应用人脐带间充质干细胞修复牙移植术后牙周组织损伤的实验研究

发布时间：2018-11-20 06:33

【摘要】：自体牙移植术是一种将自体牙从原位置移植到受区牙槽窝，用来治疗牙列缺损的一种外科治疗方法。有学者报道牙移植术的手术成功率超过了90%，提示该技术作为治疗牙列缺损的外科手段之一，具有广阔的前景和应用价值。虽然牙移植术有着较高的手术成功率，仍有许多因素影响移植牙的存活时间，如移植牙的离体时间、移植牙牙根与受区牙槽骨的外形、患者年龄等。所有因素都可以归结为牙周组织的状况和愈合程度μ。因此，改善牙周组织的状况，提高其愈合能力是提高牙移植手术成功率的主要方法之一。牙周膜干细胞是存在于牙周组织中具有多向分化潜能、在牙周组织损伤修复中起重要作用的一种成体干细胞。通过调节牙周膜干细胞的细胞活性，可以增强牙移植术后牙周组织的修复能力，提高牙移植术的手术成功率。然而牙周膜干细胞的细胞来源是一个主要问题，需要提前安排手术取材，才能得到个体化的牙周膜干细胞。骨髓间充质干细胞和牙周膜干细胞类似，也是一种来源于中胚层的成体干细胞，具有多向分化的能力，可以用作牙周组织损伤修复的种子细胞，提高牙移植术的手术成功率。但是骨髓间充质干细胞的取材也是有创μ的，并不能适用于所有病例。脐带间充质干细胞μ或许是最佳选择。来源于脐带组织的脐带间充质干细胞具有细胞来源丰富、无创取材、易于获取、无论理学限制、低免疫原性等特性，很适合作为一种新型种子细胞，，用于牙周组织损伤的修复，提高牙移植术的手术成功率。本研究拟通过体外实验，研究并细化了脐带间充质干细胞的成骨特性；通过体内实验，模拟牙周组织愈合情况，研究脐带间充质干细胞是否适合用于提高牙移植术的手术成功率。实验一人脐带间充质干细胞的原代培养及鉴定目的：通过原代培养得到本研究所需的脐带间充质干细胞，并对细胞性质进行鉴定。方法：通过酶消化法进行脐带间充质干细胞的原代培养；通过流式细胞技术对获得细胞的表面CD分子进行鉴定，包括CD29、CD146、CD105、CD34、CD44、CD45。结果：通过酶消化法可以获得大量的人脐带间充质干细胞，培养方法简单易行，结果稳定；流式鉴定结果显示人脐带间充质干细胞与其他间充质干细胞类似，表达干细胞表面标记物CD29（99.8%）、CD44（99.9%）、CD14（692.8）、CD105（98.9%），但不表达造血谱系标记物CD34(1.52%)、CD45（1.76%）。结论：通过酶消化法可以顺利的得到人脐带间充质干细胞，该细胞高表达间充质干细胞表面标记物，可以用于后期的实验研究。实验二人脐带间充质干细胞成骨分化的体外实验研究目的：研究人脐带间充质干细胞的成骨分化及其在成骨诱导过程中干细胞和成骨相关标记物的变化情况。方法：通过茜素红染色、Van Kossa染色和细胞免疫荧光技术对成骨诱导后的细胞进行鉴定；在成骨诱导过程中设立时间点（0d、7d、14d、21d），通过流式细胞技术对干细胞标记物CD146和SOX2进行检测；对碱性磷酸酶、CD146/SOX2（mRNA水平）、骨桥蛋白/骨涎蛋白/Runx2（mRNA水平和蛋白水平）进行检测。结果：对细胞进行成骨诱导21天后茜素红、Van Kossa染色阳性，免疫荧光染色检测到胞浆内大量表达骨桥蛋白和骨涎蛋白；干细胞标记物CD146和SOX2在诱导过程中呈下降趋势；碱性磷酸酶活性在第7天时即表现出具有统计学差异的升高，骨桥蛋白/骨涎蛋白（mRNA水平和蛋白水平）在成骨诱导过程中呈上升趋势，7天或14天既有显著性差异；Runx2（蛋白水平）在第7天时达到峰值，随后下降。结论：人脐带间充质干细胞的成骨分化过程是一个连续的过程；体外诱导7d后成骨分化进行已经全面启动；经过诱导的人脐带间充质干细胞比未诱导的细胞更适合用于骨缺损修复。实验三应用人脐带间充质干细胞模拟牙移植术后牙周组织再生的体内实验研究目的：通过体内实验模拟牙移植术后牙周组织的愈合情况。方法：将7d预诱导的干细胞做为实验组μ，无诱导的干细胞为对照组μ。牙本质、生物蛋白胶和人脐带间充质干细胞混合制作成一种植入复合体μ，通过植入免疫缺陷小鼠的皮下，8周后取材，观察其生长情况。结果：实验组在牙本质表面形成了一层细胞聚集区，免疫组织化学染色显示OPN呈强阳性；外侧为与牙本质垂直向平行排列的纤维样细胞层，胞核被拉长；Masson三色染色显示该新生层富含胶原纤维或类骨样结构。而在对照组并未观察到该现象的存在。结论：经过预诱导7d的人脐带间充质干细胞更适合用于牙周损伤的修复，可以用来提高牙移植术的手术成功率。
[Abstract]:Autotooth transplantation is a kind of surgical treatment for the treatment of dental defect. Some scholars have reported that the success rate of the operation of the tooth transplantation exceeds 90%, and it is suggested that the technique is one of the surgical tools for the treatment of dental defect, and has a wide application value. Although the implant has a high success rate of operation, there are still many factors that affect the survival time of the implant, such as the time of the implant, the shape of the tooth root and the alveolar bone, the patient's year Age, etc. All the factors can be attributed to the condition of the periodontal tissue and the healing range. Therefore, improving the condition of the periodontal tissue and improving the healing ability of the periodontal tissue is the main method for improving the success rate of the dental implant. one of the most important factors in the healing of the periodontal tissue. By regulating the cell activity of the periodontal ligament stem cells, the repair capacity of the periodontal tissues after the tooth transplantation can be enhanced, and the tooth transplantation can be improved. However, the cell origin of the periodontal ligament stem cells is a major problem, and it is necessary to arrange the operation materials in advance so that the individual teeth can be obtained. The peripheral membrane stem cells are similar to the mesenchymal stem cells and the periodontal ligament stem cells, but also a body stem cell derived from the mesoderm, having the ability to differentiate into multiple directions, and can be used as a seed cell for periodontal tissue injury repair, and the tooth transplantation is improved. The success rate of the operation is the same, but the material of the mesenchymal stem cells in the bone marrow is also invasive and can't be used. in all case. that mesenchymal stem cells between the umbilical cord may The umbilical cord mesenchymal stem cell derived from the umbilical cord tissue has the characteristics of abundant cell source, no invasive material acquisition, easy acquisition, no matter the limitation of the science and the like, low immunogenicity and the like, and is very suitable as a novel seed cell for repairing the periodontal tissue injury and improving the tooth