重组人BMP-2聚乳酸缓释纳米微球对体外培养兔成骨细胞增殖和矿化的影响

目的 观察重组人骨形态发生蛋白-2聚乳酸纳米微球缓释系统(rhBMP-2-PLA-Ns)对体外培养的兔成骨细胞的生物学效应. 方法 体外培养兔成骨细胞并鉴定,将rhBMP-2-PLA-Ns和第3代成骨细胞一起培养,免疫荧光法检测成骨细胞核中增殖细胞核抗原(PCNA)的表达,茜素红染色方法观察矿化结节的形成,Western blot检测rhBMP-2-PLA-Ns对成骨细胞自分泌血管内皮细胞生长因子(VEGF)的作用,并与单纯rhBMP-2组和空白组进行比较. 结果 培养5 d后,各组PCNA阳性细胞差异无统计学意义.培养10 d后,rhBMP-2-PLA-Ns组阳性细胞数明显高于单纯rhBMP-2组和空白组,表明rhBMP-PLA-Ns能够明显促进PCNA在成骨细胞核中的表达.与其他两组比较,rhBMP-2-PLA-Ns能显著促进矿化结节的形成(P<0.05);对Western blot检测结果进行半定量分析显示:三组成骨细胞均有VEGF的自分泌,加入rhBMP-2-PLA-Ns成骨细胞自分泌VEGF的量超过单纯rhBMP-2组以及空白对照组(P<0.05).结论 rhBMP-PLA-Ns有较好的生物学活性,能促进成骨细胞的增殖、矿化和VEGF自分泌的增加,具有一定的临床应用价值,为加快骨创伤的愈合提供了新的思路.

Effects of recombinant human bone morphogenetic protein-2-polylactide sustained release nanospheres on proliferation and mineralization of rabbit osteoblasts in vitro

Objective To evaluate the effects of recombinant human bone morphogenetic-2-polylactide sustained release nanospheres (rhBMP-2-PLA-Ns) on cultured rabbit osteoblasts in vitro. Methods Rabbit osteoblasts were cultured in vitro, and rhBMP-2-PLA-Ns was added into the medium of the third generation of rabbit osteoblasts. The expression of the proliferating cell nuclear antigen (PCNA) was examined by immunofluoreacence staining, and the formation of tuberculums observed with alizarin red staining. Western blot was used to detect the effects of rhBMP-2-PLA-Ns on the expression of vascular endothelial growth factor (VEGF), which was compared with that in rhBMP-2 group and blank group. Results There was no significant difference in the number of osteoblasts with positive PCNA expression among three groups five days later. Ten days later, the number of osteoblasts with positive PCNA expression in rhBMP-2-PLA-Ns group was significantly higher than that in rhBMP-2 group and blank group, which indicated that rhBMP-2-PLA-Ns could enhance the expression of PCNA. Compared with rhBMP-2 group and blank group, rhBMP-2-PLA-Ns could significantly enhance the formation of tuberculums, with statistical difference (P<0.05). The expression of VEGF was detected in all three groups, and the level of the VEGF expression in rhBMP-2-PLA-Ns group was significantly higher than that in the other 2 groups (P<0.05). Conclusion The biological activity of rhBMP-2-PLA-Ns is superior than that of rhBMP2, and rhBMP-2-PLA-Ns can promote the proliferation, mineralization of osteoblasts and the secretion of VEGF, which has a better prospect in facilitating traumatic bone healing.