CM-DiI体外标记人乳牙牙髓干细胞的可行性研究

Feasibility of in vitro Labeling Human Stem Cells from Deciduous Pulp by CM-DiI

目的:筛选CM-DiI用于人乳牙牙髓干细胞标记的最佳浓度,并对标记后细胞的生物学行为进行评价,为下一步细胞活体移植和体内示踪打下基础。方法:酶解组织块法培养人乳牙牙髓细胞并纯化,免疫组化鉴定细胞来源,进行细胞体外多向诱导分化能力实验。将第3代细胞分别使用浓度2mg/L、3mg/L及4mg/L的CM-DiI进行标记,比较标记后细胞乳酸脱氢酶释放率及细胞增殖情况,并观察经体外多次传代后细胞荧光的表达情况。结果:酶解组织块法可获得成集落状生长的人乳牙牙髓细胞;免疫组化确定细胞为间充质而非造血来源;细胞具有成脂、成骨及成牙本质等多向分化能力。3种标记物浓度均未对细胞乳酸脱氢酶释放及细胞增殖产生显著的不利影响;随体外多次传代,标记荧光强度逐渐减退。结论:CM-DiI操作简便、染色速度快,可有效的标记人乳牙牙髓干细胞,其中3mg/L的标记浓度细胞毒性极小,标记效率高,在体外多次传代后荧光信号仍能稳定表达。

Objective：To evaluate the feasibility of in vitro labeling human stem cells from deciduous pulp by CM-DiI.Methods：Enzyme tissue block method was used to culture human stem cells from pulp of deciduous teeth,and the biological characteristics of cells were identified.The third passage cells were labeled by 2μg/mL,3μg/mL and 4μg/mL,respectively.The release of lactic dehydrogenase（LDH）,proliferation and labeling rate were detected in vitro.Results：Human stem cells from deciduous pulp were harvested by enzyme tissue block method and immunohistochemistry staining confirmed the cells were derived from mesenchymal tissue,rather than from the epithelial tissue.Abilities of adipogenic and osteogenic/odontoblasts differentiation were verified after being induced.These three concentrations showed no significant adverse effect on cell proliferation and the release of LDH.Fluorescence intensity gradually decreased in vitro.Conclusion：The application of labeling human stem cells from pulp of deciduous teeth by CM-DiI is convenient and effective.3μg/mL is the optimal concentration for labeling in the present study.