地塞米松诱导联合骨形成蛋白2基因修饰兔骨髓间质干细胞的成骨转化★

Abstract

BACKGROUND:Mesenchymal stem cells (MSCs) can differentiate into osteoblasts in the action of dexamethasone; Meanwhile, bone morphogenetic protein-2 (BMP-2) can promote the cell proliferation and differentiation, and matrix secretion in the process of bone repair. BMP-2 plays an important role in treating fracture and bone defects by inducing bone formation both in vivo and in vitro.
OBJECTIVE: To analyze whether dexamethasone induction in vitro could enhance the ability of BMP-2 modified MSCs in osteogenic conversion.
DESIGN: A randomized paired design.
SETTING: Xijing Hospital of the Fourth Military Medical University of Chinese PLA.
MATERIALS: The experiments were carried out in the Orthopaedic Oncology Institute of Chinese PLA, the Fourth Military Medical University of Chinese PLA from February to August in 2004. Twenty 20-month-old New Zealand rabbits were provided by the experimental animal center of the Fourth Military Medical University of Chinese PLA [Certificate number: 2005C00117]. The rabbits were raised normally at room temperature with normal humidity. The samples were collected bilaterally, the cells from the left limb were taken as the dexamethasone-induced group, and those from the right limb as the control group.
METHODS: MSCs treated and untreated with dexamethasone were transfected with BMP-2 gene, then the BMP-2 expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical staining. The growths of the MSCs were observed, and the activity of alkaline phosphatase (ALP) and osteocalcin of MSCs were determined with ALP kit and osteocalcin radioimmunoassay kit after Ad-BMP-2 transfection.
MAIN OUTCOME MEASURES: ① BMP-2 expression in MSCs; ② Morphological changes of the MSCs; ③ ALP activity and osteocalcin content in the MSCs.
RESULTS: ① Expressions of BMP-2 gene could be observed in MSCs both treated and untreated with dexamethasone after transfection. ② The forms were irregular in most of the MSCs treated with dexamethasone, appeared as triangular or polygonal changes.They grew more slowly than those cultured with basic medium. There were no obvious changes of cellular forms after gene transfection. ③ Five days after transgene, the ALP activity in the MSCs supernatant in the dexamethasone-induced group was higher than that in the control group [(134.36±8.84), (104.02±7.83) nkat/L, t =3.350 6, P < 0.01, n =20]. The amount of osteocalcin secretion in MSCs in the dexamethasone-induced group was higher than that in the control group [(14.68±0.73), (6.52±1.21) μg/L, t =3.568 2, P < 0.01, n =20].
CONCLUSION: Dexamethasone induction before transgene can promote the proliferation of BMP-2 modified MSCs and the conversion into osteoblasts.

1Center of Rehabilitation, 2Department of Pharmacy, Xijing Hospital, Fourth Military Medical University of Chinese PLA, Xi'an 710033, Shaanxi Province, China

Li Yan★, Master, Associate chief pharmacist, Center of Rehabilitation, Xijing Hospital, Fourth Military Medical University of Chinese PLA, Xi'an 710033, Shaanxi Province, China kevin99@fmmu.
edu.cn

Received: 2006-04-26
Accepted: 2006-07-03
(06-50-4-3731/G)

Li Y, Yan L, Shen M.Dexamethasone induces osteoblast conversion of bone morphogenic protein-2 modified bone marrow mesenchymal stem cells in rabbits. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(2): 377-380(China)

[www.zglckf.com/
zglckf/ejournal/
upfiles/08-2/
2k-377(ps).pdf]

摘要
背景：骨髓间质干细胞在地塞米松等骨诱导剂的作用下能够向成骨细胞分化；同时骨形成蛋白2在骨修复过程中促进细胞增殖分化和基质分泌，在体内外均可诱导骨形成，以上二者联合应用可能会产生一定的协同效应。
目的：分析体外经地塞米松诱导是否能增强骨形成蛋白2基因修饰的骨髓间质干细胞的成骨转化能力。
设计：随机化配对设计。
单位：解放军第四军医大学西京医院。
材料：实验于2004-02/2004-08在解放军第四军医大学全军骨肿瘤研究所完成。选用2月龄新西兰白兔20只，由解放军第四军医大学实验动物中心提供（许可证号：军动管字第2005C00117号）。室温、常湿，正常喂食。双侧取材，左侧肢体来源骨髓间质干细胞为地塞米松诱导组，右侧为对照组（未经诱导）。
方法：将地塞米松诱导组和对照组兔骨髓间质干细胞分别转导含有人骨形成蛋白2基因的复制缺陷重组腺病毒Ad-BMP-2后，以反转录-聚合酶链反应和免疫组化方法检测细胞中骨形成蛋白2的表达情况。观察骨髓间质干细胞的生长情况，并应用碱性磷酸酶检测试剂盒及骨钙素放免试剂盒测定Ad-BMP-2转导5 d后两组骨髓间质干细胞的碱性磷酸酶活性和骨钙素含量。
主要观察指标：①骨髓间质干细胞中骨形成蛋白2的表达情况。②骨髓间质干细胞的形态学变化。③骨髓间质干细胞中碱性磷酸酶活性和骨钙素含量。
结果：①转导后骨形成蛋白2基因在地塞米松诱导组和对照组兔骨髓间质干细胞中均有表达。②骨髓间质干细胞经地塞米松成骨诱导后形态较不规则，呈三角形、多角形改变，较基础培养基培养细胞生长缓慢。基因转导后细胞形态无明显改变。③转基因5 d后，地塞米松诱导组骨髓间质干细胞培养上清中碱性磷酸酶活性高于对照组[(134.36±8.84，104.02±7.83) nkat/L(t =3.350 6，P < 0.01，n =20)]；地塞米松诱导组骨髓间质干细胞骨钙素分泌量高于对照组[(14.68±0.73，6.52±1.21) μg/L(t =3.568 2，P < 0.01，n =20)]。
结论：转基因前的地塞米松诱导能够促进骨形成蛋白2基因修饰的骨髓间质干细胞增殖和成骨转化。
关键词：骨髓细胞；间质干细胞；骨形态发生蛋白质类；地塞米松；骨生成

栗 艳1，闫 露1，沈 敏2
解放军第四军医大学西京医院，1康复诊疗中心，2药剂科，陕西省西安市 710033
栗 艳★，女，1960年生，山西省原平市人，汉族，解放军第四军医大学毕业，硕士，副主任药师，主要从事基因药物研究及治疗。


中图分类号: R394.2 文献标识码: A 文章编号: 1673-8225(2008)02-00377-04
栗艳，闫露，沈敏.地塞米松诱导联合骨形成蛋白2基因修饰兔骨髓间质干细胞的成骨转化[J].中国组织工程研究与临床康复,2008,12(2):377-
380 [www.zglckf.com/zglckf/ejournal/upfiles/08-2/2k-377(ps).pdf]
(Edited by Song HP/Yin YL/Wang L)