CCN2对人牙髓干细胞增殖及分化的影响

摘要

Objective To investigate the effects of CCN2 on proliferation and differentiation of human dental pulp stem cells (hDPSCs). Methods Teeth were collected from patients with orthodontics or impaction in Department of Prosthodon-tics of Affiliated Hospital of Yan’an University. The hDPSCs was separated with enzyme digestion. Morphological ob-servation of hDPSCs were observed under light microscope and the expression of surface marker protein was determined with immunofluorescence. Different concentrations of CCN2 were added into hDPSCs of logarithmic phase, and divided into different groups:CCN2 6.25, 12.5, 25, 50, 100μg/L group, meanwhile the cells not treated with CCN2 as negative control group. MTT assay was used to evaluate the proliferative effects of CCN2. The activity of alkaline phosphatase and the expression of odontoblasts marker proteins were determined to assess the effects of CCN2 on differentiation of hDPSCs. Results The human dental pulp stem cells with colony growth, Immunofluorescence test showed that the ex-pression of STRO-1 was positive. MTT assay showed that CCN2 could significantly promote the proliferation of hDP-SCs, as compared with the control group. The values of OD in CCN2 6.25, 12.5, 25, 50, 100 μg/L group were (0.78±0.05), (0.91±0.03), (1.23±0.09), (1.57±0.11), and (1.61±0.15) respectively, higher than that of the control group (0.51±0.02), the differences were statistically significant (P<0.05 or P<0.01). Furthermore, CCN2 increased alkaline phos-phatase activity of hDPSCs, exhibiting a dose-dependent manner, and the values of OD in CCN2 6.25, 12.5, 25, 50, 100 μg/L group were (0.32±0.04), (0.43±0.04), (0.61±0.03), (0.79±0.06) and (0.81±0.07) respectively, compared with the control group (0.19±0.03), the differences were statistically significant (P< 0.05 or P< 0.01). The results of West-ern blot Showed that CCN2 also could induce the expression of odontoblast marker proteins. Conclusion CCN2 can promote the hDPSCs to differentiation into odontoblasts.%目的：探讨CCN2对体外培养的人牙髓干细胞增殖、分化的影响。方法收集在延安大学附属医院口腔修复科因正畸或阻生而拔除的牙齿，酶消化法获得人牙髓干细胞，光镜下进行形态学观察，免疫荧光法检测细胞表面标志蛋白的表达。取对数生长期牙髓干细胞，加入不同浓度的CCN2将其分为CCN26.25、12.5、25、50、100μg/L组，同时设置阴性对照组；采用噻唑蓝(MTT)法检测CCN2对牙髓干细胞增殖作用的影响，通过碱性磷酸酶及Western blot检测CCN2对牙髓干细胞分化的影响。结果人牙髓干细胞呈集落状生长，免疫荧光法检测STRO-1呈阳性表达，CCN26.25、12.5、25、50、100μg/L组 OD值分别为(0.78±0.05)、(0.91±0.03)、(1.23±0.09)、(1.57±0.11)、(1.61±0.15)，均高于对照组(0.51±0.02)，差异有统计学意义(P<0.05或P<0.01)；细胞增殖实验表明，CCN2可显著促进牙髓干细胞的增殖；此外，CCN2可促进牙髓干细胞碱性磷酸酶的活性，并呈现浓度±赖关系，CCN26.25、12.5、25、50、100μg/L组OD值分别为(0.32±0.04)、(0.43±0.04)、(0.61±0.03)、(0.79±0.06)及(0.81±0.07)，与对照组(0.19±0.03)比较，差异有统计学意义(P<0.05或P<0.01)；Western blot结果表明，CCN2可诱导牙本质细胞特异性标志蛋白的表达。结论 CCN2可促进牙髓干细胞向成牙本质细胞的分化。