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背景:体外培养人牙髓细胞难度很大,国内外已有生长因子对体外培养牙髓细胞增殖分化作用的研究,而中药骨碎补对体外培养牙髓细胞的影响,经作者检索未见报道。目的:观察不同浓度骨碎补提取液对体外培养的人牙髓细胞增殖分化的影响。设计、时间及地点:对比观察实验,于2006-03/2007-05在河北医科大学第四医院科研中心完成。材料:人牙髓细胞组织选自河北医科大学第四医院口腔科要求正畸治疗需拔除的健康阻生智齿着,取得所有供者对标本采集的知情同意。产地云南的骨碎补购自河北医科大学第四医院中药房。方法:采用组织块法体外培养人牙髓细胞。水提醇沉法提取骨碎补有效成分,以每1mL药液含生药1g加入积体分数为0.02的胎牛血清培养液,稀释成质量浓度为10,50,100,500,1000mg/L。分别以此培养液作用于体外培养的人牙髓细胞,以不加骨碎补提取液培养的细胞做对照。主要观察指标:①人牙髓细胞原代培养和来源鉴定。②四甲基偶氮唑盐法检测各质量浓度组骨碎补对人牙髓细胞增殖的作用。③免疫组织化学法观察各浓度组骨碎补对人牙髓细胞纤维连接蛋白的表达。④扫描电镜和透射电镜下观察骨碎补对人牙髓细胞超微结构的影响。结果:原代培养的人牙髓细胞呈梭形或多角形。各浓度骨碎补均对体外培养的人牙髓细胞有促增殖作用,其中以100mg/L浓度组促增殖作用最明显,可诱导牙髓细胞合成、分泌纤维连接蛋白。电镜下实验组人牙髓细胞表面枝状嵴丰富,细胞周围可见细胞外基质,细胞浆内有丰富粗面内质网和游离的核糖体,核内常染色质均匀分散,异染色质少。结论:含100mg/L骨碎补培养液对体外培养的人牙髓细胞有明显的促增殖作用。
BACKGROUND: Culture of human dental pulp cells in vitro is very difficult, and growth factors have been studied for the effect of growth factors on the proliferation and differentiation of cultured dental pulp cells in vitro. However, the effects of Chinese herbal medicine, Rhizoma Drynariae, on the culture of dental pulp cells in vitro have not been reported by the authors. . OBJECTIVE: To observe the effects of different concentrations of Rhizoma Drynariae extract on the proliferation and differentiation of cultured human dental pulp cells. DESIGN, TIME AND SETTING: A comparative observation experiment was performed at the Scientific Research Center of the Fourth Hospital of Hebei Medical University from March 2006 to May 2007. MATERIALS: Human dental pulp cell tissue was selected from the Department of Stomatology, Fourth Hospital of Hebei Medical University, and was required to remove orthodontic wisdom teeth for the treatment of orthodontic treatment. All informed consents for specimen collection were obtained. Rhizome of Yunnan origin was purchased from the pharmacy of the Fourth Hospital of Hebei Medical University. METHODS: Human dental pulp cells were cultured in vitro using a tissue block method. The water extract and alcohol precipitation method was used to extract the effective components of Rhizoma Drynariae. The fetal bovine serum culture fluid with a body mass fraction of 0.02 was added to 1 g crude drug solution per 1 mL drug solution and diluted to a mass concentration of 10, 50, 100, 500, and 1000 mg/L. The human dental pulp cells cultured in vitro were treated with the culture solution, and the cells cultured without the extract of Rhizoma Drynariae as the control. MAIN OUTCOME MEASURES: Primary culture and identification of dental pulp cells. 2 Tetramethylazolazolium salt method was used to detect the effect of Rhizoma Drynariae on the proliferation of human dental pulp cells. 3 Immunohistochemical method was used to observe the expression of fibronectin in human dental pulp cells in each concentration group. 4 Scanning electron microscopy and transmission electron microscopy were used to observe the effects of Rhizoma Drynariae on the ultrastructure of human dental pulp cells. Results: The primary cultured human dental pulp cells were fusiform or polygonal. Each concentration of Rhizoma Drynariae can promote the proliferation of cultured human dental pulp cells. Among them, the concentration of 100 mg/L can promote the proliferation of dental pulp cells, and can induce the synthesis and secretion of fibronectin by dental pulp cells. Under the electron microscope, dendritic cells on the surface of human dental pulp cells were abundant in the experimental group. The extracellular matrix was observed around the cells. There were abundant rough endoplasmic reticulum and free ribosomes in the cytoplasm, and the nuclear chromatin was uniformly dispersed and the heterochromatin was less. CONCLUSION: The 100 mg/L bone sap supplementation medium can significantly promote the proliferation of cultured human dental pulp cells.