兔骨髓间充质干细胞经Ad-TGFβ1重组腺病毒载体转染定向分化为软骨细胞

Rabbit mesenchymal stem cells differentiate into chondrocytes by transfection of Ad-TGFβ1 recombinant adenovirus vector

目的:通过软骨组织工程与基因治疗相结合的技术,利用腺病毒载体将转化生长因子β1基因导入兔骨髓间充质干细胞,使其诱导靶细胞定向分化为软骨细胞。方法:实验于2004-03/2006-03在徐州医学院完成。①实验方法:兔麻醉后自胫骨上端抽取骨髓,体外分离培养骨髓间充质干细胞。取第2代细胞,以1×104/cm2接种,达70%融合时分为3组:Ad-TGFβ1转染组采用H-DMEM无血清培养基,加入刚解冻的Ad-TGFβ1液30μL,地塞米松100nmol/L,维生素C50mg/L;Ad-GFP对照组采用H-DMEM无血清培养基,加入Ad-GFP腺病毒,地塞米松100nmol/L,维生素C50mg/L;空白对照组采用H-DMEM完全培养基,不外加任何试剂或药品。②实验评估:倒置显微镜逐日观察细胞生长情况。分别于转染后7,14,21,28d检测细胞内蛋白多糖、转化生长因子β1、Ⅱ型胶原的表达。结果:①细胞形态学观察:Ad-TGFβ1转染后7d,集落中的细胞呈放射状向周围扩展,均一性好,呈长梭形,紧密排列似漩涡状,生长速度明显增快,细胞密度明显增高。②转染后细胞内蛋白多糖的检测:Ad-TGFβ1转染后14d胞浆呈紫蓝色,异染性明显,胞核清晰,可见核仁。转染后28d细胞明显老化,但胞浆中仍可见紫蓝色异染。其余两组均未见明显的异染性。③转染后转化生长因子β1的表达:Ad-TGFβ1转染组细胞中转化生长因子β1呈强阳性表达,其余两组几乎检测不到转化生长因子β1的表达。④转染后细胞内Ⅱ型胶原的表达:Ad-TGFβ1转染后7d胞浆中Ⅱ型胶原呈弱阳性表达,转染后14,21d呈强阳性表达,转染后28d表达有所降低。其余两组几乎不表达Ⅱ型胶原。结论:经Ad-TGFβ1重组腺病毒载体转染,兔骨髓间充质干细胞可定向分化为软骨细胞,是获得软骨组织工程种子细胞的方法之一。

AIM： With the combination of cartilage tissue engineering and gene therapy, to induce the differentiation of target cells into chondrocytes by transferring transforming growth factor （TGFβ1） through adenovirus into rabbit bone marrow mesenchymal stem cells （MSCs）. METHODS： The experiment was performed in Xuzhou Medical College from March 2004 to March 2006. （1）The bone marrow was drawn from the proximal tibia of anesthetized rabbit, and MSCs were separated and cultured in vitro. The second generation cells were seeded at 1× 10^4/cm^2, and when the cells were about 70% fusion, they were divided into 3 groups： Ad-TGFβ1 group was cultured with H-DMEM serum-free medium by adding 30 μL Ad-TGFβ1, 100 nmol/L dexamethasone and 50 mg/L Vitamine C; Ad-GFP group with H-DMEM serum-free medium by adding Ad-GFP gland virus, 100 nmol/L dexamethasone, and 50 mg/L Vitamine C; blank control group was with in H-DMEM total medium without any other reagent or drugs. （2）Cell growth was observed under inverted microscope every day. The expression of glycosaminoglycans （GAGs）, TGFβ1, and collagen Ⅱ was detected at 7, 14, 21 and 28 days after transferring. RESULTS： （1）Seven days after Ad-TGFβ1 transferring, the concentrated cells grew radially to the surrounding, and presented long fusiform shape; cells were arranged densely like swirl, and grew remarkably with high cell density. （2） Fourteen days after Ad-TGFβ1 transferring, the kytoplasma was blue violet, with strong metachromasia, clear nuclei and visible nucleoli. However, the cells looked in degradation 28 days after transferring, but still with blue violet staining in cytoplasma. There was no metachromasia in other two groups. （3）There was strong TGFβ1 expression in Ad-TGFβ1 group, but no expression was detected in other two groups. （4）Weak positive collagen Ⅱ expression was observed 7 days after Ad-TGFβ1 transferring, but strong positive expression at 14 and 21 days after Ad-TGFβ1 transferring; On the 28^th day, the expression was slightly decreased. No collagen Ⅱ expression was found in other two groups. CONCLUSION： The rabbit MSCs could be induced to differentiate into chondrocytes after Ad-TGFβ1 through recombinant adenoviros vector. It is one of the effective methods to obtain seed cells for cartilage tissue engineering.