山羊乳牙牙髓细胞分离培养与矿化诱导的体外研究

Isolation,cultivation and induced-mineralization of dental pulp cells from goat deciduous teeth in vitro

目的:分离培养山羊乳牙牙髓细胞,研究其矿化诱导前、后生物学特性的改变。方法:采用改良酶解组织块法培养山羊乳牙牙髓细胞,应用免疫组化方法(SAB法)对第2代细胞进行来源检测,经矿化诱导培养的第4代细胞与常规培养细胞做对照,进行相关生物学特性检测,包括细胞增殖能力、矿化能力、细胞形态改变、蛋白OCN表达、相关成骨基因(ALP、COL-I、OCN、OPN)表达水平。结果:改良酶解组织块法可较快地培养出山羊乳牙牙髓细胞,细胞爬出时间为培养的第3～4天。第2代细胞抗波丝蛋白阳性,抗角蛋白阴性,证明其间充质来源。MTT法测定显示,未诱导细胞的增殖能力明显高于矿化诱导后的细胞。与未诱导细胞相比较,矿化诱导的细胞ALP染色、钙结节茜素红染色均呈强阳性,免疫组化OCN阳性表达。诱导14d后,定时定量PCR检测证实成骨基因OCN、ALP显著上调。结论:本实验采用改良酶解组织块法成功培养出山羊乳牙牙髓细胞;连续矿化诱导培养14d后,山羊乳牙牙髓细胞分泌矿化基质,具有向成骨细胞分化和形成骨组织的潜能。

PURPOSE：To isolate and cultivate dental cells from goat deciduous teeth,and explore changes of its biological characters before and after induced-mineralization.METHODS：Pulp cells were cultivated with modified tissue block enzymolytic method,cell lineage in the second passage with SAB methods was checked out.Induced-mineralized cultivation was adopted in the fourth passage,some examinations were used to compare with normal cultivated cells ：cell proliferative capality,mineralized ability test,cell morphology change,protein（OCN） expression level,related osteogenic genes（ALP,COL-I,OCN,OPN） expression.RESULTS：Modified tissue block enzymolytic method could culture better pulp cells derived from goat deciduous teeth.Immunohistochemical staining showed that pulp cells were from mesenchyma.MTT method showed that induced-mineralization pulp cells proliferated more slowly than un-induced cells.Compared with uninduced-mineralization cells,induced-mineralization cells had stronger ALP activity and Alizarin red staining rate,its proteins（OCN） and mineralized genes（ALP,OCN） expression were significantly upregulated.CONCLUSIONS：Pulp cells can be cultivated derived from goat exfoliated deciduous teeth with modified tissue block enzymolytic method.Fourteen days after continuous induced-mineralization culture,pulp cells derived from the goat deciduous teeth might own the potential in differentiating to osteoblast and form bone-like tissue.