重组腺病毒介导外源性SOX9在正常关节软骨细胞中诱导软骨基质合成的体外表达

Induction of cartilage matrix by recombinant adenoviral vector mediated expression of SOX9 in articular chondrocytes

背景:关节软骨损害是临床一种常见的损伤,软骨形成转录因子SOX9是一种调控Ⅱ型胶原合成的关键因子。目的:观察以表达外源性SOX9的腺病毒体外成功感染关节软骨细胞后对Ⅱ型胶原和蛋白聚糖(Aggrecan)合成的影响,探讨软骨损伤后修复软骨缺损的基因治疗方法。方法:体外构建腺病毒载体AdSOX9和AdGFP,成功感染培养的人关节软骨细胞,分别以逆转录聚合酶链式反应(RT-PCR)技术检测了病毒感染前后SOX9、Ⅱ型胶原和蛋白聚糖基因mRNA的表达,同时以免疫组化技术检测了病毒感染前后关节软骨细胞中Ⅱ型胶原的表达。结果:应用AdEasy腺病毒构建专利技术体外成功构建了能高效表达外源性SOX9的腺病毒AdSOX9和只表达绿色荧光蛋白GFP的腺病毒AdGFP;以腺病毒AdSOX9和AdGFP体外成功感染人关节软骨细胞后48h,未感染对照组和AdGFP感染组,均检测到了Ⅱ型胶原和蛋白聚糖的表达;而AdSOX9感染组的细胞中,检测到了SOX9基因mRNA的表达明显增高,与未感染对照组和AdGFP感染组相比有显著性差异(P<0.05),同时,Ⅱ型胶原和蛋白聚糖的表达也较未感染对照组和AdGFP感染组明显增高,差异显著(P<0.05)。结论:以外源性SOX9为目的基因的腺病毒介导基因治疗方法,在促进关节软骨细胞Ⅱ型胶原和蛋白聚糖合成方面得出了初步满意的结果,SOX9可能是关节软骨缺损基因治疗研究领域一个新的理想靶点,值得继续深入研究。

Background ： Articular cartilage damage is a common clinical injury, the transcription factor SOX9 is a key factor in regulating the synthesis of type Ⅱ collagen. Objective： In this study, we studied the expression of type Ⅱ collagen and aggrecan after Adenovirus AdSOX9 infecting articular ehondrocytes. Methods： We constructed the Adenovirus AdSOX9, AdGFP and successfully infected cultured human articular chondrocytes with adenoviral vectors expressing SOX9 and GFP. Type Ⅱ collagen and aggrecan production were measured using RT-PCR and immunohistochemical analyses. Results ： AdSOX9 virus efficiently infected cultured human articular chondrocytes resulting in increased transcription of the type Ⅱ collagen and aggrecan progene and increased production of type Ⅱ collagen and aggrecan. Meanwhile, the significantly increased production of SOX9 mRNA was also measured. Conclusions ： Adenovirus mediated gene therapy using SOX9 represents an attractive modality for the treatment of articular cartilage defects in the knee, SOX9 may be an ideal candidate to study for gene therapy in articular cartilage defects and warrants further study in an animal model.