人转化生长因子β_1及骨形成蛋白7基因共表达载体的构建及对骨髓基质干细胞的定向诱导作用

CONSTRUCTION OF RECOMBINANT ADENOVIRUS BEARING HUMAN TRANSFORMING GROWTH FACTOR β_1 AND BONE MORPHOGENETIC PROTEIN 7 GENES AND ITS DIRECTIONAL INDUCEMENT EFFECT ON MARROW STROMAL STEM CELLS

目的构建人转化生长因子1β(transform ing grow th factor1β,TGF-1β)及骨形成蛋白7(bonem orphogenetic prote in 7,BM P-7)基因共表达腺病毒真核表达载体,并观察感染骨髓基质干细胞(m arrow strom a l stemce lls,M SC s)后目的基因的表达和对细胞生物学行为的影响。方法以复制缺陷的腺病毒A dE asy为基因载体,制备携带TGF-1β及BM P-7基因的高滴度腺病毒液感染人M SC s,通过免疫细胞化学、原位杂交、RT-PCR及己糖醛酸水平检测等方法鉴定外源基因的表达,分析外源性基因共表达对M SC s定向软骨分化的调控机制。结果腺病毒感染72 h后,TGF-1β和BM P-7免疫细胞化学染色可见大部份M SC s胞浆内均出现棕黄色粗颗粒,通过原位杂交检测出Ⅱ型胶原蛋白基因mRNA,感染10 d后细胞培养液中己糖醛酸含量为68.03±3.34μg/m l与感染前的53.20±3.70μg/m l明显升高有统计学意义(P<0.01)。结论成功构建了人TGF-1β及BM P-7共表达基因腺病毒表达载体,证实其在M SC s中的表达和具有向软骨细胞定向分化的诱导作用,为软骨缺损修复的局部基因治疗奠定实验基础。

Objective To construct the recombinant adenovirus bearing human transforming growth factor β1 （TGF-β1） and hone morphogenetie protein 7 （BMP-7） genes, and investigate its co expression in the marrow stromal stem cells （MSCs） and bioactivity effect. Methods Using the replication defective adenovirus AdEasy as a carrier, MSCs were infected by the high titer-tevel recombinant adenovirus taking TGF-β1 and BMP 7 genes. immunocytochemistry, in silu hybridization,reverse transcription polymerase chain reaction （RT-PCR）, and hexuronic acid level test were used to） detect the co expression of the exogenous genes and to analyze their effect transfection on directive differentiation of MSCs. Results The immunoeytochemistry staining showed that the brown coarse grains were situated in the cytoplasm of the most MSCs 72 h after infection. Procollagen Ⅱ mRNA in the ceils was detected by the in situ hybridization, and the content of hexuronie acid in the culture medium was significantly increased 10 days after infection compared with the level before infeeton （P〈0.01）. Conclusion The recombinant adenovirus bearing human TGF-β1 and BMP 7 genes can be constructed, and the exogenous gene can be co-expressed in MSCs, which may offer a novel approach to the local combination gene therapy for repairing joint cartilage defects.