低浓度紫杉醇对大鼠肺动脉平滑肌细胞外胶原沉积的作用及其机制研究

Effect of low-concentration paclitaxel on collagen deposition outside rat pulmonary artery smooth muscle cells and related mechanism

摘要:

目的 探讨低浓度紫杉醇（PTX）对转化生长因子β1（TGF-β1）促进大鼠肺动脉平滑肌细胞（PASMCs）外胶原沉积的作用及其机制。方法 原代培养大鼠PASMCs并分为空白对照组、模型组和干预组（n=3）。空白对照组不做任何处理，模型组施加终浓度为10 ng/mL的TGF-β1，干预组在模型组基础上施加终浓度为100 nmol/L的PTX。MTT比色法检测细胞增殖能力；实时荧光定量PCR法检测Ⅰ型胶原（COLⅠ）、Ⅲ型胶原（COLⅢ） mRNA相对表达量；ELISA法检测COLⅠ、COLⅢ蛋白的OD值；Western blot法检测COLⅠ、COLⅢ蛋白，以及TGF-β1/Smad3信号通路关键蛋白Smad3、p-Smad3的相对表达水平。结果 与空白对照组比较，模型组细胞增殖能力、COLⅠ、COLⅢ mRNA及其蛋白、p-Smad3蛋白相对表达水平均明显增高（P < 0.05）；干预组上述指标较模型组均有所下降，但仍高于空白对照组（P < 0.05）；各组Smad3蛋白相对表达水平比较差异均无统计学意义（P > 0.05）。结论 低浓度PTX对TGF-β1促进PASMCs外胶原合成具有明显抑制作用，该作用可能是通过调控Smad3蛋白的磷酸化来实现。

Abstract:

Objective To study the effect of low-concentration paclitaxel (PTX) on transforming growth factor-β1 (TGF-β1)-induced collagen deposition outside rat pulmonary artery smooth muscle cells (PASMCs) and related mechanism. Methods Primary rat PASMCs were divided into a blank control group (n=3), a model group (n=3), and a drug intervention group (n=3). No treatment was given for the blank control group. The model group was treated with TGF-β1 with a final concentration of 10 ng/mL. The drug intervention group was treated with PTX with a final concentration of 100 nmol/L in addition to the treatment in the model group. MTT colorimetry was used to measure cell proliferation. Quantitative real-time PCR was used to measure the relative mRNA expression of collagen type I (COL-I) and collagen type Ⅲ (COL-Ⅲ). ELISA was used to measure the OD value of COL-I and COL-Ⅲ proteins. Western blot was used to measure the relative protein expression of COL-I, COL-Ⅲ, and the key proteins of the TGF-β1/Smad3 signaling pathway (Smad3 and p-Smad3). Results Compared with the blank control group, the model group had significant increases in proliferation ability, relative mRNA and protein expression of COL-I and COL-Ⅲ, and relative protein expression of p-Smad3 (P < 0.05). Compared with the model group, the drug intervention group had significant reductions in the above indicators, but which were still higher than those in the blank control group (P < 0.05). There was no significant difference in the relative protein expression of Smad3 among the three groups (P > 0.05). Conclusions Low-concentration PTX exerts a marked inhibitory effect on TGF-β1-induced collagen deposition outside PASMCs, possibly by regulating the phosphorylation of Smad3 protein.