脱落乳牙干细胞骨向分化相关分子Runx2动态表达研究

发布时间：2018-06-28 17:03

本文选题：脱落乳牙干细胞 + 骨向分化　；参考：《天津医科大学》2011年硕士论文

【摘要】：目的：体外分离、培养人脱落乳牙干细胞(SHEDs),分析其生物学特征,探讨SHEDs骨向分化潜能,分不同时间点观察成骨相关转录因子Runx2的动态表达规律,为研究SHEDs成骨调控因素及其组织再生临床应用提供研究基础。方法：依据Miura等的实验方法,选取6～10岁健康儿童因滞留拔除的乳牙,采用酶消化法分离、有限稀释法纯化,获得人脱落乳牙干细胞。观察原代和传代细胞生长、增殖情况,测定细胞克隆形成率,HE染色,观察原代及传代细胞形态学特征。流式细胞仪及细胞免疫荧光染色检测SHEDs表面标志。培养的第3代SHEDs,在体外经含有IBMX、胰岛素、消炎痛和氢化可的松条件培养液,向脂肪组织进行诱导分化,观察细胞形态变化及细胞内形成脂滴情况,4周后油红-O染色。第3代SHEDs,在含有10%胎牛血清、1×10-8mol/1地塞米松、50 mg/lβ-甘油磷酸钠和10mmol/1维生素C的成骨诱导条件培养液诱导3周,观察细胞形态和生长规律的变化,分别于成骨诱导第0天和第21天做茜素红染色,检测矿化结节形成情况,鉴定其成骨分化的生物学特征。于成骨诱导的第0、3、6、9、12、15、18和21天,用RT-PCR技术检测Runx2的动态表达。结果： 1.通过酶消化法和有限稀释法获得了人脱落乳牙干细胞,原代培养的细胞一般在接种2天后,倒置显微镜下见细胞贴壁生长,形态呈现多形性。随着生长传代,细胞较多的出现多角形、梭形,成纤维细胞样,并且大小趋近一致。原代细胞爬片HE染色,大部分细胞呈梭形,体积较小,轮廓清晰,细胞核为圆形或卵圆形,体积较大。少数细胞内可见多个核仁存在。 2.计算细胞克隆形成率为22-26个/103细胞。流式细胞仪和细胞免疫荧光染色检测细胞表面标志显示,CD146+占总细胞量的92.51±1.34%,STRO-1+占总细胞量的20.01±1.62%。在成脂诱导体系的的作用下,可见个别细胞胞浆内出现高强度的折射点,油红－O染色可见橙红色阳性颗粒。在矿化诱导液的作用下,细胞形态变为多角形,形成钙化结节,经茜素红色呈阳性。 3.RT-PCR技术检测SHEDs成骨诱导过程中Runx2的动态表达规律显示：0天已有表达,0～6天成明显增强趋势,6～12天显著下降,12～18天表达再次上升,以后相对平稳。结论： 1.SHEDs可体外分离、培养,表达间充质干细胞表面标志,具有较强的倍增能力及克隆形成能力,具有成脂和成骨向分化能力。 2.Runx2在SHEDs成骨分化过程中成动态表达,成骨分化各阶段均有表达,早期和晚期表达上升,中期表达有下降趋势。
[Abstract]:Objective: to isolate and culture human deciduous dental stem cells (Sheds) in vitro, analyze their biological characteristics, explore the potential of bone differentiation and observe the dynamic expression of osteoblastic transcription factor Runx2 at different time points. To provide a basis for the study of osteogenesis regulation factors and clinical application of tissue regeneration in Sheds. Methods: according to Miura et al.'s experimental method, the deciduous teeth of 6 ~ 10 years old healthy children were isolated by enzyme digestion and purified by limited dilution method, and human deciduous tooth stem cells were obtained. The growth and proliferation of primary and passage cells were observed, the cell clone formation rate was determined by HE staining, and the morphological characteristics of primary and passage cells were observed. The surface markers of Sheds were detected by flow cytometry and immunofluorescence staining. The third generation of SHEDs was induced to differentiate into adipose tissue in vitro with IBM X, insulin, indomethacin and hydrocortisone conditioned medium. The morphological changes of cells and lipid droplets in the cells were observed after 4 weeks of oil red-O staining. The third generation of Sheds was induced in osteogenic conditioned medium containing 10% fetal bovine serum of 1 脳 10-8mol/1 dexamethasone 50 mg/l 尾 -glycerophosphate and 10mmol/1 vitamin C for 3 weeks. The changes of cell morphology and growth pattern were observed. Alizarin red staining was performed on day 0 and day 21 of osteogenesis to detect the formation of mineralized nodules and to identify the biological characteristics of osteogenic differentiation. The dynamic expression of Runx2 was detected by RT-PCR on the 18th and 21st days after osteogenesis induction. Results: 1. Human deciduous tooth stem cells were obtained by enzyme digestion and finite dilution method. The cells cultured in primary culture generally showed adherent growth under inverted microscope 2 days after inoculation. The morphology of the cells showed pleomorphism. With the growth and passage, the cells appeared polygonal, fusiform, fibroblast-like, and the size of the cells tended to be the same. He staining showed that most of the cells were fusiform, small in volume, clear in outline, round or oval in nucleus and larger in volume. Several nucleoli can be seen in a few cells. 2. The cell clone formation rate was calculated to be 22-26 / 103 cells. Flow cytometry and immunofluorescence staining showed that CD146 accounted for 92.51 卤1.34% of the total cell count. STRO-1 accounted for 20.01 卤1.62% of the total cell count. High intensity refraction points were observed in the cytoplasm of individual cells under the action of lipid induction system, and orange red positive granules were observed by oil red-O staining. Under the action of the mineralizing inducer, the cells become polygonal and form calcified nodules. The dynamic expression of Runx2 was detected by RT-PCR during osteogenic induction of Sheds. The results showed that the expression of Runx2 was significantly increased at 0 day and increased at 6 days. The expression of Runx2 increased again on day 1218 after 612 days, and the expression of Runx2 increased again at 1218 days after ossification induced by SHEDs. The future is relatively stable. Conclusion: 1. Sheds can be isolated, cultured and expressed the surface markers of mesenchymal stem cells in vitro. 2. Runx2 was expressed dynamically in the process of osteogenic differentiation of Sheds. The expression of Runx2 was found in all stages of osteogenic differentiation. The expression of Runx2 increased in early and late stages, and decreased in middle stage.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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