腺病毒介导的BMP-2基因转染兔骨髓基质干细胞及对其增殖、成骨分化的影响-东南大学学报医学版

腺病毒介导的BMP-2基因转染兔骨髓基质干细胞及对其增殖、成骨分化的影响

单位：1. 中国医科大学附属盛京医院脊柱创伤外科, 辽宁沈阳 110004;
2. 中国医科大学附属第一医院骨科, 辽宁沈阳 110001

分类号：Q786; R329.28

出版年·卷·期（页码）：2009·28·第一期（22-26）

摘要：

目的: 用骨形态发生蛋白-2(BMP-2)腺病毒表达载体(Ad-BMP-2)转染兔骨髓基质干细胞(BMSCs),研究转染对其增殖、成骨分化的影响。方法: 用Ad-BMP-2转染兔BMSCs,利用免疫细胞化学法、原位杂交染色和蛋白印迹法检测转染后细胞BMP-2的表达。流式细胞仪观察细胞周期的变化,分析转染对BMSCs增殖的影响。酶标法检测ALP活性;免疫荧光检测骨钙素(OCN)、Ⅰ型胶原表达,分析转染对BMSCs成骨分化的影响。结果: BMP-2基因在转染细胞内mRNA水平和蛋白水平均有较高表达,蛋白印迹法检测到培养液中有BMP-2蛋白阳性表达。流式细胞仪检测G₁期、S期细胞比例与空白对照组无差异。ALP活性明显增高,骨钙素、Ⅰ型胶原免疫荧光检测,见胞浆内有大量显绿色荧光的阳性物质。结论: 在腺病毒载体介导下BMP-2基因成功导入BMSCs,细胞增殖未因转染而受影响,并可持续表达基因产物,向成骨细胞分化。

Objective To study the effects of adenovirus-BMP-2(Ad-BMP-2) transfection on proliferation and differentiation of rabit bone marrow stromal cells (BMSCs) in vitro.Methods Ad-BMP-2 transfected rabbit BMSCs.mRNA and protein expression of BMSCs were detected by BMP-2 hybridization in situ and immunocytochemical technique and Western blotting analysis.The change of BMSCs cell cycle after transfection was observed by flow cytometry.Alkaline phosphatase activity and staining,immunofluorescence analysis of osteocalcin and collagen Ⅰ were used to analyze the effect of BMP-2 gene transfection on BMSCs proliferation and differentiation.Results In the hybridization in situ and immunocytochemical analysis,we observed positive signal in the plasma of transfected BMSCs.In the Western blotting analysis,positive signal lane of BMP-2 was observed in transfected group,whereas in the control group no signal was seen.Flow cytometry showed there was no difference of cell proportion of G₁ phase and S phase between tansfected BMSCs group and the control group.Alkaline phosphatase activity of transfected cells was significantly higher than that in the control group.Positive green flurescent substance in plasma was seen in the immunofluorescence analysis of osteocalcin and collagenⅠ and immunocytochemical analysis.Conclusion Ad-BMP-2 can efficiently infect BMSCs and promote cell proliferation and osteoblast conversion.

参考文献：

[1] YAMAGUCHI A,ISHIZUYA T,KINTOU N,et al.Effects of BMP-2,BMP-4,and BMP-6 on osteoblastic differentiation of bone marrow-derived stromal cell lines,ST2 and MC3T3-G2/PA6[J].Biochem Biophys Res Commun,1996,220:366-371.
[2] BODEN S D,GROB D,DAMIEN C.Ne2-steo bone growth factor for posterolateral lumbar spine fusion:results from a nonhuman primate study and a prospective human clinical pillot study[J].Spine,2004,29(5):504-514.
[3] CAMPER Y.Bone grafts and substitutes[J].Orthop Network,1995,6:7-9.
[4] 吴宗键,王继芳,卢世璧.诱导成骨的局部基因治疗[J].中华骨科杂志,2001,21(12):760-762.
[5] RAWADIG,VAYSSIERE B,DUNN F,et al.BMP-2 controls alkaline phosphatase expression and osteoblast mineralization by a Wnt autocrine loop[J].J Bone Miner Res,2003,18(10):1842-1853.
[6] HOSOI T.Bone and bone related biochemical examinations.Bone and collagen related metabolites.Structure and metabolisms of collagen[J].Clin Calcium,2006,16(6):971-977.
[7] KAO P C,RIGGS B L,SCHRYVER P G.Development and evaluation of an osteocalcin chemiluminoimmunoassay[J].Clin Chem,1993,39(7):1369-1374.
[8] EBARA S,NAKAYAMA K.Mechanism for the action of bone morphogenetic proteins and regulation of their activity[J].Spine,2002,27(16Suppl 1):s10-s15.

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