BMP-2和地塞米松对人牙髓细胞增殖和分化的影响

Effect of bone morphogenetic protein 2 and dexamethason on proliferation and differentiation of human dental pulp cells in vitro

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 effect of bone morphogenetic protein 2 （BMP-2） and dexamethason （DXM） on proliferation and differentiation of human dental pulp cells in vitro. Methods Primary human dental pulp cells were cultured in vitro by tissue culture method. The 3rd generation cells were used to identify cell phenotype for vimentin and cytokeratin by immunocytochemistry staining. The 3-5 generations of human dental pulp cells were randomly divided into 4 groups： 100 ng/mL BMP-2 （group A）, 1×10^-8 mol/L DXM （group B）, and both 100 ng/mL BMP-2 and 1×10^-8 mol/L DXM （group C） were added; neither BMP-2 nor DXM was added in group D as control group. The cell growth curve was drawn at 1, 3, 5, and 7 days after culture. The expressions of osteo/dentanogenic genes including alkaline phosphatase （ALP）, dentin sialophoshoprotein （DSPP）, and dentin matrix protein 1 （DMP-1） were detected by RT-PCR analysis at 5 and 7 days after culture, the ratio between the positive staining area and the total area by ALP staining at 14 days, and absorbance （A） value at 562 nm by alizarin red staining at 21 days after culture. Results Human dental pulp cells were successfully isolated and cultured, which were long fusiform and showed a positive reaction for vimentin and a negative reaction for cytokeratin. The growth curve indicated that cells increased with the extending of incubation time, reached a peak at 5 days, then reduced at 7 days to the level at 3 days. At 5 days after culture, the cells were significantly more in groups A, B, and C than group D （P〈0.05）, in group C than group A （P〈0.05）, and in group A than group B （P〈0.05）. RT-PCR analysis showed that the mRNA expressions of ALP, DSPP, and DMP-1 at 5 days were significantly higher in groups A, B, and C than group D （P〈0.05）, and in group C than groups A and B （P〈0.05）, but no significant difference was found between groups A and B （P〉0.05）; the mRNA expression of DSPP in groups A, B, and C was significantly higher than that in group D （P〈0.05）, but there was no significant difference in mRNA expressions between other groups at 7 days （P〉0.05）. At 14 days, positive staining in varying degrees was observed in each group, especially in group C; the ratio between the positive staining area and the total area was significantly higher in group C than groups A, B, and D （P〈0.05）, and in groups A and B than group D （P〈0.05）, but there was no significant difference between groups A and B （P〉0.05）. At 21 days, there were a variety of mineralized nodules in groups A, B, and C in nonuniformly scattered or clustered distribution, but no mineralized nodules were observed in group D. The A values of mineralized nodules showed significant difference between groups （P〈0.05）. Conclusion BMP-2 may be more effective in promoting proliferation of human dental pulp cells than DXM. Combined application of BMP-2 and DXM can remarkably promote the proliferation and differentiation of human dental pulp cells.