17β-雌二醇对人牙髓细胞增殖影响的体外研究

摘要

目的：观察不同浓度17β-雌二醇对体外培养人牙髓细胞增殖的影响。方法选取年轻恒牙，采用组织块酶消化法获取人牙髓细胞并进行原代培养。取第4代对数生长期人牙髓细胞SABC法进行波形蛋白和角蛋白免疫组化染色鉴定细胞来源。将处于对数生长期的第4代人牙髓细胞随机分为5个实验组、1个对照组和1个空白对照组，96孔培养板每组选6个孔。实验组：在有细胞孔内分别加入含2％活性碳吸附胎牛血清的无酚红高糖DMEM培养液配置的终末浓度为10－5、10－6、10－7、10－8、10－9 mol／L的17β-雌二醇。对照组：在有细胞孔内仅加入培养液。空白对照组：在无细胞孔内仅加入培养液。分别于培养48 h和72 h时用MTT比色法检测17β-雌二醇对人牙髓细胞增殖的影响。采用流式细胞术检测各浓度组17β-雌二醇对人牙髓细胞的细胞周期的影响。结果与对照组比较，在一定浓度范围内17β-雌二醇能促进人牙髓细胞的增殖和细胞周期进程，以10－8 mol／L的17β-雌二醇作用最为明显（ P＜0．05）。结论17β-雌二醇对人牙髓细胞细胞增殖有一定的诱导作用。%Objective To investigate the effects of 17 beta estradiol ( 17β-E2 ) on proliferation activity , alkaline phosphates’(ALP) activity, the cell cycle of human dental pulp cells (HDPCs) in vitro.Methods The dental pulp tissue was taken from healthy young people'molars or premolar ,then the dental pulp cells were obtained by tissue explant-collagenase digestion method and cultured in vitro .The fourth generation dental pulp cells were identified by vimentin and keratin immunohistochemisty staining for cell origin .The fourth generation dental pulp cells at logarithmic phase were digested and made into cell suspension with 0.25%trypsin,and the cell suspension was placed into 96-well microplates, then the HDPCs at logarithmic phase were randomly divided into 5 experimental groups ,1 control group and 1 blank control group ,with 6 holes in each group.The experimental groups: 17 beta estradiol 150μl with terminal concentration being 10 -9 ,10 -8 ,10 -7 ,10 -6 , 10 -5 mol/L,respectively was added into corresponding cell holes ,and 17 beta estradiol was placed into phenol red free high sugar DMEM medium containing 2% activated carbon adsorption fetal calf serum .The control group: only DMEM nutrient solution was added into corresponding cell holes . The blank control group: DMEM nutrient solution was added into corresponding holes without cells.The effects of 17β-E2 on the proliferation of HDPCs were detected by MTT at 48h,72h, respectively .The effects of 17β-E2 on the cell cycle of HDPCs were detected by flow cytometry .Results In contrast with control group, 17β-E2 could promote proliferation of HDPCs and cell cycle process within a certain range of concentration . The effects of 17β-E2 in concentration of 10 -8 mol/L were the most significant ( P <0.05).Conclusion The 17β-E2 can induce proliferation of HDPCs at some extent in vitro .