【作者】 张国英；

【导师】 余占海；

【作者基本信息】 兰州大学 ， 口腔临床医学， 2008， 硕士

【摘要】 目的:1、研究转化生长因子β₁（transforming growth factor-β₁,TGF-β₁）对原代培养人牙周膜细胞（human periodontal ligament cells,HPDLCs）生物学活性的影响;2、研究TGF-β₁对大鼠实验性牙周炎模型的牙周组织中IL-6、BGP表达的影响。方法:1、利用组织培养技术,从健康的牙周组织中获取牙周膜细胞,采用MTT法观察细胞增殖情况,考马斯亮蓝法和流式细胞仪技术检测细胞蛋白总含量和细胞周期变化,酶动力学法和RT-PCR检测碱性磷酸酶（alkaline phosphatase,ALP）活性和ALPmRNA表达的变化。2、在成功建立大鼠实验性牙周炎模型的基础上,实验动物注射TGF-β₁后分别于第1、2、3、4周处死（每次每组10只）,取牙周组织标本做切片,行常规HE染色,观察牙周组织病理变化状况,并采用免疫组织化学法检测IL-6、BGP水平的变化。结果:1、牙周膜细胞生长情况原代培养细胞从组织块中游出时间为3～14天,镜下细胞呈长梭形或星形,胞突细长,放射状排列,胞体丰满,胞浆均匀,核圆形或卵圆形者为成纤维细胞。2、TGF-β₁对牙周膜细胞生长增殖的影响2.0μg/L、5.0μg/L、10.0μg/L的TGF-β₁明显促进细胞增殖（P＜0.05）,呈剂量、时间依赖性,且10.0μg/L的TGF-β₁作用细胞72h时促进细胞增殖作用效果最显著,0.5μg/L的TGF-β₁与对照组相比差异无统计学意义。3、TGF-β₁对牙周膜细胞蛋白总含量的影响2.0μg/L、5.0μg/L、10.0μg/L组的TGF-β₁作用48h时细胞蛋白总含量均高于对照组（P＜0.05）,且10.0μg/L的TGF-β₁作用下细胞蛋白总含量增高最显著,0.5μg/L的TGF-β₁与对照组相比差异无统计学意义。4、TGF-β₁对牙周膜细胞ALP活性变化的影响2.0μg/L、5.0μg/L、10.0μg/L的TGF-β₁处理48h,酶动力学检测结果显示细胞ALP活性均高于对照组（P＜0.05）,0.5μg/L的TGF-β₁与对照组相比差异无统计学意义。5、TGF-β₁对牙周膜细胞ALP mRNA表达的影响RT-PCR检测显示经2.0μg/L、5.0μg/L、10.0μg/L的TGF-β₁作用48h后,ALPmRNA的表达明显增高（P＜0.05）,图像分析光密度,与空白对照组比较,分别增高1.34、1.66、1.75倍。6、TGF-β₁对牙周膜细胞周期的影响FCM检测显示经10.0μg/L TGF-β₁作用48h后,G₁期细胞降低（P＜0.05）,S期细胞和细胞增殖指数PrI值（S+G₂/M）%增高（P＜0.05）。7、动物实验中各牙周炎组牙周组织中IL-6表达明显高于正常组（P＜0.05）而BGP呈下降趋势（P＜0.05）;各时间段牙周炎治疗组中IL-6表达明显低于牙周炎组（P＜0.05）,BGP明显高牙周炎组（P＜0.05）,且牙周炎治疗组各时间段之间BGP表达有明显差异（P＜0.05）,而IL-6表达只有部分时间段有明显差异（P＜0.05）。结论:2.0～10.0μg/L的TGF-β₁体外具有促进人牙周膜细胞增殖和诱导分化的作用,其作用可能与TGF-β₁促进细胞DNA、蛋白质合成和ALPmRNA的表达及ALP酶活性增强有关。大鼠实验性牙周炎模型牙周组织中IL-6表达明显高于正常组而BGP表达低于正常组,经TGF-β₁治疗后,实验性牙周炎模型牙周组织中IL-6表达明显低于同期的牙周炎组而BGP含量明显增加。根据上述结果证实TGF-β₁可促进牙周创伤愈合和组织再生,从而为牙周病临床的合理用药提供科学依据。更多还原

