【作者】 赵玲；

【导师】 高毅；

【作者基本信息】 河北医科大学 ， 口腔医学（专业学位）， 2015， 硕士

【摘要】 牙周炎是口腔常见疾病之一,其对人体健康的伤害仅次于癌症及心脑血管疾病。除了影响患者的咀嚼功能和颌面部美观,它还是糖尿病、肾病、心血管疾病及新生儿早产的高危因素。牙周炎晚期牙槽骨重度吸收常常导致牙齿缺失,如何防止牙槽骨的吸收,稳定以及重建是治疗牙周炎的关键。中医药注重整体观念,辨证施治。我科临床上应用补肾活血固齿方治疗牙周炎取得了良好的疗效,但其作用机制尚不清楚。碱性磷酸酶(Alkaline Phosphosphatase,ALP)是一种同源二聚体的糖蛋白,参与骨代谢过程,被认为是骨细胞外基质成熟的早期标志。骨组织中含有80%~90%的Ⅰ型胶原蛋白(Collagen TypeⅠ,COLl),它构成骨组织的蛋白框架,对维持骨组织的生物力学特性和结构的完整性发挥着重要作用,是成骨细胞向基质成熟方向分化的标志。矿化结节由胶原沉积后基质矿化形成,标志着成骨细胞进入分化的最后阶段。本实验通过观察补肾活血固齿方对小鼠颅顶前成骨细胞(MC3T3-E1)超微结构及分化的影响,研究该方剂成骨的作用机制,为临床应用该药治疗牙周炎提供理论依据。目的:连续灌胃SD大鼠补肾活血固齿方一周,提取大鼠的血液制备含药血清,用以体外培养MC3T3-E1细胞,观察该方剂对MC3T3-E1细胞的超微结构、ALP活性,COLⅠ表达及矿化结节形成的影响,探讨补肾活血固齿方在骨形成中的作用机制。方法:1 MC3T3-E1细胞株的培养。2依照体表面积折算成等效剂量的补肾活血固齿方灌胃大鼠,制备含药血清;同等剂量的蒸馏水灌胃大鼠,制备无药血清作为对照组,10%胎牛血清作为空白对照组。3透射电镜观察补肾活血固齿方对MC3T3-E1细胞超微结构的影响。用浓度为10%的含药血清、无药血清、胎牛血清分别培养MC3T3-E1细胞,培养第1d、4d、7d、14d时分别制作标本于透射电镜下对比观察MC3T3-E1细胞的超微结构。4检测补肾活血固齿方对MC3T3-E1细胞ALP活性的影响。调节MC3T3-E1细胞浓度为1×105/ml接种于96孔板中,每孔130ul。10%含药血清、无药血清、胎牛血清分别培养细胞,检测第1d、4d、7d、14d时细胞上清液中ALP活性。5免疫细胞染色法检测补肾活血固齿方作用MC3T3-E1细胞72h时,对COLⅠ表达的影响。6观察补肾活血固齿方对MC3T3-E1细胞矿化结节形成数量的影响。用10%含药血清、无药血清、胎牛血清分别连续培养MC3T3-E1细胞14d,利用茜素红染色法观察细胞矿化结节的形成并计数。结果:1补肾活血固齿方对MC3T3-E1细胞超微结构的影响MC3T3-E1细胞经含药血清培养1d、4d、7d、14d时观察发现,随着时间的延长MC3T3-E1细胞细胞质中细胞器逐渐丰富;14d时MC3T3-E1细胞呈成骨细胞样表型,细胞表面突起较多,核呈卵圆形,较大,核膜曲折凹陷;胞质丰富,胞浆中糖元明显可见;粗面内质网扩张明显,网腔内充满低电子密度絮状物;线粒体嵴的密度较大,细胞合成功能旺盛。无药血清组、胎牛血清组细胞的细胞器与含药血清组细胞相比较不丰富,14d时MC3T3-E1细胞表面突起较少。2补肾活血固齿方对MC3T3-E1细胞培养上清液ALP活性的影响3组细胞培养上清液中ALP活性随着培养时间的延长而逐渐提高。无药血清与胎牛血清两组间ALP活性无统计学差异(P>0.05);含药血清组上清液ALP活性强于无药血清组、胎牛血清组,有统计学差异(P<0.05)。3补肾活血固齿方含药血清作用于MC3T3-E1细胞72h对细胞COLⅠ表达的影响镜下见含药血清组细胞的细胞质呈深棕黄色染色,无药血清组细胞的细胞质呈浅棕黄色染色。细胞质染色强度分析采用χ2检验,两组染色强度差别有统计学意义(P<0.05)。4补肾活血固齿方对MC3T3-E1细胞矿化结节形成数量的影响3组细胞在14d时均形成矿化结节。无药血清与胎牛血清两组间矿化结节数量差别无统计学意义(P>0.05);含药血清组与无药血清组、胎牛血清组相比,其矿化结节数量显著增多,差别有统计学意义(P<0.05)。结论:补肾活血固齿方可以促进MC3T3-E1细胞向成骨细胞样表型发展,增加MC3T3-E1细胞ALP活性及COLⅠ和矿化结节的形成,促进MC3T3-E1细胞分化成熟,促进骨组织的形成。补肾活血固齿方可通过促进牙槽骨的再生而对牙周炎的治疗发挥作用。更多还原

