activity of NF- kappa B pathway.
The results showed that the Western blot and fluorescence staining of the key protein of the NF- kappa B pathway showed that the transposition of the P65 nucleus was obvious. The expression results showed that P65 was gradually transferred from the cytoplasm to the nucleus; the expression of p-P65 in 0.25h increased significantly, and continued to rise at 0.5h, but then began to decline; I kappa B- alpha decreased slightly and then recovered; p-I kappa B- alpha was 0.2 The expression of 5h is the highest, while 0.5h begins to decline but is still high, suggesting that the NF- kappa B pathway is activated by the fluorescence staining of.NF- kappa B P65 and indicates that P65 is transferred into the nucleus to start the related transcriptional program.
Conclusion: MTA induced SCAPs tooth / bone differentiation and morphogenesis may be achieved by activating NF- kappa B pathway.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R783.1

【共引文献】

相关期刊论文 前5条

1 张庆鸿;梁星;;脱乙酰壳多糖支架在牙周组织工程中的应用[J];国际口腔医学杂志;2012年04期

2 刘家祺;亓发芝;;Beta-磷酸三钙复合材料支架的研究进展[J];中国美容医学;2011年04期

3 陈敏;聂敏海;;牙周组织工程研究的新进展[J];西南军医;2013年02期

4 葛柳莹;赵利芬;段开文;;牙周引导组织的再生膜材料[J];中国组织工程研究与临床康复;2010年42期

5 李玮;孟雪梅;;牙周组织工程种子细胞及其体内移植方法的研究进展[J];中国修复重建外科杂志;2010年10期

相关硕士学位论文 前3条

1 李娜;双黄补对牙周膜细胞在玉米醇溶蛋白支架材料上黏附生长的影响[D];河北医科大学;2011年

2 张文;人脂肪干细胞在水凝胶内三维培养的研究[D];大连理工大学;2010年

3 刘家祺;纳米级多孔β-磷酸三钙支架材料的制备、表征及细胞相容性研究[D];复旦大学;2012年

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