Experimental Studies of a Bone Worphogenetic Protein-2 Expressing Adenoviral Vector to the Reconstruction of Bone Deffect.

表达腺病毒载体骨形态发生蛋白2对骨缺损重建作用的实验研究。

• 批准号: 12671934
• 负责人: FUJIMURA Kazuma
• 金额: $ 2.18万
• 依托单位: Kanazawa Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671934/
• 关键词: Adenoviral vector; Osteoinduction; Bone Morphogenetic Protein-2; rhBMP-2; 骨誘導

1. 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 actiyity in vitro and in vivo.2. 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 x10^8 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.3. Abundant BMP-2 expression in C2C12 cells infected with this viral vector was confirmed by immunofluorescence. C2C12 cell 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.4. This study demonstrated the osteoinductive activity in vitro and in vivo by an adenoviral vector carrying the BMP-2 gene. Gene therapy with AxCAOBMP-2 under transient immunosuppression may be useful for bone reconstruction.Reference: Okubo Y, Bessho K, Fujimura K, Iizuka T, Miyatake SL, J Bone Joint Surg Am. 83-A Suppl 1 (Pt 2) : S99-104.2001.)

1.为了检查基因转移程序递送BMP-2的有效性，我们构建了表达人BMP-2的复制缺陷型腺病毒载体AxCAOBMP-2，并评估了其体外和体内的骨诱导活性。2.用该病毒载体或表达大肠杆菌LacZ基因的对照腺病毒载体（AxCALacZ）体外感染C2 C12成肌细胞。感染后24小时，进行间接免疫荧光。在感染后第5天，测量细胞中的碱性磷酸酶（ALP）和培养基中的骨钙素。此外，为了检查BMP-2在体内的基因转移的有效性，我们评估了AxCAOBMP-2的骨诱导作用，在注射腺病毒载体的前一天，在环磷酰胺的瞬时免疫抑制下，以125 mg/kg的剂量腹膜内给药。将25微升AxCAOBMP-2（8.75 × 10^8空斑形成单位[pfu]，组I）和AxCALacZ（1.75 × 10^8 pfu，对照组）以及5微升AxCAOBMP-2（1.75 × 10^8 pfu，组II）注射到Wistar大鼠的右小腿肌肉中。在第21天，通过放射学和组织学观察各组的骨形成情况.通过免疫荧光证实了用该病毒载体感染的C2 C12细胞中BMP-2的丰富表达。用该载体转染BMP-2基因的C2 C12细胞在细胞内产生ALP，并在培养基中产生和分泌骨钙素。仅在免疫抑制的AxCAOBMP-2处理组中发现骨诱导作用。组I的骨诱导活性高于组II。4.本研究证明了携带BMP-2基因的腺病毒载体在体外和体内的骨诱导活性。在瞬时免疫抑制下用AxCAOBMP-2进行基因治疗可用于骨重建。参考文献：Okubo Y，Bessho K，Fujimura K，Iizuka T，Miyatake SL，J Bone Joint Surg Am. 83-A增刊1（第2部分）：S99-104.2001。）

项目成果

Okubo Y, Bessho K, Fujimura K, Kaihara S, Iizuka T, Miyatake S: "The time course study of osteoinduction by bone morphogenetic protein-2 via adenoviral vector"Life Sci. 70(3). 325-336 (2001)

Okubo Y、Bessho K、Fujimura K、Kaihara S、Iizuka T、Miyatake S：“骨形态发生蛋白 2 通过腺病毒载体进行骨诱导的时间过程研究”生命科学。

Y Okubo, K Bessho, K Fujimura, T lizuka, SI Miyatake: "Osteoinductioh by morphogenetic protein-2 via adenoviral vector under transient immunosupression."Biochem. Biophys. Res. Commun.. 267 (1). 382-387 (2000)

Y Okubo、K Bessho、K Fujimura、T lizuka、SI Miyatake：“在瞬时免疫抑制下通过腺病毒载体通过形态发生蛋白 2 进行骨诱导。”Biochem。

Okubo Y, Bessho K, Fujimura K, lizuka T, Miyatake SI: "In vitro and in vivo studies of a bone morphogenetic protein-2 expressing adenoviral vector."J Bone Joint Surg Am. 83-A Suppl 1 (Pt 2). S99-104 (2001)

Okubo Y、Bessho K、Fujimura K、lizuka T、Miyatake SI：“表达骨形态发生蛋白 2 的腺病毒载体的体外和体内研究。”J Bone Joint Surg Am。

K Bessho, Y Konishi, S Kaihara, K Fujjmujra, Y Okubo, T Iizuka: "Bone induction by Escherichia colli derived recombinant human bone morphogenetic protein-2 compared with chinese hamster ovary cell-derived recombinant human bone morphogenetic protein-2."Br

K Bessho、Y Konishi、S Kaihara、K Fujjmujra、Y Okubo、T Iizuka：“与中国仓鼠卵巢细胞来源的重组人骨形态发生蛋白 2 相比，大肠杆菌来源的重组人骨形态发生蛋白 2 的骨诱导作用。”Br

Fujimura K, Bessho K, Kusumoto K, Konishi K, Ogawa Y, lizuka T: "Experimental osteoinduction by recombinant human bone morphogenetic protein 2 in tissue with low Wood flow : a study in rats."Brit. J. Oral. Maxillofac. Surg.. 39(4). 294-300 (2001)

Fujimura K、Bessho K、Kusumoto K、Konishi K、Okawa Y、lizuka T：“重组人骨形态发生蛋白 2 在低木流组织中进行的实验性骨诱导：一项大鼠研究。”