慢病毒介导C-1—1基因维持“自组装”工程化软骨永久性表型的研究

Perpetual phenotype of self-assembled tissue-engineered cartilages transferred with lentiviral-mediated C-1-1

目的观察慢病毒介导C-1—1基因对维持“自组装”工程化软骨永久性表型的影响。方法构建携带C-1-1基因的重组慢病毒表达载体并转染成人骨髓间充质干细胞（hMSCs），用嘌呤霉素筛选获得阳性细胞。逆转录-聚合酶链反应（RT—PCR）和免疫印迹试验（Westernblot）观察C-1—1基因的表达效果。用含有生长分化因子-5（GDF-5）的成软骨培养基诱导培养3周，3周后重悬细胞，以5×10^9个／L的细胞终质量浓度接种于2％琼脂糖包被的24孔板，行自组装培养3周后取材。通过Ⅱ型、x型胶原免疫组织化学，甲苯胺蓝染色鉴定分化结果，并与空载体转染组和未转染组比较。结果测序证实成功构建携带C-1-1基因的重组慢病毒表达载体，转染hMSCs后，C-1—1基因在转录水平和翻译水平都有明显表达。自组装培养3周后，3组标本经甲苯胺蓝染色均可见广泛蓝染并带有异染型着色。C-1-1基因转染组的Ⅱ型胶原平均吸光度（A）值为（0．3754±0．0255），与空载体转染组和未转染组比较，差异无统计学意义（P〉0．05）；X型胶原平均A值为（0．0115±0．0062），显著低于空载体转染组和未转染组（P〈0．01）。结论慢病毒介导C-1—1基因转染后，可增强“自组装”工程化软骨表型的稳定性，抑制其成熟肥大。

Objective To observe the effect of lentiviral-mediated C-l-1 on maintenance of per- petual phenotype of self-assembled engineered cartilages. Methods The lentiviral expression vector carry- ing C-1-1 gene was constructed and transfected into cultured human bone marrow mesenchymal stem cells （hMSCs）, and then resistance clones were acquired by puromycin screening. The expression of C-l-1 gene was detected by using semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting, hMSCs at passage 3 were induced with chondrogenic medium containing 200 p,g/L growth differentiation factor 5 （GDF-5） for 3 weeks. Three weeks later, the cells were suspended and then inocu-lated into each well of 2% agarose-coated 24-well plates at a density of 5 x 106/mL. Another 3 weeks later, the differentiating effect was identified by histological staining. Results The successful construction of the lentiviral expression vector carrying C-l-1 gene was identified by sequencing. After the lentiviral ex- pression vector was transfected into the hMSCs, the expression of C-l-1 mRNA and protein was enhanced. After self-assembly culture for 3 weeks, toluidine blue staining was positive. The mean absorbance （A） values of Collagen Ⅱ in C-1-1 gene transfection group was （0. 3754 ±0. 0255）, which was not higher than that in the control group （P 〉 0. 05）. The mean A values of Collagen X in C-1-1 gene transfection group was （0. 0115± 0. 0062）, which was lower than that in the control group （P 〈 0. 001 ）. Conclusion After C-l-1 gene was transfected through lentiviruses into the hMSCs, the phenotype of self-assembled engineered cartilages was more stable while their maturation and hypertrophy was inhibited.