Effects of RNAi-mediated inhibition of aggrecanase-1 and aggrecanase-2 on rat costochondral chondrocytes in vitro  

作　　者：Zheng-hui WANG Zhuang-qun YANG Xi-jing HE Li WANG Li-xia LI Jun-bo TU 

机构地区：[1]Department of Oral and Maxillofacial Plastic Surgery, Stomatological Hospital Xi-an Jiao Tong University, Xi-an 710004 China [2]Department of Orthopaedics, the Second Hospital, Xi-an Jiao Tong University, Xi-an 710004, China

出　　处：《Acta Pharmacologica Sinica》2008年第10期1215-1226,共12页中国药理学报（英文版）

基　　金：This study was supported by the National Natural Science Foundation of China （No 30572052） and the International Cooperation Project of Shaan＇xi Province, China （No 2007KW-07）. Acknowledgement The excellent technical assistance of Jian-feng SHI （Research Center of Stomatological Hospital） is gratefully acknowledged.Author contribution Zhuang-qun YANG, Xi-jing HE, Zheng-hui WANG, Li WANG, and Li-xia LI designed research; Zheng-hui WANG performed research; Zheng-hui WANG, Li WANG, and Li-xia LI analyzed data; Zheng-hui WANG and Jun-bo TU wrote the paper.

摘　　要：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-1β （IL-1β）. 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 α-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-1β. 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.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-1β （IL-1β）. 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 α-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-1β. 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.

关 键 词：AGGRECANASE CHONDROCYTE AGGRECAN RNAi extracellular matrix 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]