【Abstract】 Periodontitis is one of the most common oral diseases, second only to the damage of human’s health caused by cancer and cardiovascular. Other than its effect on the chewing function and maxillofacial appearance, Periodontitis can also bring danger thus triggering diabetes,kidney disease,cardiovascular disease, and premature newborn. The severe absorption of alveolar bone in the late stage of periodontitis often leads to tooth loss, therefore the key to cure periodontal disease is to prevent alveolar absorption, meanwhile, stabilize and rebuild the alveolar. Chinese medicine pays more attention to holism and dialectical therapy. Recently Stomatological Department of Hebei Chinese Medicine Hospital has obtained the satisfied clinical effect by tonifying kidney, activating blood and strengthening teeth to cure periodontitis. However,the relevant detailed mechanism has not been explored.Alkaline Phosphosphatase(ALP),a homologous dimeric glycoprotein, takes part in the process of bone metabolism, and also is regarded as the early sign of mature bone extracellular matrix.Collagen Type Ⅰ(COL I),of which 80%~90% is contained in bore tissue, builds up the protein frame in bone tissue, and plays an important role in maintaining bone biomechanics characteristic and structural integrity, meanwhile,deemed as the maturation sign of matrix differentiated from osteoblast.Mineralized nodules were formed through matrix mineralization after collagen deposition,marks the last stage of osteoblastic differentiation.In this study,we researched on the mechanism involving in the effect of this prescription on accelerating bone formation by observing the influence of BUSHENHUOXUEGUCHI on ultrastructure and differentiation of MC3T3-E1 cells,providing the theoretical basis for treatment of periodontitis using this prescription.Objective: This study is to explore mechanism of periodontitis treatment using this prescription by performing a series of experiments:feeding the mouse with BSHXGC for a week,the serum containing this prescription obtained from the mouse was added to cultivate MC3T3-E1 cells in vitro. Observing the effect of BUSHENHUOXUEGUCHI on ultrastructure,ALP activity,the expression of COLI and mineralized nodule formation in MC3T3-E1 cells.Thus completing investigate of action mechanism involving in the effect of this prescription on bone formation.Methods:1 Culture MC3T3-E1 cell line in vitro.2 Contained-herb serum was prepared from rats by gavage with BUSHENHUOXUEGUCHI of body surface area converted equivalent dose; obtaining blank-contained serum as control group by rate gavage with normal saline and 10% FBS as blank control group.3 To observe the effects of BUSHENHUOXUEGUCHI on ultramicrostructure of MC3T3-E1 cell.MC3T3-E1 cells were cultured by blank-contained serum, contained-herb serum and blank control group,respectively.The ultrastructure changes in MC3T3-E1 cells were observed by means of electron transmission at 1d、4d、7d and 14 d.4 ALP activity test was carried out to evaluate the effect of BUSHENHUOXUEGUCHI with kites on the ALP expression of MC3T3-E1 cell.MC3T3-E1 cells were seeded on 96-well plates at 1300 cells per well and cultured by blank-contained serum,contained-herb serum and blank control group, respectively. ALP detection kits was employed to detect ALP activity in the clear supernatant liquid at 1d、4d、7d and 14 d.5 MC3T3-E1 cells were cultured by blank-contained serum,contained-herb serum and blank control group,respectively. Immunohistochemical assay was employed to test the protein level of COLⅠat 72 hs.6 The effects of BUSHENHUOXUEGUCHI on the mineralized nodules formation of MC3T3-E1 cells were evaluated by alizarin red staining.MC3T3-E1 cells were cultured by blank-contained serum,contained-herb serum and blank control group, respectively. Alizarin red staining was used to observe and count the formation of mineralized nodules.Results:1 The effects of BUSHENHUOXUEGUCHI on ultramicrostructure of MC3T3-E1 cells.With the culture time going on,the amount of mitochondria,Golgi bodies and endoplasmic reticulum showed a slight increase,but were not well developed in MC3T3-E1 cells cultured with blank-contained serum and blank controlled group, respectively. Compared with the two controlled groups, not only the number of protuberances increased,inducing the osteoblast differentiation of MC3T3-E1 cells, the nucleus was also egg- shaped,in addition, nuclear membrane was tortuous and invaginates, and mitochondria, Golgi bodies and endoplasmic reticulum were abundant meanwhile well developed,besides, intracellular secretory more matrix vesicles were found, and metabolic activity developed in MC3T3-E1 cells cultivated with contained-herb serum.2 The influence of BUSHENHUOXUEGUCHI on the ALP activity in the clear supernatant liquid of MC3T3-E1 cell.The activity of ALP increased gradually with the prolonging cultivation time in all three groups. There was no difference for statistics significance between blank-contained serum and blank controlled groups(P<0.05).While the activity of ALP had stronger activity in MC3T3-E1 cells cultivated by contained-herb serum than that in blank-contained serum and blank controlled groups, and difference had statistical significance(P<0.05).3 The effects of BUSHENHUOXUEGUCHI on the expression of COL I in MC3T3-E1 cell.Checked by microscope after immunohistochemical staining,we discovered that cytoplasm stained with dark brown yellow in MC3T3-E1 cells cultivated with contained-herb serum at 72 hs, while light brown yellow in blank-contained serum group. Difference had statistical significance between two groups analyzed with χ2 test(P<0.05).4 The influence of BUSHENHUOXUEGUCHI on the number of mineralized nodules forming in MC3T3-E1 cells.Mineralized nodules were formed in all three groups after cultivated at 14 d.There was no difference for statistical significance between blank-contained serum and blank control groups(P<0.05).While the average volume of mineralized nodules were enlarged manifestly in MC3T3-E1 cells cultivated in contained-herb serum than that in blank-contained serum and blank controlled groups, and difference had statistical significance(P<0.05).Conclusion: BUSHENHUOXUEGUCHI can accelerate the differentiation of MC3T3-E1 cells into osteoblast in vitro, enhancing the activity of ALP, increasing mineralized nodule formation, and also promoting cells differentiation and maturation of MC3T3-E1, meanwhile bone formation in vitro. This may provide new insights into the treatment of periodontitis by promoting the regeneration of alveolar bone.更多还原