【Abstract】 Objective:1.To study the influence of TGF-β₁ on biologic activity of HPDLCs in primary culture.2.To determine the effects of TGF-β₁on the expressions ofIL-6、BGP in periodontal tissue of rat periodontitis model.Methods:1.Human periodontal ligament cells were obtained from healthy periodontium using the explant technique.The effect of TGF-β₁ on the proliferative ability of HPDLCs was evaluated with MTT assay;The effects of TGF-β₁on the total amount of protein and the cell cycle were detected by coomassie brilliant blue（CBB）method and flow cytometry;The effects of TGF-β₁ on the alkaline phosphatase（ALP）activity and expression of ALP mRNA were measured with the p-nitrophenylphosphate（PNPP）and reverse -transcription polymerase chain reaction（RT-PCR）.2.Based on the successful rat periodontitis model,the experimental rats were randomized into different group followed by oral treatment with TGF-β₁ for 1,2,3,4 weeks and then the rats were sacrificed and analyzed.Pathological assay and HIE staining were used to detect the general conditions and pathological changes of rats periodontal tissues.And immunohistochemical staining was conducted to determine the expressions ofIL-6、BGP in rats periodontitis model.Result:1.The growth condition of the human periodontal ligament cellsThe time was 3～14 days for the primarily cultured cells growing out from tissue. Elongated fusiform cells or astrocytes could be seen through inverted microscope.The cell which was sudden slender,radial array,and its body fullness,uniform cytoplasm,and nuclear round or oval was consided as the fibroblast.2.The effect of TGF-β₁ on the proliferation of HPDLCsTreatment with TGF-β₁ in 2.0μg/L、5.0μg/L and 10.0μg/L increased cell proliferation significantly compared with the control group（P＜0.05）.At 3 days 10.0μg/L TGF-β₁ showed a higher cell proliferation compared with the other groups.0.5μg/L TGF-β₁did not positively influnce the cell proliferation in this study.3.The effect of TGF-β₁ on the total amount of protein of HPDLCsCompared with the control group,the total amount of protein of HPDLCs treated with TGF-β₁ in 2.0μg/L、5.0μg/L and 10.0μg/L was increased dramatically(（P＜0.05）.The cells with 10.0μg/L TGF-β₁ treated produced statistically more protein than with other treatments. 4.The effect of TGF-β₁ on the alkaline phosphatase（ALP）activity of HPDLCs The ALP activity of HPDLCs was positively affected by TGF-β₁ in 2.0μg/L、5.0μg/L and 10.0μg/L at 48 hours compared with the control group,and 10.0μg/L TGF-β₁ treated was enhanced more significantly than with other treatments.5.The effect of TGF-β₁on the expression of ALP mRNA of HPDLCsThe expression of ALPmRNA was significantly up-regulated by TGF-β₁ in 2.0μg/L、5.0μg/L and 10.0μg/L at 48 hours(（P＜0.05）.Compared with the control group,optical density respectively increased 1.34、1.66、1.75 times through image analysis.6.The effect of TGF-β₁ on cell cycle of HPDLCsEmploying the FCM technique,we observed the decreased G1%,the increased S%and（S +G2/M）%when the cells were stimulated by TGF-β₁（10.0μg/L）after 48 hours.7.The levels of IL-6 in the periodontitis tissues were significantly higher than that in control group and the level of BGP was lower.Treatment with TGF-β₁ decreased the levels of IL-6 and increased the level of BGP in periodontitis tissue(（P＜0.05）.The general conditions and pathological changes in the control group and groups treated with TGF-β₁ were much better than that in periodontitis group.Conclusion:These findings suggested that TGF-β₁ was capable of producing an accelerative effect on the proliferation and differentiation functions on HPDLFs,It may be related to TGF-β₁ enhancing cell DNA and protein synthesis,ALP mRNA expression and ALP activity.Moreover,it inhibited the expressions of IL-6 and increased the level of BGP in periodontal tissues of rats periodontitis model and promoted the regeneration of the periodontal tissues.In a word,our studies suggested that TGF-β₁ may have potential clinical application to periodontal disease.更多还原