发育期牙根端复合体细胞条件培养液对脂肪干细胞增殖、分化的影响

The effects of developing apical complex cells conditioned medium on the proliferation and differentiation of ADSCs

目的:初步探讨发育期根端复合体(developing apical complex,DAC)条件培养基(DAC conditioned medium,DACCM)对脂肪干细胞(adipose tissue-derived stem cells,ADSCs)增殖和分化的影响。方法:取出生20 d SD大鼠分离其下颌磨牙DAC,并采用组织块联合酶消化法培养DAC细胞;经HE、免疫组化染色鉴定DAC后,收集原代培养的DAC细胞上清,分别用过滤和不过滤的方式制备不同条件培养基并用其对ADSCs进行培养;分别于培养3、5、7 d各时间点,MTT法检测ADSCs增殖能力;用碱性磷酸酶(ALP)试剂盒及反转录酶-聚合酶链反应(RT-PCR)检测ADSCs的ALP蛋白活性及其基因的表达水平。结果:免疫组化染色显示,培养的DAC细胞CK-14、vimentin均为阳性表达;DACCM-未滤培养组ADSCs的增殖活性、ALP蛋白活性以及ALP基因表达水平均高于单纯α-MEM培养组和DACCM-过滤培养组,差异均有统计学意义(P<0.05)。结论:不经过滤的DAC细胞上清制备的条件培养基对ADSCs增殖、分化的促进作用更明显。

AIM： To study the effects of the developing apical complex（ DAC） cells conditioned medium（ DACCM） on the proliferation and differentiation of adipose tissue-derived stem cells（ ADSCs）. METHODS： DAC cells,separated from the 20 d postnatal SD rat,were cultured by tissue block combined with enzyme digestion method.DACs was identified by HE staining and immumohistochemical staining. DACCM was collected and prepared by filtration and none-filtration（ DACCM-F and DACCM-UF） respectively. The proliferation of ADSCs was detected by MTT assay. The mRNA and protein expression of alkaline phosphatase（ ALP） were detected by RT-PCR and ALP detection Kit respectively. SPASS13. 0 was used for data analysis. RESULTS： DAC cells were obtained from the developing roots of the SD rat. The cells were CK-14 and vimentin positive. The proliferation,ALP mRNA and protein expression of ADSCs cultured in DACCM-UF exceeded those of the cells cultured in α-MEM or DACCM-F（ P〈0. 05） CONCLUSION： DACCM-UF may stimulate the proliferation and differentiation of ADSCs.