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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 the regulatory effect of AcSDKP on the collagen synthesis of rat cardiac fibroblasts induced by transforming growth factor-β1 (TGF-β1) in N-acetyl-seryl-aspartyl-lysyl-proline. METHODS: Rat cardiac fibroblasts were isolated by differential adherence method. The synthesis of cardiac fibroblasts was detected by 3H-proline incorporation. Immunocytochemical staining and Western blotting were used to detect the expression of type Ⅰ and type Ⅲ collagen in cardiac fibroblasts. Results The proline content (CPM) increased gradually with the increase of TGF-β1 concentration (2.5μg / L, 2.5μg / L, 5μg / L, 7.5μg / L) , 427.0 ± 40.6 and 454.8 ± 26.1 respectively), which were 1.61 times, 2.50 times, 4.66 times and 4.97 times respectively in the control group (CPM 91.6 ± 9.8), the difference was significant (P <0.05). When different concentration of AcSDKP (10-10mol / L, 5 × 10-10mol / L, 10-9mol / L, 10-8mol / L) was added, the content of proline decreased gradually (CPM value was 378.8 ± 6.4, 292.8 ± 14.4,130.6 ± 17.6 and 230.6 ± 19.4), which were 88.7%, 68.6%, 30.6% and 54.0% of the TGF-β1 group (5μg / L) respectively. The difference was significant (P <0.05). The results of immunocytochemistry showed that the average values of collagen type Ⅰ and Ⅲ in TGF-β1 group (5μg / L) were 1.36-fold and 2.12-fold higher than that of the control group respectively (P <0.05); Western The results of blotting showed that the expression of type Ⅰ and type Ⅲ collagen in TGF-β1 group were 1.09 and 1.29 times of the control group, respectively. The difference was significant (P <0.05). When AcSDKP (10-9mol / L) was given for intervention, the results of immunocytochemistry showed that the expression intensity of intracellular type I and type III collagens was weaker than that of TGF-β1 group, and the average absorbance values were 61.3 % And 68.5%, the difference was significant (P <0.05). The results of Western blotting showed that the absorbance values of type Ⅰ and type Ⅲ collagen bands were 83% and 54% of the TGF-β1 group, respectively, with significant difference (P <0.05). Conclusion AcSDKP can significantly inhibit collagen synthesis and collagen type Ⅰ and Ⅲ expression induced by TGF-β1 in cardiac fibroblasts, which may be related to its anti-cardiac fibrosis effect.