辛伐他汀纳米脂质体对骨髓间充质干细胞成骨分化的影响

摘要/Abstract

摘要： 目的：通过对骨形成蛋白-2（bone morphogenetic protein 2，BMP-2）和碱性磷酸酶（alkaline phosphatase，ALP）的表达进行测定，研究纳米脂质体剂型对辛伐他汀（simvastatin，SMV）在骨髓间充质干细胞（bone marrow stromal cells，BMSC）形成骨过程中的促进作用。方法：采用全骨髓贴壁培养法体外培养小鼠原代BMSC。以SMV原料药处理组作为对照组，分别设SMV药物浓度1 μmol/L和2 μmol/L为SMV低剂量组（SMV low dosage group，S1）和SMV高剂量组（SMV high dosage group，S2）；将辛伐他汀纳米脂质体（simvastatin nano-liposomes，SMV-liposome）处理组作为实验组，根据SMV药物包载浓度1 μmol/L和2 μmol/L分别设为SMV-liposome低剂量组（SMV-liposome low dosage group，SL1）和SMV-liposome高剂量组（SMV-liposome high dosage group，SL2）；此外，实验中所有涉及到不做任何处理的组，都作为空白组（blank）。将SMV和SMV-liposome分别与BMSC共孵育48 h后，用碱性磷酸酶比色法测定其ALP比活性，用碱性磷酸酯酶显色试剂盒（BCIP/NBT alkaline phosphatase color development kit，BCIP/NBT Kit）检测其ALP蛋白表达量，用Western Blot检测其BMP-2蛋白的表达量。结果：细胞毒性实验结果显示，对照组与实验组在与BMSC共孵育48 h后，各组细胞生存率均高于84%，且组间差异无统计学差意义（P>0.05）。在给药浓度相同时，实验组ALP比活性显著高于对照组，其中S1组和SL1组ALP比活性分别为0.27±1.11和0.21±2.15（P<0.001），S2组和SL2组ALP比活性分别为0.63±1.64和0.72±2.88（P<0.05）。ALP BCIP/NBT染色结果表明，与对照组相比，实验组ALP染色更深，且组内呈现剂量依赖性。实验组BMP-2蛋白表达量显著高于对照组。同时，ALP比活性、ALP BCIP/NBT染色及BMP-2蛋白表达结果表明，对照组和实验组内均呈现剂量依赖性，即高剂量组结果优于低剂量组。结论：纳米脂质体剂型能显著提高SMV促进BMSC的成骨分化作用。

关键词: 辛伐他汀, 间充质干细胞, 脂质体, 骨形态发生蛋白质类, 碱性磷酸酶

Abstract: Objective：To investigate the effects of liposomal formulation on simvastatin nano-liposomes (SMV-liposome) promoting the osteogenic differentiation of mice bone marrow stromal cells (BMSC) analyzed by the expressions of bone morphogenetic protein 2 (BMP-2) and alkaline phosphatase (ALP). Methods: Primary BMDC were cultured in vitro using adherence and culture of whole bone marrow method. SMV dosage was set as control group and had two different dosages in this group on the basis of the concentration of SMV. 1 μmol/L and 2 μmol/L SMV concentration were represented by SMV low dosage group (S1) and SMV high dosage group (S2), respectively. Similarly, SMV-liposome dosage was set as experimental group including two different dosages, 1 μmol/L SMV capsuled concentration as SMV-liposome low dosage group (SL1) and 2 μmol/L SMV capsuled concentration as SMV-liposome high dosage group (SL2). Besides, groups with no drug intervention in the experiments were set as blank. BMSC were treated with different concentrations of SMV and SMV-liposome for 48 h, the activity of ALP was measured using p-nitropheny-phosate method, and ALP expression in the BMSC cells was stained by BCIP/NBT alkaline phosphatase color development kit (BCIP/NBT Kit). Furthermore, BMP-2 expression in the BMSC was determined by Western Blot. Results: MTT assay showed, after incubated with different concentrations of SMV and SMV-liposome, the cell viabilities of BMSC were all above 85% and had no significant difference in the groups. Compared with the same dosage of SMV in these groups, control group and experimental group had significantly elevated the specific activity of ALP, the staining of BCIP/NBTKit as well as the protein expression of BMP-2. Besides, the data showed dosedependent elevation in the control group and experimental group, namely the high dose group had better results than the low dose group. Conclusion: Nano-liposomal formulation significantly enhanced SMV effects on the osteogenetic differentiation of BMSC.

Key words: Simvastatin, Mesenchymal stromal cells, Liposomes, Bone morphogenetic proteins, Alkaline phosphatase

徐璐, 王超, 沈文文, 祁荣. 辛伐他汀纳米脂质体对骨髓间充质干细胞成骨分化的影响[J]. 北京大学学报（医学版）, 2014, 46(6): 883-888.

XU Lu, WANG Chao, SHEN Wen-Wen, QI Rong. Effects of simvastatin nano-liposomes on osteogenic differentiation of bone marrow stromal cells[J]. Journal of Peking University(Health Sciences), 2014, 46(6): 883-888.

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