体外共培养人牙髓干细胞对人牙囊干细胞增殖、成骨分化的作用

Role of in vitro co-culture of human dental pulp stem cells in proliferation and osteogenic differentiation of human dental follicle stem cells

目的通过体外建立人牙囊干细胞(human dental follicle stem cells,HDFSCs)和人牙髓干细胞(human dental pulp stem cells,HDPSCs)共培养体系,研究HDPSCs对HDFSCs增殖、成骨分化的作用。方法 1利用改良的组织块-消化联合法、免疫组化方法、流式细胞术以及多向诱导方法对HDFSCs和HDPSCs进行分离、培养、鉴定;2采用0.4μm孔径的Transwell小室构建HDFSCs和HDPSCs体外共培养体系;3CCK-8测定HDFSCs增殖活性的变化;4与HDPSCs体外共培养1周,qRTPCR检测HDFSCs几个标志性成骨基因:碱性磷酸酶(alkaline phosphatase,ALP)、人类相关转录因子2(runt-related transcription factor 2,Runx2)、骨桥蛋白(osteopontin,OPN)、骨涎蛋白(bone sialoprotein,BSP)以及Ⅰ型胶原(collagen type 1,Col-1)的表达情况。结果 1成功分离、培养、鉴定HDFSCs和HDPSCs;2从第4天开始,共培养组HDFSCs的增殖活力明显高于对照组(P<0.05);3与对照组相比,共培养组ALP、Runx2、OPN、BSP、Col-1的相对表达量均明显增高(P<0.05)。结论与HDPSCs共培养能增强HDFSCs增殖活力及成骨分化。

Objective To determine the role of human dental pulp stem cells（ HDPSCs） in the proliferation and osteogenic differentiation of human dental follicle stem cells（ HDFSCs） under co-culture in vitro. Methods 1HDFSCs and HDPSCs were isolated,cultured and identified by a modified tissue block digestion,immunohistochemistry, flow cytometry and multilineage inducement. 2 Cell culture chamber（ Transwell） with pore size of 0. 4 μm in diameter were used to facilitate the co-culture system of HDFSCs and HDPSCs in vitro. 3CCK-8 assay was used to test the changes of proliferative activity of HDFSCs; 4 In 1 week after in vitro co-culture system of HDFSCs and HDPSCs,the expression of several osteoblast marker genes including the alkaline phosphatase（ ALP）,runt-related transcription factor 2（ Runx2）,osteopontin（ OPN）,bone sialoprotein（ BSP） and collagen type 1（ Col-1） were measured with qRT-PCR. Results 1HDFSCs and HDPSCs were isolated,cultured and identified successfully. 2Starting from the fourth day,HDFSCs in the co-culture group showed a higher proliferative activity than that in the control group（ P 0. 05）.3Compared with the control group,there were significant increases in the relative mRNA levels of ALP,Runx2,OPN,BSP and Col-1（ P 0. 05） in the co-culture group. Conclusion Co-culture with HDPSCs stimulates the proliferous activity and osteogenic differentiation in HDFSCs.