bFGF纳米控释载体对骨髓间充质干细胞的生物学作用

Enhancement of Proliferation and Differentiation of Bone Mesenchymal Stem Cells by Basic Fibrous Growth Factor Controlled Release Nanoparticles

本研究旨在探讨甲基丙烯酸缩水甘油酯右旋糖酐(dex-GMA)载碱性成纤维细胞生长因子(bFGF)凝胶纳米微球(dex-GMA-bFGF-NPs)对骨髓间充质干细胞(BMSCs)生物学作用。采用改良乳液聚合技术合成纳米微球,将单纯bFGF(A组)、空白dex-GMA凝胶纳米微球(B组)和载bFGF凝胶纳米微球(C组)加入骨髓间充质干细胞培养液中,不添加任何添加物的单纯MSCs为空白对照组(D),用细胞计数法、噻唑蓝比色法(MTT法)、流式细胞术观察细胞增殖情况;并检测细胞ALP活性,以反映dex-GMA-bFGF-NPs对BMSCs分化情况的影响。结果显示:dex-GMA-bFGF-NPs大小均匀,包封率高达88%,85%的bFGF在前14 d释放。dex-GMA-bFGF-NPs对bFGF缓释有效促进BMSCs的增殖,在培养12 d后,A、B、C和D组细胞数分别为(21.97±0.25)×104个细胞/ml(、12.43±0.13)×104个细胞/ml(、27.45±0.78)×104个细胞/ml和(12.03±0.43)×104个细胞/ml,差异有统计学意义(P<0.05),培养3 d后,A组的G2/M+S期百分数最高,7 d后,C组的G2/M+S期百分数最高。结论:dex-GMA-bFGF-NPs可控制活性bFGF的长时间持续释放,作为bFGF的缓释载体,可明显促进骨髓间充质干细胞的增殖和分化。

This research was carried out to investigate the effect of basic fibrous growth factor（bFGF） controlled release hydrogel nanoparticles on the proliferation and differentiation of mesenchymal stem cells.The dex-GMA-bFGF-NPs were prepared by an improved emulsion polymerization method;their morphology,size and encapsulated ratio were assessed by routine procedure.Dynamic dialysis method was used to determine the release characteristics of dex-GMA-bFGF-NPs in vitro.The secondary culture MSCs were divided into four groups according the different ingredients being added into the DMEM culture medium： free bFGF group（A）,blank dex-GMA nanoparticles group（B）,dex-GMA-bFGF nanoparticles group（C）,nothing group（D）.The proliferation of cultured MSCs was measured by using cell counting method,MTT method and flow cytometry.ALP kit was used to evaluate the ALP activity of the MSCs to show the differentiation of the cells by adding the dex-GMA-bFGF-NPs to the DMEM culture medium（C group） or bFGF only（A group）.B group and D group were taken as the controls.The results were ana lyzed by statistical analysis software（SPSS11.0）.All results showed that the shape of dex-GMA-bFGF-NPs was spherical.The encapsulated ratio was 88% and more than 85% of the encapsulated bFGF could be released during 14 days.The in vitro cellular study showed the control release of bFGF from nanoparticles could promote the profliferation of MSCs.After 12 days,the cell number in groups A、B and C was（21.97±0.25）×10^4 cells/ml,（12.43±0.13）×10^4 cells/ml,（27.45±0.78）×10^4 cells/ml and（12.03±0.43）×10^4 cells/ml,with the difference being statistically significant among them（P〈0.05） The flow cytometry revealed that the G2/M＋S percentage in group C was the highest at 4-8 days after plate culture（P0.05）.During the first 3 days,the proliferation and differentiation of BMSCs between group A and group B were of no significance（P〉0.05）,but were much faster than those of group C and D.After 7 days,dex-GMA-bFGF-NPs could enhance BMSCs proliferation and differentiation continually,but bFGF had no enhancement any more,the difference between group A and group B became more significant（P〈0.05）.So we made the conclusion： the bFGF loading dex-GMA hydrogel nanoparticles can release bFGF more than 21 days and can promote the proliferation and differentiation of the BMSCs through a long period of controlled release of bFGF.Dex-GMA-bFGF-NPs may be an ideal controlled release carrier for bioactive growth factors.