Effects of RNAi-mediated inhibition of aggrecanase-1 and aggrecanase-2 on rat costochondral chondrocytes in vitro
Effects of RNAi-mediated inhibition of aggrecanase-1 and aggrecanase-2 on rat costochondral chondrocytes in vitro
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DOI：
10.1111/j.1745-7254.2008.00856.x
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发表时间：
2008-10-01
影响因子：
8.2
通讯作者：
Tu, Jun-bo
中科院分区：
文献类型：
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作者：
Wang, Zheng-hui;Yang, Zhuang-qun;Tu, Jun-bo
目的：免疫排斥反应和软骨降解是软骨移植或同种异体软骨细胞移植失败的主要原因。主要特征是聚集蛋白聚糖从软骨基质中的损失，主要是由于特异性蛋白酶聚集蛋白聚糖酶-1和聚集蛋白聚糖酶-2的作用。该体外研究的目的是确定通过RNAi特异性抑制聚集蛋白聚糖酶-1和聚集蛋白聚糖酶-2是否会减轻培养的软骨细胞的聚集蛋白聚糖损失。研究方法：构建针对聚集蛋白聚糖酶-1和聚集蛋白聚糖酶-2的shRNA表达质粒载体，转染培养的大鼠肋软骨细胞。用白细胞介素-1 β（IL-1 β）诱导转染的细胞。通过RT-PCR分析基因mRNA水平。用免疫组化和Western blotting法检测各组大鼠血清中聚集蛋白聚糖和II型胶原的含量。结果：随着软骨细胞的去分化，聚集蛋白聚糖酶-1的表达明显增加。RNA干扰对聚集蛋白聚糖酶-1和聚集蛋白聚糖酶-2的特异性抑制对软骨细胞的形态和生长速度没有负面影响。聚集蛋白聚糖酶-1和聚集蛋白聚糖酶-2的mRNA表达显著降低。聚集蛋白聚糖酶-1特异性的shRNA增加了α-2-巨球蛋白的表达水平。软骨细胞外基质的其他基因不受影响。RNAi显著增加了IL-1 β处理的软骨细胞的聚集蛋白聚糖和II型胶原含量。结论：结果表明，通过RNAi抑制聚集蛋白聚糖酶-1和聚集蛋白聚糖酶-2可以减轻聚集蛋白聚糖降解，而不干扰软骨细胞基因表型恢复。RNAi技术可以成为研究软骨退行性过程的有用工具。
Aim: Failure of transplanted cartilage or allogenic chondrocytes is attributed mainly to immunological rejection and cartilage degradation. A major feature is the loss of aggrecan from the cartilage matrix, primarily due to the action of the specific proteinases aggrecanase-1 and aggrecanase-2. The aim of this in vitro study was to determine whether the specific inhibition of aggrecanase-1 and aggrecanase-2 by RNAi would mitigate aggrecan loss from cultured chondrocytes. Methods: Expression plasmid vectors of shRNA targeting aggrecanase-1 and aggrecanase-2 were constructed and transfected into cultured rattus costochondral chondrocytes. The transfected cells were induced with interleukin-1 beta (IL-1 beta). Gene mRNA levels were analyzed by RT-PCR. Aggrecan and collagen II content were measured by immunohistochemistry and Western blotting. Results: As the chondrocytes underwent dedifferentiation, agggrecanase-1 increased significantly. The specific inhibition of aggrecanase-1 and aggrecanase-2 by RNAi had no negative effect on the morphology and growth velocity of the chondrocytes. The mRNA of aggrecanase-1 and aggrecanase-2 decreased significantly. The alpha-2-macroglobulin expression level was increased by the shRNA specific for aggrecanase-1. Other genes of the chondrocytic extracellular matrix were not affected. RNAi significantly increased the aggrecan and collagen II content of chondrocytes treated with IL-1 beta. Conclusion: The results suggest that inhibition of aggrecanase-1 and aggrecanase-2 by RNAi can mitigate aggrecan degradation, without interfering with chondrocytic gene phenotype recovery. RNAi technology can be a useful tool for studying degenerative processes in cartilage.