中国修复重建外科杂志

中国修复重建外科杂志

载基因脂多糖胺纳米囊泡诱导BMSCs定向成骨分化

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目的 制备并优化阳离子载体脂多糖胺纳米囊泡(lipopolysaccharide-amine nanopolymersomes,LNPs)性能,探讨其负载靶基因后体外诱导 BMSCs 定向成骨分化能力。 方法 采用接枝共聚的方法合成 LNPs,探讨合成时不同 pH(7.5、8.0、8.5、9.0)对 LNPs 及 LNPs/pBMP-2-绿色荧光蛋白(green fluorescent protein,GFP)复合物理化性能的影响;通过体外大鼠 BMSCs 细胞毒性及转染实验,筛选出具低毒高转染效能的 LNPs,再用其负载 pBMP-2-GFP 基因转染大鼠 BMSCs,通过定期观察细胞形态、测定细胞表达 BMP-2 蛋白水平和 ALP 活性、钙结节生成情况来评价 LNPs/pBMP-2-GFP 对 BMSCs 成骨分化的影响。 结果 pH 为 8.5 时合成的 LNPs 含氮量、粒径及 zeta 电位均低于其余 pH 值组,其细胞毒性最低(细胞成活率 96.5%±1.4%)、转染效率最高(98.8%±0.1%)。BMSCs 经 LNPs/pBMP-2-GFP 转染诱导处理后,在前 4 d 内,其 BMP-2 蛋白表达量均明显高于 Lipofectamine2000(Lipo)/pBMP-2-GFP 组、支链型聚乙烯亚胺 25K/pBMP-2-GFP 组和空白对照组(P<0.05)。诱导后 14 d 其 ALP 活性高于 Lipo/pBMP-2-GFP 组和空白对照组(P<0.05),与成骨诱导液处理组相当(P>0.05);茜素红染色显示其和成骨诱导液处理组细胞出现明显钙结节,而 Lipo/pBMP-2-GFP 组和空白对照组细胞出现凋亡、未见明显钙结节;诱导后 21 d 镜下观察见细胞中出现透明块状结节,呈成骨细胞形态特点。 结论 pH 为 8.5 时合成的 LNPs 具有低细胞毒性、高转染效率,能高效介导 BMP-2 基因转染大鼠 BMSCs,并成功诱导 BMSCs 成骨定向分化,其成骨诱导效果与成骨诱导液处理相当。LNPs 介导的基因转染可能是诱导干细胞定向分化的一种简便有效的手段。

Objective To investigate the ability of gene-loaded lipopolysaccharide-amine nanopolymersomes (LNPs) in inducing osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by in vitro gene transfection, where LNPs were used as a non-viral cationic carrier, and their properties were optimized during synthesis. Methods LNPs were synthesized by a graft-copolymerization method, and the effects of different pH environments during synthesis on physicochemical properties of LNPs and LNPs/plasmid of bone morphogenetic protein 2-green fluorescent protein (pBMP-2-GFP) complexes were explored. Then, optimized LNPs with maximum transfection efficiency and safe cytotoxicity in rat BMSCs were identified by cytotoxicity and transfection experiments in vitro. Thereafter, the optimized LNPs were used to mediate pBMP-2-GFP to transfect rat BMSCs, and the influences of LNPs/pBMP-2-GFP on osteogenic differentiation of BMSCs were evaluated by monitoring the cell morphology, concentration of BMP-2 protein, activity of alkaline phosphatase (ALP), and the formation of calcium nodules. Results The nitrogen content, particle size, and zeta potential of LNPs synthesized at pH 8.5 were lower than those of the other pH groups, with the lowest cytotoxicity (96.5%±1.4%) and the highest transfection efficiency (98.8%±0.1%). After transfection treatment, within the first 4 days, BMSCs treated by LNPs/pBMP-2-GFP expressed BMP-2 protein significantly higher than that treated by Lipofectamine2000 (Lipo)/pBMP-2-GFP, polyethylenimine 25K/pBMP-2-GFP, and the blank (non-treated). At 14 days after transfection, ALP activity in BMSCs treated by LNPs/pBMP-2-GFP was higher than that treated by Lipo/pBMP-2-GFP and the blank, comparable to that induced by osteogenic medium; with alizarin red staining, visible calcium nodules were found in BMSCs treated by LNPs/pBMP-2-GFP or osteogenic medium, but absent in BMSCs treated by Lipo/pBMP-2-GFP or the blank with apoptosis. At 21 days after transfection, transparent massive nodules were discovered in BMSCs treated by LNPs/pBMP-2-GFP, and BMSCs exhibited the morphologic features of osteoblasts. Conclusion LNPs synthesized at pH 8.5 has optimal transfection efficiency and cytotoxicity, they can efficiently mediate pBMP-2-GFP to transfect BMSCs, and successfully induce their directional osteogenic differentiation, whose inducing effect is comparable to the osteogenic medium. The results suggest that gene transfection mediated by LNPs may be a convenient and effective strategy in inducing directional differentiation of stem cells.

关键词: 基因载体; 脂多糖胺纳米囊泡; 基因转染; 定向分化; BMSCs

Key words: Gene carrier; lipopolysaccharide-amine nanopolymersomes; gene transfection; directional differentiation; bone marrow mesenchymal stem cells

引用本文: 李菁, 陈盈, 滕伟, 王琴梅. 载基因脂多糖胺纳米囊泡诱导BMSCs定向成骨分化. 中国修复重建外科杂志, 2018, 32(11): 1469-1476. doi: 10.7507/1002-1892.201804125 复制

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