力生长因子重组腺病毒载体构建及其在成骨细胞中的表达

背景：研究表明，力生长因子(mechano-growth factor，MGF)能激活卫星细胞，促进成肌细胞增殖，在治疗肌损失、预防心肌损伤和修复神经损伤等方面有重要的作用。机械拉伸可使成骨细胞表达MGF，但是MGF对骨组织生理生化行为的影响机制尚不清楚。目的：应用携带MGF基因的重组腺病毒载体转染成骨细胞，观察MGF在成骨细胞中的表达。方法：将MGF基因插入到腺病毒穿梭质粒pAdTrack-CMV中，构建pAdTrack-MGF重组体。pAdTrack-MGF与腺病毒骨架质粒pAdEasy-1在BJ5183菌中进行同源重组，生成重组腺病毒质粒pAdEasy-MGF。脂质体介导pAdEasy-MGF转染293A细胞，包装成整的重组腺病毒Ad/MGF。用Ad/MGF感染原代培养的大鼠成骨细胞，荧光示踪计数法测定感染效率，RT-PCR法对重组腺病毒感染结果进行鉴定。结果与结论：实验构建的重组腺病毒载体Ad/MGF滴度可达8.5×108 pfu/mL。Ad/MGF能高效感染体外培养的Wistar大鼠成骨细胞并表达目的基因，当感染复数为100时，感染效率最高。说明实验构建的Ad/MGF重组腺病毒可在大鼠成骨细胞中有效表达。

Construction of recombinant adenovirus vector containing mechano-growth factor gene and its expression in rat osteoblasts

BACKGROUND: Studies have confirmed that mechano-growth factor (MGF) can activate satellite cell and facilitate myoblast proliferation, which play an important role in treating muscle loss, preventing myocardial damage and repairing nerve injury. Mechanical stretch can accelerate MGF expression in osteoblast, however, the influence mechanisms of MGF on physiological and biochemical behavior of bone tissues remain poorly understood. OBJECTIVE: Osteoblasts were transfected with recombinant adenovirus vector containing MGF gene to observe the expression of MGF in osteoblasts. METHODS: The MGF coding sequence was cloned to the pAdtrack -CMVplasmid to construct pAdtrack-MGF. Then pAdtrack-MGF was transformed into E. Coli BJ5183 carrying backbone plasmid already, and in which following co-transformation with the backbone vector pAdEasy-1. The homologous recombinant was transfected into human embryo kidney cells 293A through the lipofectamine to pack the adenovirus. The identified recombinant adenovirus (Ad/MGF) was amplified in 293A cells. The infection efficiency was measured by fluorescent tracer technique, and the results of recombinant adenovirus vector infection were identified by RT-PCR. RESULTS AND CONCLUSION: The title of recombinant adenovirus was 8.5×108 pfu/ mL. Osteoblasts infected by Ad/MGF could over express MGF. When multiplicity of infection was equal to 100, the vector had the best efficacy of infection. The recombinant adenovirus vector containing MGF gene could transfect the rat osteoblasts successfully.