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

1.构建了表达BMP-2的复制缺陷型腺病毒载体AxCAOBMP-2，并对其体外和体内诱导成骨活性进行了研究。用该病毒载体和表达LacZ基因的对照腺病毒载体(AxCALacZ)体外感染C2C12成肌细胞。感染后24小时进行间接免疫荧光检测。感染后第5天检测细胞内碱性磷酸酶(ALP)和培养上清液中骨钙素。此外，为了检测体内BMP-2基因转移的有效性，我们评估了AxCAOBMP-2在环磷酰胺短暂免疫抑制下的成骨诱导作用，在注射腺病毒载体的前一天，腹腔注射剂量为125 mg/kg。将25微升AxCAOBMP-2(8.75×10~(-8)pfu，组I)和AxCALacZ(1.75×10~(^8)pfu，对照组)和5微升AxCAOBMP-2(1.75×10~(^8)pfu，组II)注入Wistar大鼠右侧小腿肌肉。第21天，对各组的骨形成进行放射学和组织学观察。免疫荧光检测证实BMP-2在C2C12细胞中大量表达。通过该载体转导BMP-2基因的C2C12细胞在细胞内产生碱性磷酸酶，并在培养上清液中产生和分泌骨钙素。只有AxCAOBMP-2免疫抑制组有骨诱导。I组的成骨活性高于II组。本研究证实了携带BMP-2基因的腺病毒载体在体内外的骨诱导活性。暂时性免疫抑制下的AxCAOBMP-2基因治疗可能有助于骨重建。参考文献：Okubo Y，Bessho K，Fujimura K，Iizuka T，Miyatake SL，J bone Joint Surg83-A附录1(铂2)：S99-104.2001。)

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.)