目的通过将BMP-2和地塞米松分别及联合作用于体外培养的人牙髓细胞,探讨两者对牙髓细胞增殖和分化能力的影响。方法取25岁以下患者因正畸原因拔出的无病变牙牙髓组织,采用组织块法体外培养人牙髓细胞并传至第3代,行波形蛋白和角蛋白免疫细胞化学染色鉴定。取生长状况良好的第3~5代牙髓细胞分为4组,A、B、C组分别加入100 ng/m L BMP-2、1×10–8 mol/L地塞米松、100 ng/m L BMP-2以及1×10–8 mol/L地塞米松,D组不添加诱导剂作为对照组。培养1、3、5、7 d绘制细胞生长曲线;培养5、7 d行RT-PCR检测成牙本质细胞形成标记基因表达情况,包括牙本质涎磷蛋白(dentin sialophoshoprotein,DSPP)、牙本质基质蛋白1(dentin matrix protein 1,DMP-1)、ALP;培养14 d行ALP染色并计算阳性染色面积占总面积比值;培养21 d行茜素红染色并测定562 nm波长处吸光度(A)值。结果成功分离培养呈长梭形的人牙髓细胞,经波形蛋白和角蛋白免疫细胞化学染色鉴定显示符合牙髓细胞的生物学特征。随培养时间延长,各组细胞均逐渐增加,5 d时达峰值,7 d时降至3 d水平。培养5 d,A、B、C组细胞显著多于D组,C组多于A组,A组多于B组,比较差异均有统计学意义(P<0.05)。RT-PCR检测示,培养5 d,A、B、C组ALP、DSPP、DMP-1 m RNA相对表达量均显著高于D组,C组显著高于A、B组,差异均有统计学意义(P<0.05);A、B组间比较差异无统计学意义(P>0.05)。培养7 d,除A、B、C组DSPP m RNA相对表达量显著高于D组(P<0.05)外,其余各组间各基因相对表达量比较差异均无统计学意义(P>0.05)。培养14 d ALP染色示,各组可见紫黑色沉淀,其中C组着色程度最深;经半定量测定,C组阳性染色面积占总面积比值显著高于A、B、D组,A、B组高于D组,差异均有统计学意义(P<0.05);A、B组间差异无统计学意义(P>0.05)。培养21 d茜素红染色示,C组矿化结节呈片状大面积分布,A、B组呈散在分布,D组无矿化结节形成;各组间A值比较差异均有统计学意义(P<0.05)。结论 BMP-2对牙髓细胞的增殖作用比地塞米松更明显,两者对牙髓细胞分化作用差异不明显;但与单独应用相比,两者联合应用对牙髓细胞的增殖和分化有明显促进作用。 OBJECTIVE: To investigate the effects of BMP-2 and dexamethasone on proliferation and differentiation of human dental pulp cells in vitro and in vitro respectively. Methods Twenty-four-year-old non-diseased dental pulp tissues were obtained from patients under 25 years of age. Human dental pulp cells were cultured in vitro by tissue block method and passaged to passage 3 for immunohistochemical identification of vimentin and keratin. The 3rd to 5th generation of dental pulp cells with good growth condition were divided into 4 groups. Group A, B and C were given 100 ng / m L BMP-2, 1 × 10-8 mol / L dexamethasone, 100 ng / m L BMP-2 and 1 × 10-8 mol / L dexamethasone, and the group D did not add the inducer as the control group. The cell growth curve was drawn on the 1st, 3rd, 5th and 7th day. The expression of marker gene in odontoblasts was detected by RT-PCR on day 5 and 7, including dentin sialophoshoprotein (DSPP), dentin matrix (DMP-1) and ALP. ALP staining was performed 14 days after culture and the area of ​​positive staining was calculated. The ratio of total area was calculated by staining with alizarin red and the absorbance at 562 nm . RESULTS: Human dental pulp cells with long fusiform shape were successfully isolated and cultured. Immunocytochemical staining with vimentin and keratin showed that they were in accordance with the biological characteristics of dental pulp cells. With the prolongation of culture time, the number of cells in each group increased gradually, reached its peak on the 5th day and dropped to the 3rd day on the 7th day. After cultured for 5 days, the number of cells in group A, B and C was significantly more than that in group D, more in group C than in group A, and more in group A than in group B (P <0.05). RT-PCR results showed that the relative expression of ALP, DSPP and DMP-1 mRNA in group A, B and C were significantly higher than that in group D at 5 d after culture, and the difference was statistically significant between group C and group A (P <0.05). There was no significant difference between A and B groups (P> 0.05). After 7 days of culture, the relative expression levels of DSPP m RNA in groups A, B and C were significantly higher than those in group D (P <0.05) . The ALD staining on day 14 showed that purple-black precipitate was observed in each group, of which the staining degree in group C was the deepest. The ratio of positive staining area to total area in group C was significantly higher than that in groups A, B and D In group D, the differences were statistically significant (P <0.05); there was no significant difference between group A and group B (P> 0.05). Alizarin red staining of 21 d showed that the mineralized nodules in C group showed flaky distribution with scattered distribution in group A and B and no mineralized nodules in group D. The difference of A value between groups was statistically significant Significance (P <0.05). Conclusion The proliferation of dental pulp cells by BMP-2 is more obvious than that of dexamethasone, and there is no obvious difference between the two groups on the differentiation of dental pulp cells. However, compared with the single application, the combination of the two have a significant effect on the proliferation and differentiation of dental pulp cells Significantly promote the role.