真核表达人Wnt7b基因建立软骨细胞退变模型

Establishment of a chondrocyte degeneration model by over-expression of human Wnt7b gene in 293ft cell line

目的在真核细胞中表达人Wnt7b基因并且观察对大鼠原代软骨细胞的退变作用。方法取出生24 h SD大鼠关节处软骨,Ⅱ型胶原酶多次消化后获得原代软骨细胞,取P1代细胞进行实验。扩增人Wnt7b基因及克隆至PCDH-GFP上,转染PCDH-GFP和PCDH-Wnt7b至293ft细胞中,48 h后收集细胞上清及转染细胞,Western blot鉴定Wnt7b在293ft细胞中的表达。收集的上清分别稀释10倍和50倍培养大鼠软骨细胞,24 h后观察细胞形态并收集细胞蛋白和抽提RNA,Western blot和定量PCR检测软骨退变指标MMP13、MMP3、Ⅱ型胶原、A-can、ADAMTS5、ColⅩ和SOX9的表达。结果成功克隆人Wnt7b基因至PCDH-GFP载体上且在293ft细胞中得到有效表达。含Wnt7b培养基干预软骨细胞24 h后,软骨细胞形态由原来的多角形变成长梭形,Wnt7b处理组软骨细胞MMP13和MMP3表达显著上调,Ⅱ型胶原的表达下调。PCR结果表明A-can和Sox9表达下降,ColⅩ和ADAMTS5表达增强。结论有效在真核细胞中表达Wnt7b基因并且促进大鼠软骨细胞退变,建立软骨细胞退变的体外模型。

Objective To investigate the role of human Wnt7 b gene in rat chondrocyte degeneration. Methods Wnt7 b gene obtained by PCR was cloned to PCDH- GFP. 293 ft cell line was transfected with PCDH- GFP and PCDH- Wnt7 b, and the supernatant and transfected cells were collected. The expression level of Wnt7 b in 293 ft cells was detected by Western blotting.The first passage of chondrocytes were isolated from articular cartilages of newborn born（within 24 h） SD rats were cultured in the supernatants from the transfected cells（at 10- and 50-fold dilutions）. The cell morphology of the rat chondrocytes was observed under inverted microscope, and the protein expressions of MMP13, MMP3 and type II collagen and m RNA expressions of A-can, ADAMTS5, Col X and Sox9 were examined by Western blotting or real-time PCR. Results Human Wnt7 b gene cloned to PCDH- GFP was expressed efficiently in 293 ft cell line. Rat chndrocytes cultured for 24 h in the supernatants from PCDH-Wnt7b-transfected 293 ft cells underwent changes from a polygonal to a spindle-shaped morphology. The protein expression levels of MMP13 and MMP3 increased while type II collagen decreased significantly, and the m RNA levels of A-can and Sox9 were down-regulated while Col X and ADMATS5 up-regulated in ratchondrocytes after incubation in supernatants from PCDH- Wnt7b- transfected 293 ft cells. Conclusion Human Wnt7 b gene can be expressed efficiently in 293 ft cell line and can induce rat chondrocyte degeneration in vitro.