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

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.

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

Objective To investigate whether AcSDKP can inhibit collagen synthesis in cultured rat cardiac fibroblasts mediated by transforming growth factor beta 1(TGF-βSUB>1/SUB>).Methods Neonatal rat cardiac fibroblasts were isolated. The synthesis of collagen was measured by SUP>3/SUP> H proline incorporation assay. The expressions of type Ⅰ and Ⅲ collagen protein were detected by immunocytochemistry and Western blotting. Results SUP>3/SUP> H 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-βSUB>1/SUB> concentration of 1μg/L, 2.5μg/L, 5μg/L and 7.5μg/L. Compared with TGF-βSUB>1/SUB> group (5μg/L), SUP>3/SUP> H 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-β1 group with increasing of AcSDKP concentration(10SUP>-10/SUP>mol/L, 5×10SUP>-10/SUP>mol/L, 10SUP>-9/SUP>mol/L, 10SUP>-8/SUP>mol/L). Compared with control group, the expression of collagen type Ⅰ and type Ⅲ in rat cardiac fibroblasts increased significantly in TGF-βSUB>1/SUB> group(5μg/L). Obsorbance values