一种表达骨形态发生蛋白-2的腺病毒载体的体外和体内研究。

In vitro and in vivo studies of a bone morphogenetic protein-2 expressing adenoviral vector.

作者信息

Okubo Y, Bessho K, Fujimura K, Iizuka T, Miyatake S I

机构信息

Graduate School of Medicine, Kyoto University, Japan.

出版信息

J Bone Joint Surg Am. 2001;83-A Suppl 1(Pt 2):S99-104.

PMID:11314802

Abstract

BACKGROUND

Bone morphogenetic proteins (BMPs) play important roles in the migration of osteoblast progenitor cells, the proliferation of mesenchymal cells, and their differentiation into chondrogenic and osteogenic cells. However, the optimum procedure to deliver BMPs remains unknown. To examine the effectiveness of a gene transfer procedure for the delivery of BMP-2, we constructed a human BMP-2-expressing replication-deficient adenoviral vector, AxCAOBMP-2, and evaluated its osteoinductive activity in vitro and in vivo.

METHODS

C2C12 myoblasts were infected in vitro with this viral vector or an Escherichia coli LacZ gene-expressing control adenovirus vector (AxCALacZ). Twenty-four hours after the infection, indirect immunofluorescence was performed. On day 5 after the infection, alkaline phosphatase (ALP) in the cells and osteocalcin in the culture medium were measured. Furthermore, to examine the effectiveness of gene transfer of BMP-2 in vivo, we evaluated osteoinduction by AxCAOBMP-2, under transient immunosuppression with cyclophosphamide, given at a dose of 125 mg/kg intraperitoneally the day before injection of the adenoviral vector. Twenty-five microliters of AxCAOBMP-2 (8.75 x 10(8) plaque-forming units [pfu], Group I) and AxCALacZ (1.75 x 10(8) pfu, control group) and 5 microl of AxCAOBMP-2 (1.75 x 10(8) pfu, Group II) were injected into a right calf muscle of Wistar rats. On day 21, bone formation in each group was investigated radiologically and histologically.

RESULTS

Abundant BMP-2 expression in C2C12 cells infected with this viral vector was confirmed by immunofluorescence. C2C12 cells transferred with the BMP-2 gene by this vector produced ALP in the cells and also produced and secreted osteocalcin in the culture medium. Osteoinduction was found only in the AxCAOBMP-2 treated groups with immunosuppression. Osteoinduction activity was higher in Group I than in Group II.

CONCLUSION

This study demonstrated the osteoinductive activity in vitro and in vivo by an adenoviral vector carrying the BMP-2 gene.

CLINICAL RELEVANCE

Gene therapy with AxCAOBMP-2 under transient immunosuppression may be useful for bone reconstruction.

摘要

背景

骨形态发生蛋白（BMPs）在成骨细胞祖细胞迁移、间充质细胞增殖以及它们向软骨细胞和成骨细胞分化过程中发挥重要作用。然而，递送BMPs的最佳方法仍不明确。为了检测基因转移方法递送BMP - 2的有效性，我们构建了一种表达人BMP - 2的复制缺陷型腺病毒载体AxCAOBMP - 2，并在体外和体内评估其骨诱导活性。

方法

将C2C12成肌细胞在体外感染该病毒载体或表达大肠杆菌LacZ基因的对照腺病毒载体（AxCALacZ）。感染后24小时，进行间接免疫荧光检测。感染后第5天，检测细胞中的碱性磷酸酶（ALP）和培养基中的骨钙素。此外，为检测BMP - 2基因转移在体内的有效性，我们在注射腺病毒载体前一天腹腔注射剂量为125 mg/kg的环磷酰胺进行短暂免疫抑制的情况下，评估AxCAOBMP - 2的骨诱导作用。将25微升AxCAOBMP - 2（8.75×10⁸ 空斑形成单位 [pfu]，第一组）和AxCALacZ（1.75×10⁸ pfu，对照组）以及5微升AxCAOBMP - 2（1.75×10⁸ pfu，第二组）注射到Wistar大鼠的右小腿肌肉中。在第21天，对每组的骨形成进行放射学和组织学研究。