transplantation. The results of this study are to study and refine the bone-forming characteristics of the mesenchymal stem cells between the umbilicals by in-vitro experiments. By the in-vivo experiment, the healing of the periodontal tissue is simulated, and whether the mesenchymal stem cells of the umbilical cord are suitable for the purpose of improving the tooth movement The success rate of the operation of implantation. Primary culture and identification of the quality stem cells: the umbilical cord between the umbilical cords required for this study was obtained by primary culture The method comprises the following steps of: performing primary culture of the mesenchymal stem cells of the umbilical cord through an enzyme digestion method; and identifying the surface CD molecules of the obtained cells by flow cytometry, including the CD29, the CD146 and the CD105, Results: A large number of human umbilical cord mesenchymal stem cells can be obtained by the enzyme digestion method, and the method is simple and feasible, and the result is stable; the flow identification results show that the human umbilical cord mesenchymal stem cells are similar to the other mesenchymal stem cells, and the surface marker CD29 (92.8%) and the CD44 (99.9) of the stem cell surface marker are expressed.%), CD14 (692.8), CD105 (98.9%), but not hematopoietic lineage marker CD34 (1.52 Conclusion: The human umbilical cord mesenchymal stem cells can be successfully obtained by the enzyme digestion method, and the high-expression mesenchymal stem cell surface marker of the cell can be obtained. It can be used in later experimental studies. In vitro experimental study of the differentiation of mesenchymal stem cells into bone: the study of the osteogenic differentiation of the mesenchymal stem cells between human umbilical cord and the induction of osteogenesis Changes of stem cells and bone-related markers in the lead-in process. Methods: Bone-induced cells were identified by fluorescein-red staining, Van Kossa staining and cellular immunofluorescent techniques; time points (0d, 7d, 14d, 21d) were set up in the bone-inducing process, and by flow cytometry Stem cell markers CD146 and SOX2 were tested; alkaline phosphatase, CD146/ SOX2 (mRNA level), osteopontin/ sialoprotein/ Ru Nx2 (mRNA level and protein level) were tested. The results showed that after 21 days of bone induction, the cells were stained with fluorescein red and Van Kossa. Immunofluorescence staining was used to detect the expression of osteopontin and sialoprotein in the cytoplasm. The marker of stem cell C D146 and SOX2 showed a decreasing trend during induction; the activity of alkaline phosphatase showed a statistically significant increase on day 7, and the osteopontin/ sialoprotein (mRNA level and protein level) increased in the process of bone induction, and there was a significant difference between 7 and 14 days. Runx2 (protein level) reached the peak at day 7 and then decreased. Conclusion: The process of bone differentiation of the mesenchymal stem cells between human umbilical cord is a continuous process, and the differentiation of bone in vitro after 7days in vitro has been fully initiated, and the induced human umbilical cord charge Stem cells are more suitable for bone defect repair than uninduced cells. In vivo experimental study of the regeneration of the periodontal tissues after the artificial tooth-grafting of the stem cells Objective: To study the healing of the periodontal tissues after the tooth transplantation in vivo. the pre-induced stem cells were made into the experimental group, and the non-induced stem cells were in the control group. The results showed that in the experimental group, a layer of cell accumulation area was formed on the surface of the dentine, and the immunohistochemical staining showed that the OPN was positive. a fiber-like cell layer that is arranged in parallel with the dentin, and the nucleus is elongated; Ma sson three-color staining showed that the nascent layer was rich in gum The presence of this phenomenon was not observed in the control group.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R782.12

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