CAJ | 학술논문

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(Western blot)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5×109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.
목적 관찰만병독개도C-1-1기인대유지“자조장”공정화연골영구성표형적영향.방법 구건휴대C-1-1기인적중조만병독표체재체병전염성인골수간충질간세포(hMSCs),용표령매소사선획득양성세포.역전록-취합매련반응(RT-PCR)화면역인적시험(Western blot)관찰C-1-1기인적표체효과.용함유생장분화인자-5(GDF-5)적성연골배양기유도배양3주,3주후중현세포,이5×109개/L적세포종질량농도접충우2％경지당포피적24공판,행자조장배양3주후취재.통과Ⅱ형、X형효원면역조직화학,갑분알람염색감정분화결과,병여공재체전염조화미전염조비교.결과 측서증실성공구건휴대C-1-1기인적중조만병독표체재체,전염hMSCs후,C-1-1기인재전록수평화번역수평도유명현표체.자조장배양3주후,3조표본경갑분알람염색균가견엄범람염병대유이염형착색.C-1-1기인전염조적Ⅱ형효원평균흡광도(A)치위(0.3754±0.0255),여공재체전염조화미전염조비교,차이무통계학의의(P＞0.05)；X형효원평균A치위(0.0115±0.0062),현저저우공재체전염조화미전염조(P＜0.01).결론 만병독개도C-1-1기인전염후,가증강“자조장”공정화연골표형적은정성,억제기성숙비대.
Objective To observe the effect of lentiviral-mediated C-1-1 on maintenance of perpetual phenotype of self-assembled engineered cartilages.Methods The lentiviral expression vector carrying 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-1-1 gene was detected by using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Westem blotting.hMSCs at passage 3 were induced with chondrogenic medium containing 200 μg/L growth differentiation factor 5 (GDF-5) for 3 weeks.Three weeks later,the cells were suspended and then inoculated into each well of 2％ agarose-coated 24-well plates at a density of 5 × 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-1-1 gene was identified by sequencing.After the lentiviral expression vector was transfected into the hMSCs,the expression of C-1-1 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-1-1 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.