- I - 摘要 目的 本课题旨在分析细胞在氧化石墨烯(graphene oxide，GO)支架上附着、生长 和钙化能力，探索 GO 支架的生物相容性及其作为新型牙再生支架的可行性。 方法 1 采用改良组织块法培养大鼠牙髓干细胞(dental pulp stem cells，DPSCs)并 采用 CD34、CD44 和 STRO-1 抗体进行免疫荧光染色鉴定。采用酶消化法培养胎 鼠下颌突的牙源性上皮细胞(dental epithelial cells，DECs)并采用 CK14 抗体进行免 疫荧光染色鉴定。2 采用 Hummers 法和梯度冷冻干燥法制备三维 GO 支架，通过 扫描电镜观察支架表面形态，拉曼光谱仪进行材料成分测定。3 将 DPSCs 分别接 种到α-MEM 培养基(对照组)和含有不同浓度 GO 分散液的培养基(实验组)内，24 小时后通过 MTT 法检测各组吸光度，计算出细胞成活率，以分析 GO 是否有细胞 毒性。4 将 GO 支架放入有 DPSCs 细胞的培养基培养 3 天，用免疫荧光染色法检 测 tubulin-β，以观察细胞在支架上的附着情况。5 按照分层接种法将 DPSCs 和 DECs 按照 1:1 的比例接种到 GO 支架(DPCSs/DECs/GO 组)和明胶海绵支架 (DPCSs/DECs/GS 组)上，在体外构建上皮—间充质干细胞立体培养模型并移植到 大鼠大网膜内。4 周后取材，行组织学观察和牙本质涎蛋白(Dentin sialoprotein， DSP)的免疫组化染色，检测移植物内组织的生成状况。采用扫面电镜观察移植物 内组织形态，能谱仪测定移植物内钙磷元素含量，X 线衍射仪测定移植物组织结 构，与正常牙齿进行对比，检测矿化程度。动物体内实验是为了活体分析细胞在 GO 支架上的分化能力。 结果 1 培养所得牙髓细胞不表达造血干细胞表面标记物 CD34，阳性表达间充质 干细胞表面标记物 CD44 和 STRO-1，证实该细胞为 DPSCs。培养所得下颌突细胞 阳性表达上皮细胞标记物 CK14，证实该细胞为 DECs。2 GO 支架表征学测定结果 表明，该支架孔径为 50-100μm，孔隙率高达 95%，表面有皱褶结构；拉曼光谱结 果显示，D 峰出现在 1355cm-1，G 峰出现在 1582cm-1，符合 GO 的特点。3 MTT 实验结果显示，GO 分散液组细胞活性与α-MEM 组无显著性差异(P>0.05)，说明 GO 无细胞毒性。4 免疫荧光结果显示 DPSCs 细胞在 GO 支架上生长良好，能够 很好地黏附和伸展。说明 GO 具有良好的生物相容性。5 动物体内实验结果：HE 染色显示每组支架均有新生组织长入，细胞生长良好，DPCSs/DECs/GO 组可见均 质红染的钙化物质形成；免疫组化染色可见 DPCSs/DECs/GS 组 DSP 阴性表达，个 别地方呈弱阳性。而 DPCSs/DECs/GO 组 DSP 呈强阳性；扫描电镜图像可见华北理工大学硕士学位报告 - II - DPCSs/DECs/GO 组内有胶原和血管形成，细胞生长良好。能谱仪检测结果显示移 植物内有少量钙磷物质表达，X 线衍射仪结果可见移植物尚未达到正常牙齿的矿化 水平，说明矿化程度较低。动物实验结果表明与 GS 相比 GO 支架更有助于 DPSCs 分化。 结论 1 采用改良组织块法能够获得大鼠 DPSCs；采用酶消化法可以培养出胎鼠 DECs。2 GO 支架具有良好的生物相容性，有利于 DPSCs 的生长与附着。3 GO 作 为牙再生新型支架，对 DPSCs 体内牙向分化具有一定的促进作用。 图 14 幅；表 1 个；参 110 篇。 关键词：氧化石墨烯；牙再生；牙髓干细胞；细胞分化 分类号：R781- III - Abstract Objectives The purpose of this study was to analyze the cell abilities of adhesion, growth and calcification on the graphene oxide (GO) scaffold, and to explore the biocompatibility and feasibility as a new scaffold for tooth regeneration. Methods 1 Rat dental pulp stem cells (DPSCs) were cultured by modified tissue block method and were identified by immunofluorescence staining with CD34, CD44 and STRO-1 antibodies. The rat dental epithelial cells (DECs) which was obtained from mandibular process of fetal rats were cultured by enzyme digestion method and were identified by immunofluorescence staining with CK14 antibiody. 2 GO scaffold was prepared by Hummers method and gradient freeze-drying method. The surface morphology of the scaffold was observed by scanning electron microscope and the composition of the scaffold was determined by Raman spectrometer. 3 DPSCs were inoculated respectively into α-MEM medium (control group) and medium containing GO dispersion at diffewas detected by MTT method after 24 hours, and then the cell viability was calculated to rent concentrations (experimental group). The absorbance of each group evaluate the cytotoxicity of GO. 4 The DPSCs were co-cultured with GO scaffold for 3 days, and the immunofluorescence staining was used to detect the tubulin-β on GO scaffold in order to evaluting the adhesion staus of DPSCs on it. 5 The DECs and DPSCs were taken as seed cells, and implanted on GO scaffolds (DPCSs/DECs/GO group) and gelatin sponge scaffolds (DPCSs/DECs/GS group) at 1:1 ratio according to the stratified inoculation method. Then the three-dimensional culture models were transplanted into omentum majus of rats. After 4 weeks, the graft tissues were detected by histological observation and immunohistochemical staining. The content of calcium and phosphorus in the graft was measured