發(fā)布時間：2018-11-20 06:33

【摘要】：自體牙移植術(shù)是一種將自體牙從原位置移植到受區(qū)牙槽窩，用來治療牙列缺損的一種外科治療方法。有學(xué)者報道牙移植術(shù)的手術(shù)成功率超過了90%，提示該技術(shù)作為治療牙列缺損的外科手段之一，具有廣闊的前景和應(yīng)用價值。 雖然牙移植術(shù)有著較高的手術(shù)成功率，仍有許多因素影響移植牙的存活時間，如移植牙的離體時間、移植牙牙根與受區(qū)牙槽骨的外形、患者年齡等。所有因素都可以歸結(jié)為牙周組織的狀況和愈合程度μ。因此，改善牙周組織的狀況，提高其愈合能力是提高牙移植手術(shù)成功率的主要方法之一。 牙周膜干細(xì)胞是存在于牙周組織中具有多向分化潛能、在牙周組織損傷修復(fù)中起重要作用的一種成體干細(xì)胞。通過調(diào)節(jié)牙周膜干細(xì)胞的細(xì)胞活性，可以增強(qiáng)牙移植術(shù)后牙周組織的修復(fù)能力，提高牙移植術(shù)的手術(shù)成功率。然而牙周膜干細(xì)胞的細(xì)胞來源是一個主要問題，需要提前安排手術(shù)取材，才能得到個體化的牙周膜干細(xì)胞。骨髓間充質(zhì)干細(xì)胞和牙周膜干細(xì)胞類似，也是一種來源于中胚層的成體干細(xì)胞，具有多向分化的能力，可以用作牙周組織損傷修復(fù)的種子細(xì)胞，提高牙移植術(shù)的手術(shù)成功率。但是骨髓間充質(zhì)干細(xì)胞的取材也是有創(chuàng)μ的，并不能適用于所有病例。臍帶間充質(zhì)干細(xì)胞μ或許是最佳選擇。來源于臍帶組織的臍帶間充質(zhì)干細(xì)胞具有細(xì)胞來源豐富、無創(chuàng)取材、易于獲取、無論理學(xué)限制、低免疫原性等特性，很適合作為一種新型種子細(xì)胞，，用于牙周組織損傷的修復(fù)，提高牙移植術(shù)的手術(shù)成功率。 本研究擬通過體外實驗，研究并細(xì)化了臍帶間充質(zhì)干細(xì)胞的成骨特性；通過體內(nèi)實驗，模擬牙周組織愈合情況，研究臍帶間充質(zhì)干細(xì)胞是否適合用于提高牙移植術(shù)的手術(shù)成功率。 實驗一人臍帶間充質(zhì)干細(xì)胞的原代培養(yǎng)及鑒定 目的：通過原代培養(yǎng)得到本研究所需的臍帶間充質(zhì)干細(xì)胞，并對細(xì)胞性質(zhì)進(jìn)行鑒定。方法：通過酶消化法進(jìn)行臍帶間充質(zhì)干細(xì)胞的原代培養(yǎng)；通過流式細(xì)胞技術(shù)對獲得細(xì)胞的表面CD分子進(jìn)行鑒定，包括CD29、CD146、CD105、CD34、CD44、CD45。結(jié)果：通過酶消化法可以獲得大量的人臍帶間充質(zhì)干細(xì)胞，培養(yǎng)方法簡單易行，結(jié)果穩(wěn)定；流式鑒定結(jié)果顯示人臍帶間充質(zhì)干細(xì)胞與其他間充質(zhì)干細(xì)胞類似，表達(dá)干細(xì)胞表面標(biāo)記物CD29（99.8%）、CD44（99.9%）、CD14（692.8）、CD105（98.9%），但不表達(dá)造血譜系標(biāo)記物CD34(1.52%)、CD45（1.76%）。結(jié)論：通過酶消化法可以順利的得到人臍帶間充質(zhì)干細(xì)胞，該細(xì)胞高表達(dá)間充質(zhì)干細(xì)胞表面標(biāo)記物，可以用于后期的實驗研究。 實驗二人臍帶間充質(zhì)干細(xì)胞成骨分化的體外實驗研究 目的：研究人臍帶間充質(zhì)干細(xì)胞的成骨分化及其在成骨誘導(dǎo)過程中干細(xì)胞和成骨相關(guān)標(biāo)記物的變化情況。方法：通過茜素紅染色、Van Kossa染色和細(xì)胞免疫熒光技術(shù)對成骨誘導(dǎo)后的細(xì)胞進(jìn)行鑒定；在成骨誘導(dǎo)過程中設(shè)立時間點（0d、7d、14d、21d），通過流式細(xì)胞技術(shù)對干細(xì)胞標(biāo)記物CD146和SOX2進(jìn)行檢測；對堿性磷酸酶、CD146/SOX2（mRNA水平）、骨橋蛋白/骨涎蛋白/Runx2（mRNA水平和蛋白水平）進(jìn)行檢測。結(jié)果：對細(xì)胞進(jìn)行成骨誘導(dǎo)21天后茜素紅、Van Kossa染色陽性，免疫熒光染色檢測到胞漿內(nèi)大量表達(dá)骨橋蛋白和骨涎蛋白；干細(xì)胞標(biāo)記物CD146和SOX2在誘導(dǎo)過程中呈下降趨勢；堿性磷酸酶活性在第7天時即表現(xiàn)出具有統(tǒng)計學(xué)差異的升高，骨橋蛋白/骨涎蛋白（mRNA水平和蛋白水平）在成骨誘導(dǎo)過程中呈上升趨勢，7天或14天既有顯著性差異；Runx2（蛋白水平）在第7天時達(dá)到峰值，隨后下降。結(jié)論：人臍帶間充質(zhì)干細(xì)胞的成骨分化過程是一個連續(xù)的過程；體外誘導(dǎo)7d后成骨分化進(jìn)行已經(jīng)全面啟動；經(jīng)過誘導(dǎo)的人臍帶間充質(zhì)干細(xì)胞比未誘導(dǎo)的細(xì)胞更適合用于骨缺損修復(fù)。 實驗三應(yīng)用人臍帶間充質(zhì)干細(xì)胞模擬牙移植術(shù)后牙周組織再生的體內(nèi)實驗研究 目的：通過體內(nèi)實驗?zāi)M牙移植術(shù)后牙周組織的愈合情況。方法：將7d預(yù)誘導(dǎo)的干細(xì)胞做為實驗組μ，無誘導(dǎo)的干細(xì)胞為對照組μ。牙本質(zhì)、生物蛋白膠和人臍帶間充質(zhì)干細(xì)胞混合制作成一種植入復(fù)合體μ，通過植入免疫缺陷小鼠的皮下，8周后取材，觀察其生長情況。結(jié)果：實驗組在牙本質(zhì)表面形成了一層細(xì)胞聚集區(qū)，免疫組織化學(xué)染色顯示OPN呈強(qiáng)陽性；外側(cè)為與牙本質(zhì)垂直向平行排列的纖維樣細(xì)胞層，胞核被拉長；Masson三色染色顯示該新生層富含膠原纖維或類骨樣結(jié)構(gòu)。而在對照組并未觀察到該現(xiàn)象的存在。結(jié)論：經(jīng)過預(yù)誘導(dǎo)7d的人臍帶間充質(zhì)干細(xì)胞更適合用于牙周損傷的修復(fù)，可以用來提高牙移植術(shù)的手術(shù)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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R782.12

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