摘要
目的探究A型肉毒素(BTXA)对肌腱成纤维细胞增殖和TGF-β1、bFGF、MMP-2表达的影响。方法采用组织块法提取培养成年新西兰兔脚趾深屈肌腱成纤维细胞,分为对照组(Ctrl组)、低剂量组、中剂量组及高剂量组,分别给予0、10、30、50 U/mL BTXA。MTT检测细胞体外增殖活性,流式细胞术检测细胞周期分布;免疫荧光检测细胞黏附及形态变化;ELISA和qRT-PCR分别检测细胞内TGF-β1、bFGF、MMP-2蛋白及mRNA表达变化。结果经BTXA处理的各组细胞培养48 h后,细胞增殖活力均显著低于对照组,差异有统计学意义(F=95.573,P<0.05);0、10、30、50 U/mL BTXA阻滞细胞周期停滞在G0/G1期的比例分别为(38.74±4.21)%、(43.13±6.36)%、(48.37±7.58)%、(61.03±7.63)%,组间差异有统计学意义(F=76.684,P<0.05)。免疫荧光结果显示BTXA显著抑制细胞内Actin-tracker Green表达。ELISA及qRT-PCR结果显示,高剂量组细胞TGF-β1表达水平显著低于其他三组,中、高剂量组细胞MMP-2含量高于对照组和低剂量组;与对照组相比,中、高剂量组细胞TGF-β1、bFGF mRNA相对表达量显著降低,且高剂量组细胞TGF-β1 mRNA表达水平显著低于其他3组;各组细胞MMP-2 mRNA表达水平随BTXA浓度增加而升高;差异均有统计学意义(P<0.05)。结论BTXA可能通过抑制TGF-β1、bFGF表达促进MMP-2活性,发挥抑制肌腱成纤维细胞增殖的作用。
Objective To explore the effects of botulinum toxin type A(BTXA)on proliferation and expression levels of TGF-β1,bFGF,and MMP-2 of tendon fibroblasts.Methods Fibroblasts from the deep flexor tendon of the toes of adult New Zealand rabbits were extracted and cultured by tissue block method and divided into control group(ctrl group),low-dose group,medium-dose group,and high-dose group,which were given 0,10,30,and 50 U/mL BTXA,respectively.MTT was used to detect the proliferation activity of cells in vitro.Flow cytometry was used to detect the distribution of the cell cycle.Immunofluorescence was used to detect cell adhesion and morphological changes.Intracellular TGF-β1,bFGF,MMP-2 protein,and mRNA expression changes were detected by ELISA and qRT-PCR,respectively.Results After 48 hours of culture,the cell proliferation activity of each group treated with BTXA was significantly lower than that of the control group,and the difference was statistically significant(F=95.573,P<0.05).0,10,30 and 50 U/mL BTXA blocked the proportion of cell cycle arrest in the G0/G1 phase was(38.74±4.21)%,(43.13±6.36)%,(48.37±7.58)%,and(61.03±7.63)%and the differences between groups were statistically significant(F=76.684,P<0.05).Immunofluorescence results showed that BTXA significantly inhibited the expression of actin tracker green in cells.ELISA and qRT-PCR results showed that the expression level of TGF-β1 in the high-dose group was significantly lower than that in the other three groups,and the MMP-2 content in the medium and high-dose groups was higher than that in the control and low-dose groups.Compared with the control group,the relative expression levels of TGF-β1 and bFGF mRNA in the medium and high dose groups were significantly decreased,and the TGF-β1 mRNA expression in the high dose group was significantly lower than that in the other three groups.The expression level of MMP-2 mRNA in cells of each group increased with BTXA concentration.The differences were all statistically significant(P<0.05).Conclusions BTXA may
作者
王楠
许良
吴国明
WANG Nan;XU Liang;WU Guoming(Department of Orthopedics,Hangzhou Xiaoshan District Hospital of Traditional Chinese Medicine,Hangzhou 311201,China)
出处
《健康研究》
CAS
2022年第4期432-436,共5页
Health Research
基金
杭州市科技计划项目(20171226Y94)。
