小鼠牙髓干细胞培养及诱导分化

Isolation, cultivation and differentiation of stem cells from mouse dental pulp

目的 :分离、培养小鼠牙髓干细胞并诱导分化 .方法 :采用酶消化培养法获得小鼠的单个牙髓细胞悬液 ,细胞密度调整为 1× 10 4/孔细胞 ,用 2 0 0mL·L -1胎牛血清的α MEM培养液培养 14d ,挑出细胞克隆消化传代 ,TGF β、BMP2 、bFGF细胞因子诱导 ,PCR检测DSPP的mRNA的表达 .结果 :小鼠牙髓细胞呈集落状生长 ,克隆形成率为 1.6 -2 .5个 / 10 4细胞 ,所形成的集落细胞结合紧密 ,细胞体积小、胞核大 ,经细胞因子刺激后的细胞表达DSPP的mRNA .结论 :培养的小鼠牙髓集落状生长的细胞具有干细胞增殖快等特性 。

AIM: To culture, identify and differentiate dental pulp stem cells. METHODS: Pulp tissue was separated from the mouse teeth and digested by collagenase type I. Single cell suspensions of dental pulp were seeded into 6 well plates with alpha modification of Eagles medium supplemented with 200 mL·L -1 FCS. Colony forming efficiency was assessed in 14 day culture. Subconfluent cultures (first passage) of colony forming cells were induced with TGF β, BMP2 and bFGF. Transcripts for DSPP and BSP were detected by reverse transcription PCR by using total RNA isolated from these induced cells. RESULTS: There were clonogenic cells in dental pulp and the incidence of colony forming cells derived from mouse dental pulp cells was 1.6-2.5 colonies/10 4 plate. Mouse specific DSPP mRNA was expressed in colony forming cells after 8 d of induction with BMP2, TGF β and bFGF in vitro . CONCLUSION: Colony forming cells derived from mouse dental pulp have high rate of proliferation and differentiation potentials.