体外培养牙周膜细胞增殖分化与中药双黄补水提液的调控效应

背景:牙周组织的修复再生取决于牙周膜细胞的数量和增殖分化能力。牙周膜细胞具有多向的分化潜能,可分化成成牙骨质细胞、成骨细胞和成纤维细胞,形成牙骨质、牙槽骨和牙周膜,实现牙周组织再生。目的:观察体外培养人牙周膜细胞增殖和分化过程中传统中药双黄补水提液对其功能的影响。设计:观察性实验。单位:无锡市第三人民医院中心实验室。材料:牙周膜组织(由健康青少年患者因矫正畸形门诊就诊时自愿提供);黄连、黄岑、骨碎补(中国药品检定所提供)。方法:实验于2003-07/10在无锡市第三人民医院中心实验室完成。分别将粉碎的黄连、黄岑、骨碎补和蒸馏水1∶10(m∶V)混合,沸水回流5h。收集提取液,粗滤后残渣再次沸水回流3h。合并2次提取液,旋转蒸发浓缩,所得中药水提液浓度为3kg/L。分黄连组、黄芩组、骨碎补组、黄连加黄岑组、黄连加骨碎补组、黄岑加骨碎补组、双黄补组和对照组8组进行实验。通过体外培养人牙周膜细胞,应用双黄补提取液作为辅助因子,采用MTT比色法测定细胞增殖情况,羟脯氨酸法测定胶原蛋白占总蛋白比值。主要观察指标:牙周膜细胞增殖的吸光度值及胶原蛋白占总蛋白的比值。结果:①牙周膜细胞增殖的测定结果:除黄连组外其他各组均明显促进人牙周膜细胞增殖,与对照组比较,差异明显(P<0.05)。双黄补组细胞的吸光度值增大最为明显。随着作用时间的延长,细胞的吸光度值逐渐增大,在第5天达到最大。不同时间各组间比较,差异有显著性意义(P<0.05)。②各组牙周膜细胞胶原蛋白占总蛋白的比值:除黄连组外其他各组均明显促进比值增大,与对照组比较,差异明显(P<0.05)。双黄补组比值增大最为明显。随着作用时间的延长,比值逐渐增大,在第5天达到最大。不同时间各组间比较,差异有显著性意义(P<0.05)。结论:双黄补水提液可明显促进人牙周膜细胞增殖,明显提高胶原蛋白在总蛋白中的比值,可作为人牙周膜细胞增殖的理想的中药辅助因子。

Effects of Shuanghuangbu on the proliferation and differentiation of human periodontal ligament cells in vitro

BACKGROUND: The repair of periodontal tissue is dependent on the number and proliferation and differentiation abilities of periodontal ligament (PDL) cells. PDL cells have the potentiality of multi-directional differentiation such as cementoblast,osteoblast and fibroblast to fonn cement, alveolar bone and periodontal ligament and finally achieve periodontal tissue regeneration.OBJECTIVE:To observe the effects of Shuanghuangbu extract on the proliferation and differentiation of PDL cells.DESIGN:Observation trail.SETTING:Central Laboratory of Wuxi Third People's Hospital. MATERIALS: Periodontal tissue was provided voluntarily by the healthy young patients with deformity correction, and golden thread,skullcap,and rhizoma drynariae by the National Institute for the Control of Pharmaceutical and Biological Products.METHODS: The experiment was conducted in the Central Laboratory of Wuxi Third People's Hospital from July to October 2003.The crushed golden thread.skullcap,and rhizoma drynariae were mixed with distilled water at ratio of 1:10 (m:V),and refluxed in boiling water for 5 hours.The extract was collected,and after colation,the residue was refluxed in boiling water for another 3 hours. Both extract was blended, rotary evaporated and condensed, finally the liquid extract of 3 kg/L was obtained.There were 8 groups in the study including golden thread group, skullcap group,rhizoma drynariae group,golden thread plus skullcap group,golden thread plus rhizoma drynariae group, skullcap plus rhizoma drynariae group, Shuanghuangbu group and control group. PDL cells were cultured/n vitro assisted with Shuanghuangbu.The proliferation of cells was detected with MTT method and the ratio of collagen content in total protein was evaluated with hydroxyproline (HP).MAIN OUTCOME MEASURES:A value of proliferated PDL cells and the proportion of collagen protein in total protein.RESULTS:①Proliferation of PDL cells:Except golden thread group,all Chinese medicine promoted the proliferation of PDL cells significantly compared with control group (P＜0.05). The A value of Shuanghuangbu group was significantly increased.A value was increased with time and reached the peak on day 5.There were significant differences among each group at different time (P＜0.05).②Ratio of collagen content in total protein:Except golden thread group,the percentage was significantly increased by other Chinese medicines compared with control group (P＜0.05), especially Shuanghuangbu. The percentage was increased with time and reached the peak on day 5. There were significant differences among each group at different time (P＜0.05). CONCLUSION:The findings suggest that as a traditional Chinese herb,Shuanghuangbu can significantly stimulate the proliferation and differentiation of PDL cells.and increase the proportion of collagen content in total protein.It may act as an ideal Chinese medicine helper factor for the regeneration of PDL cells.