中药双黄补对体外培养人牙周膜细胞增殖活性的影响

背景：目前的研究大多为单味中药或单一中药成分对体外培养人牙周膜细胞的影响，而中药组方提取液对体外培养细胞影响的研究较少。 目的：观察中药双黄补对体外培养的人牙周膜细胞增殖活性的影响。 设计、时间及地点：对比观察实验，于2008-11/2009-02 在河北医科大学第四医院科研中心完成。 材料：人牙周膜细胞组织选自河北医科大学第四医院口腔科要求手术拔除的埋伏多生牙者。黄连、黄芩、骨碎补均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周膜细胞。水提醇沉法制备双黄补提取液。以每 1 mL药液含生药 1 g加入体积分数20%的胎牛血清培养液，稀释成质量浓度为10，25，50，100，150，200，250，500，750，1 000 mg/L，分别作用于体外培养的人牙周膜细胞，以不加双黄补提取液的培养细胞为对照。 主要观察指标：采用四甲基偶氮唑蓝法测定培养细胞增殖活性的变化，应用流式细胞术检测100 mg/L的双黄补提取液对牙周膜细胞周期的变化和对成纤维细胞生长因子18水平的影响。 结果：双黄补对人牙周膜细胞的增殖活性的影响存在质量浓度和时间效应，各质量浓度双黄补均能促进体外培养人牙周膜细胞的增殖活性，其中以100 mg/L质量浓度促增殖活性作用最明显(P < 0.01)，相同质量浓度不同作用时间对细胞促增殖活性作用也不同，以48 h作用最明显(P < 0.05)。与对照组相比，100 mg/L的双黄补促进牙周膜细胞成纤维细胞生长因子18水平增高，S期和G2M期细胞数目增多，在48 h最明显。 结论：中药双黄补可增强人牙周膜细胞的增殖活性，具有明显的浓度效应和时间效应。

Effects of Shuanghuangbu on proliferation activity of human periodontal ligament cells cultured in vitro

BACKGROUND: It is rather less to investigate the effects of complex Chinese herbal medicine extract on human periodontal ligament cells cultured in vitro in existing study, nevertheless, most studies focus on the effects of alone Chinese herbal medicine or component on the cells. OBJECTIVE: To investigate the effects of Shuanghuangbu on proliferation activity of human periodontal ligament cells cultured in vitro. DESIGN, TIME AND SETTING: A controlled observation was performed at the Scientific Research Center in the Fourth Hospital of Hebei Medical University between November 2008 and February 2009. MATERIALS: Human periodontal ligament cells were sourced from the patient who acquired therapy through pulling out ambush accessory tooth at the Department of Stomatology in the Fourth Hospital of Hebei Medical University. Coptidis rhizome, scutellaria and rhizoma drynariae were provided by the Dispensary of Traditional Chinese Medicine in the Fourth Hospital of Hebei Medical University. METHODS: Human periodontal ligament cells were cultured in vitro using method of tissue piece. Shuanghuangbu extract was prepared with water extraction and alcohol precipitation method, 1 mL herb liquor contained 1 g crude drug and added with 20% fetal bovine serum, then diluted into decoctions of 10, 25, 50, 100, 150, 200, 250, 500, 750, 1 000 mg/L, given to the experiment group. Cells cultured with only culture fluid without Shuanghuangbu extract served as the control group. MAIN OUTCOME MEASURES: Proliferation activity of human periodontal ligament cells cultured in vitro was determined by MTT. The effect of 100 mg/L Shuanghuangbu extract on the content of fibroblast growth factor 18 and the cycle of human periodontal ligament cells cultured in vitro was detected by flow cytometry. RESULTS: Shuanghuangbu could reinforce proliferation activity of human periodontal ligament cells cultured in vitro and showed evident effectiveness of concentration and time, especially at the 100 mg/L (P < 0.01). The proliferation promotion on cultured cells was different on alike medicine concentration and different action time, and result showed best at 48 hours (P < 0.05). Compared with control group, Shuanghuangbu at the 100 mg/L had the best effect on the promotion of fibroblast growth factor 18 of human periodontal ligament cells, the number of cells at S phase and G2M phase were remarkably increased at 48 hours. CONCLUSION: Shuanghuangbu can reinforce proliferation activity of human periodontal ligament cells cultured in vitro in an obvious manner of concentration- and time-dependence.