AcSDKP对TGF-β_1诱导的大鼠心脏成纤维细胞胶原蛋白合成及表达的调节作用  

EFFECT OF AcSDKP ON THE COLLAGEN SYNTHESIS AND EXPRESSION IN CULTURED RAT CARDIAC FIBROBLASTS MEDIATED BY TRANSFORMING GROWTH FACTOR BETA-1

作　　者：吴芳[1] 杨方[1] 王小君[1] 刘丽[1] 杨嫣[2] 王瑞敏[1] 马文东[1] 罗玲[1] 户万秘[1] 裴鑫[1] 张丽娟[1] 

机构地区：[1]华北煤炭医学院实验研究中心 [2]北京大学医学部临床医学系,03级1班北京100083

出　　处：《解剖学报》2007年第6期708-712,共5页Acta Anatomica Sinica

基　　金：河北省科技厅博士基金资助项目(04547002D-5);河北省教育厅基金资助项目(2003112);唐山市新药基础研究重点实验室项目(04362001B)

摘　　要：目的探讨N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸(AcSDKP)对转化生长因子β1(TGF-β1)诱导的大鼠心脏成纤维细胞胶原合成的调节作用。方法差速贴壁法获取大鼠心脏成纤维细胞;采用3H-脯氨酸掺入法检测心脏成纤维细胞胶原蛋白的合成。采用免疫细胞化学染色和Western blotting法检测心脏成纤维细胞Ⅰ型与Ⅲ型胶原蛋白的表达。结果随着TGF-β1浓度的增加(1μg/L,2.5μg/L,5μg/L,7.5μg/L),细胞脯氨酸含量(CPM值)逐渐增加(分别为147.6±10.2,229.2±16.4,427.0±40.6,454.8±26.1),分别是对照组(CPM值为91.6±9.8)的1.61倍、2.50倍、4.66倍、4.97倍,差异均有显著性(P<0.05)。当加入不同浓度的AcSDKP(10-10mol/L,5×10-10mol/L,10-9mol/L,10-8mol/L)时,细胞脯氨酸含量逐渐下降(CPM值为378.8±6.4,292.8±14.4,130.6±17.6,230.6±19.4),分别是TGF-β1组(5μg/L)的88.7%,68.6%,30.6%,54.0%。差异均具有显著性(P<0.05)。免疫细胞化学结果显示,TGF-β1组(5μg/L)细胞内Ⅰ型与Ⅲ型胶原蛋白平均吸度值分别是对照组的1.36倍和2.12倍,差异具有显著性(P<0.05);Western blotting法结果显示,TGF-β1组的Ⅰ型与Ⅲ型胶原蛋白表达条带吸光度值分别是对照组的1.09倍和1.29倍,差异具有显著性(P<0.05)。当给予AcSDKP(10-9mol/L)进行干预时,免疫细胞化学结果显示,细胞内Ⅰ型与Ⅲ型胶原蛋白表达强度较TGF-β1组减弱,其平均吸光度值分别是TGF-β1组的61.3%和68.5%,差异具有显著性(P<0.05)。Western blotting法结果显示,Ⅰ型与Ⅲ型胶原蛋白表达条带吸光度值分别是TGF-β1组的83%和54%,差异具有显著性(P<0.05)。结论AcSDKP对TGF-β1介导的心脏成纤维细胞胶原合成与Ⅰ、Ⅲ型胶原蛋白的表达有明显抑制作用,这可能与其抗心脏纤维化的作用相关。Objective To investigate whether AcSDKP can inhibit collagen synthesis in cultured rat cardiac fibroblasts mediated by transforming growth factor beta-1（TGF-β1 ）. Methods Neonatal rat cardiac fibroblasts were isolated. The synthesis of collagen was measured by ^3 H-prohne incorporation assay. The expressions of type Ⅰ and Ⅲ collagen protein were detected by immunocytochemistry and Western blotting. Results ^3H-proline content increased gradually （147.6 ± 10.2, 229.2 ± 16.4, 427.0 ± 40.6,454.8 ± 26.1 CPM） and was 1.61, 2.50,4.66 and 4.97 times of control group（91.6 ± 9.8 CPM） respectively in rat cardiac fibroblasts mediated by TGF-β, concentration of 1μg/L, 2.5μg/L, 5μg/L and 7.5μg/L. Compared with TGF-β, group （5μg/L）, ^3H-proline content decreased gradually（378.8 ± 6.4,292.8 ± 14.4,130.6 ± 17.6 and 230.6 ± 19.4 CPM） and was 88.7%, 68.6%, 30.6% and 54.0% of TGF-β1 group with increasing of AcSDKP concentration（10^-10mol/L, 5 × 10^-10mol/L, 10^-9mol/L, 10^-8mol/L）. Compared with control group, the expression of collagen type Ⅰ and type m in rat cardiac fibroblasts increased significantly in TGF-β1 group（5μg/L）. Obsorbance values of collagen type Ⅰ and type Ⅲ in rat cardiac fibroblasts in TGF-β1, group were respectively 1.36 and 2.12 times of control group by immunocytochemistry assay and 1.09 and 1.29 times of control group by Western blotting. However, the expression of collagen type Ⅰ and type Ⅲ in rat cardiac fibroblasts decreased significantly in AcSDKP group（ 10^-9 mol/L） compared with TGF-β1 group（5μg/L）. Obsorbance values of collagen type Ⅰ and type Ⅲ in rat cardiac fibroblasts in AcSDKP group were respectively 61.3% and 68.5% of TGF-β1 group by immunohistochemistry assay and 83% and 54% of TGF-β, group by Western boltting. Conclusion AcSDKP can inhibit collagen synthesis and expression of type Ⅰ and Ⅲ collagen protein in cultured rat cardiac fibroblasts mediated by TGF-β , which is possibly related with the effect of AcSDKP

关 键 词：N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸 转化生长因子 成纤维细胞 胶原蛋白 免疫印迹 大鼠 

分 类 号：R542.23[医药卫生—心血管疾病]