腺病毒介导细胞外信号调节激酶2表达对去分化软骨细胞增殖及胞外基质合成的影响  

Effects of expression of extracellular signal-regulated kinase 2 mediated by adenovirus on proliferation and extracelluar matrix synthesis of dedifferentiated chondrocytes

作　　者：季煜华[1] 曾耀英[1] 俞瑜[1] 邢飞跃[1] 王会营[1] 江颖娟[1] 

机构地区：[1]暨南大学组织移植与免疫研究中心,广东省广州市510632

出　　处：《中国临床康复》2006年第17期82-84,i0005,共4页Chinese Journal of Clinical Rehabilitation

基　　金：国家基础研究发展规划项目(G1999054303);广东省"十五"重大科技专项(A302020204)~~

摘　　要：目的:观察腺病毒介导细胞外信号调节激酶2的过度表达对去分化软骨细胞增殖和细胞外基质合成的影响,以及腺病毒介导细胞外信号调节激酶2用于修饰去分化软骨细胞的可行性。方法:实验于2004-09/2005-05在暨南大学组织移植与免疫研究中心进行。SD大鼠5只,周龄为3～5周,此处请核对体质量为(150±20)g,分离、培养大鼠肋生长板软骨细胞,利用Adeasy系统构建携带细胞外信号调节激酶2基因的腺病毒载体。流式细胞仪检测不同感染复数的携带细胞外信号调节激酶2基因的腺病毒载体感染第5代大鼠肋生长板软骨细胞的效率,Westernblot检测携带细胞外信号调节激酶2基因的腺病毒载体感染大鼠肋生长板软骨细胞中细胞外信号调节激酶1/2和磷酸化细胞外信号调节激酶2的表达,3H-TdR,3H-proline和35S-sulfate同位素掺入检测携带细胞外信号调节激酶2基因的腺病毒载体感染对大鼠肋生长板软骨细胞增殖、胶原和蛋白多糖合成的影响。结果:①成功构建携带细胞外信号调节激酶2基因的腺病毒载体重组腺病毒。②100感染复数的携带细胞外信号调节激酶2基因的腺病毒载体感染第5代大鼠肋生长板软骨细胞的效率大于90%。③携带细胞外信号调节激酶2基因的腺病毒载体感染的第5代大鼠肋生长板软骨细胞中细胞外信号调节激酶1/2和磷酸化细胞外信号调节激酶2的表达均显著增加,分别是Ad-CMV组的15.58和1.85倍。④同位素检测结果表明携带细胞外信号调节激酶2基因的腺病毒载体的感染促进3H-TdR、3H-proline和35S-sulfate的掺入,分别是对照组的2.52,1.61和1.53倍(P<0.05)。结论:腺病毒介导细胞外信号调节激酶2基因的过度表达促进去分化大鼠肋生长板软骨细胞的增殖和细胞外基质成分的合成。AIM： To investigate the effects of overexpression of extraeellular signalregulated kinase （ERK） 2 mediated by adenovirus on the proliferation and extracelluar matrix synthesis of dedifferentiated chondrecytes, and evaluate the feasibility of ERK2 in modifying dedifferentiated chondrecytes. METHODS： The experiment was carried out in the Center of Tissue Transplantation and Immunology, Jinan University from September 2004 to May 2005. Five SD rats of 3-5 weeks old and （150±20） g weight were selected to isolate and primarily culture the rat costochondral growth plate chondrecytes （GPCS）. The adenovirus vector carrying ERK2 gene was constructed with Adeasy system, and the infective efficiency of constructed adenovirus vector of different multiplicity of infection （MOI） on the 5^th rat costochondral GPCS was detected by flow cytometer; Western blot was used to examine the expressions of ERK1/2 and phosphated ERK2 of adenovirus vector carrying ERK2 infected by rat costochondral GPCS. The influences of adenovirus vector carrying ERK2 infection on the proliferation and extracelluar matrix synthesis of collagen and proteoglycan in GPCS were measured by ^3H-TdR, ^3H-proline and ^35S-suffate isotope. RESULTS： ①The recombinant adenovirus carrying ERK2 gene was successfully constructed. ②The infective efficiency of 100 MOI adenovirus vector carrying ERK2 on the 5^th GPCS was more than 90%. ③The expressions of ERK1/2 and phosphated ERK2 in 5^th GPCS infecting adenovirus vector carrying ERK2 gene were markedly increased, 15.58 and 1.85 times more than those of control group. ④The isotope detection indicated, the infection of adenovirus vector carrying ERK2 gene promoted the ^3H-TdR, ^3H-proline and ^35S-sulfate mixture, 2.52, 1.61 and 1.53 times more than those of control group （P 〈 0.05）. CONCLUSION： The overexpression of ERK2 gene mediated by adenovirus promotes the proliferation and extracelluar matrix synthesis of dedifferentiated rat costechondral GPCS.

关 键 词：软骨细胞 腺病毒科 细胞外基质 动物 基因修饰 

分 类 号：R681.3[医药卫生—骨科学]