低剂量紫杉醇抑制成纤维细胞增殖、迁移及硬膜外纤维化研究

Low-dose paclitaxel inhibits fibroblast proliferation,migration and epidural fibrosis

目的:探究低剂量紫杉醇(PTX)对转化生长因子-β1(TGF-β1)激活的人成纤维细胞(HFs)增殖、迁移能力和硬膜外纤维化的影响及机制。方法:HFs(购自中国科学院上海细胞库)分为对照组、TGF-β1(5μg/L)、TGF-β1(5μg/L)+PTX(10 nmol/L)和PTX(10 nmol/L)组。使用倒置显微镜观察细胞形态,划痕和Transwell实验检测细胞迁移,流式细胞仪分析细胞周期百分比及凋亡率;蛋白印迹法(Western blot)检测各组Smad3、磷酸化Smad3(p-Smad3)、Ⅰ型胶原蛋白(COL1A1)、α-平滑肌肌动蛋白(α-SMA)蛋白表达水平;使用随机数字法将36只成年雄性SD大鼠分为假手术组、对照组和PTX组,每组12只。术后4周使用苏木精-伊红(HE)和Masson染色观察各组硬膜外纤维性情况,多组间采用单因素方差分析。结果:(1)划痕实验结果显示,TGF-β1组HFs 12 h和24 h迁移率为[(31.50±3.98)%、(77.73±3.03)%,F=60.312,P<0.05]显著高于对照组[(23.47±3.19)%、(50.31±5.31)%],而PTX组[(14.59±0.58)%、(20.93±0.72)%,F=52.385,P<0.05]则显著低于对照组。(2)流式细胞学结果显示,TGF-β1组S期细胞百分比显著高于对照组[(30.07±0.39)%比(25.62±1.06)%,F=27.335,P<0.05],PTX组G 2期细胞百分比显著高于对照组[(37.55±1.71)%比(24.94±0.51)%,F=149.725,P<0.05],而PTX组与对照组之间的细胞凋亡率差异无统计学意义(F=1.263,P>0.05)。(3)Western blot实验结果显示,TGF-β1组p-Smad3/Smad3显著高于对照组[(129.05±7.66)%,F=39.615,P<0.05],而PTX组则显著低于对照组[(42.30±1.73)%,F=1215.471,P<0.05],各组α-SMA和COL1A1的蛋白表达水平与p-Smad3/Smad3趋势一致。(4)动物实验结果表明,对照组观察到更显著的硬膜外纤维化,PTX组成纤维细胞数目和胶原密度[78.00±13.00)、(456.94±112.69)μm 2]显著低于对照组[(194.67±7.09)、(1456.00±71.84)μm 2,F=186.170、168.671,P<0.05]。结论:低剂量PTX抑制HFs增殖、迁移能力及TGF-β1/Smad3信号途径的激活,改善椎板切除术后硬膜外纤维化。

Objective To observe the effects and possible mechanism of low-dose paclitaxel(PTX)on the proliferation,migration and epidural fibrosis of human fibroblasts(HFs)activated by transforming growth factor-β1(TGF-β1).Methods HFs were divided into control group,TGF-β1(5μg/L)group,TGF-β(5μg/L)+PTX(10 nmol/L)group and PTX(10 nmol/L)group.The morphology,viability,migration rate,cell cycle and apoptosis rate were measured using fluorescence microscope,cell counting kit-8(CCK-8)assay,scratch assay and flow cytometry.Western blotting was used to measure the protein expression level of anti-αsmooth muscle actin(α-SMA),collagen typeⅠAlpha 1(COL1A1)and the phosphorylation level of Smad family member 3(Smad3).Using the random number method,36 adult male SD rats were divided into sham operation group,control group and PTX group,with 12 rats in each group.Hematoxylin-eosin(HE)staining and Masson staining were used to observe the epidural fibrosis in each group after 4 weeks.One-way analysis of variance was used among multiple groups.Results(1)The results of the scratch test showed that the 12 h and 24 h HFs mobility in the TGF-β1 group was(31.50±3.98)%and(77.73±3.03)%respectively(F=60.312,P<0.05),significantly higher than that in the control group[(23.47±3.19)%and(50.31±5.31)%respectively],and that in the PTX group[(14.59±0.58)%and(20.93±0.72)%respectively,F=52.385,P<0.05]was significantly lower than in the control group.(2)The results of flow cytometry showed that the percentage of cells in the S phase in the TGF-β1 group was significantly higher than that in the control group[(30.07±0.39)%vs.(25.62±1.06)%,F=27.335,P<0.05],while that in the G2 phase in the PTX group was significantly higher than that in the control group[(37.55±1.71)%vs.(24.94±0.51)%,F=149.725,P<0.05],and there was no significant difference in the apoptosis rate between the PTX group and the control group(F=1.263,P>0.05).(3)The results of Western blotting showed that p-Smad3/Smad3 expression in the TGF-β1 group was significantly higher than that in the control group[(129.05±7.66)%,F=39.615,P<0.05],while that in the PTX group was significantly lower than in the control group[(42.30±1.73)%,F=1215.471,P<0.05].The protein expression levels ofα-SMA and COL1A1 in each group showed a consistent trend with p-Smad3/Smad3.(4)The results of animal experiments showed that more significant epidural fibrosis was observed in the control group.The number of fibroblasts and collagen density in PTX group[(78.00±13.00),(456.94±112.69)μm2]were significantly reduced as compared with those in the control group[(194.67±7.09),(1456.00±71.84)μm2,F=186.170,F=168.671,P<0.05].Conclusion Low-dose PTX inhibits the proliferation,migration,and the activation of TGF-β1/Smad3 signaling pathway of HFs,and improves the condition of epidural fibrosis after laminectomy.