by energy disperse pectroscopy, the tissue structure of the graft was determined by X-ray diffraction, which were used to detect the degree of mineralization. The animal studies was used to detect the differentiation ability of cells on GO scaffold in vivo. Results 1 The cultured dental pulp cells were negative for CD34, and positive for CD44 and STRO-1, which conformed that these cells were DPSCs. The cultured mandibular process cells were positive for CK14, which conformed that these cells were DECs. 2 The results of characterization of GO scaffolds showed that the pore size was 50-100 μm, the porosity was as high as 95%, and there were many wrinkle structures on the surface of the scaffold. Raman spectra showed that D peak appeared at 1355 cm-1 and G peak appeared at 1582 cm-1, which was consistent with the identification of graphene oxide. 3 MTT results showed that the cell viability had no significant difference between the experimental group and the control group (P>0.05). This confirmed that GO has no toxicity of DPSCs proliferation. 4 The results of immunofluorescence showed the GO scaffold wad positive for tubulin-β, which indicating that the cells grew well on GO scaffolds and had good cell adhesion, indicating that GO had good biocompatibility. 5 HE staining showed that new tissue grew into the scaffolds in every group, and the cells grew well. Immunohistochemical staining showed that the dentin sialprotein (DSP) was positive in DPCSs/DECs/GO group, negative in DPCSs/DECs/GS group. Scanning electron microscopy showed that there are collagen and angiogenesis in DPCSs/DECs/GO group.The content of calcium and phosphorus in the graft was significantly lower than that in the normal teeth, X-ray diffraction showed that the implant had not reached the 华北理工大学硕士学位报告华北理工大学硕士学位报告 - IV - mineralized level of normal teeth. These indicated that the GO scaffold is more conducive to DPSCs differentiation than GS. Conclusions 1 Rat DPSCs could be obtained by modified tissue block method. Fetal rat DECs could be cultured by enzyme digestion. 2 The GO scaffold has good biocompatibility, which is beneficial to the growth and attachment of cells. 3 GO, as a new scaffold for tissue engineering tooth regeneration, can promote tooth differentiation of DPSCs. Figure14; Table1; Reference 110 Keywords: graphene oxide, tooth regeneration, dental pulp stem cells, cell differentiation Chinese books catalog: R781目 次 - V - 目 次 引 言........................................................................................................................1 第 1 章 大鼠牙髓干细胞和牙源性上皮细胞的培养和鉴定......................................3 1.1 试剂与仪器......................................................................................................3 1.1.1 实验动物................................................................................................3 1.1.2 主要试剂................................................................................................3 1.1.3 主要设备................................................................................................4 1.2 实验方法..........................................................................................................4 1.2.1 大鼠 DPSCs 的培养和鉴定.................................................................. 4 1.2.2 胎鼠 DECs 的培养和鉴定.................................................................... 7 1.3 结果............................................................