摘要
目的:本研究旨在探究Pamrevlumab(FG-3019)对人Tenon’s囊成纤维细胞(HTFs)的增殖、移行和表型转化的影响。方法:应用组织块培养法进行HTFs的原代培养,通过波形蛋白(Vimentin)和细胞角蛋白(Cytokeratin)免疫荧光染色鉴定HTFs。首先将HTFs分为9组:Control组加入等量DMEM作为阴性对照组,其他组加入不同浓度的FG-3019,使其终浓度分别为10、20、50、100、200、300、400、500μg/m L,通过MTT法检测FG-3019对HTFs的毒性。然后将HTFs分为4组:Control组(加入等量DMEM培养48 h)、TGF-β1组(10 ng/mL的TGF-β1培养48 h)、TGF-β1+FG-3019组(10 ng/mL的TGF-β1和100μg/m L FG-3019培养48 h)、TGF-β1+FG-3019+anisomycin组(10 ng/mL的TGF-β1、100μg/m L FG-3019和10μg/m L anisomycin培养48 h)。通过MTT法和EdU法检测HTFs增殖,通过伤口愈合实验评价FG-3019对HTFs移行的影响通过qRT-PCR或Western blotting检测CTGF、p38 MAPK、p-p38 MAPK、α-SMA、FN和collagen I的表达变化。结果:免疫荧光染色显示,HTFs中波形蛋白阳性表达(98.17%),细胞角蛋白阴性表达(1.83%)。与Control组相比,200、300、400和500μg/m L组的HTFs的细胞活力均显著降低(P<0.05)。与TGF-β1组相比,TGF-β1+FG-3019组的OD_(490nm)降低了19.64%,增殖指数降低了57.87%,伤口愈合率降低了56.46%(P<0.05)。与TGF-β1组相比,TGF-β1+FG-3019组的α-SMA、FN和collagen I蛋白相对表达量依次降低了70.78%、70.99%和70.04%,CTGF m RNA和蛋白相对表达量分别降低了75.83%和60.73%(P<0.05),p-p38 MAPK蛋白相对表达量降低了70.22%(P<0.05)。与TGF-β1+FG-3019组相比,TGF-β1+FG-3019+anisomycin组的增殖、移行、表型转化、CTGF m RNA和蛋白相对表达量、p-p38MAPK蛋白相对表达量均显著增加(P<0.05)。结论:FG-3019部分通过抑制p38 MAPK信号通路来抑制TGF-β1诱导的HTFs增殖、移行和表型转化。
Objective:To reveal the effects of Pamrevlumab(FG-3019)on the proliferation,migration and phenotypic transformation of human Tenon’s cystic fibroblasts(HTFs).Methods:The primary culture of HTFs was carried out by tissue mass culture,and HTFs was identified by Vimentin and Cytokeratin immunofluorescence staining.Firstly,HTFs was divided into 9 groups:control group added the same amount of DMEM as negative control group,and other groups added different concentrations of FG-3019 with the final concentration of 10,20,50,100,200,300,400,500μg/m L,respectively.The toxicity of FG-3019 on HTFs was detected by MTT method.Then HTFs were divided into 4 groups:Control group(cultured with the same amount of DMEM for 48 h),TGF-β1 group(cultured with 10 ng/mL TGF-β1 for 48 h),TGF-β1+FG-3019 group(cultured with 10 ng/mL TGF-β1 and 100μg/m L FG-3019 for 48 h),TGF-β1+FG-3019+anisomycin group(cultured with 10 ng/mL TGF-β1,100μg/m L FG-3019 and 10μg/m L anisomycin for 48 h).The proliferation of HTFs was detected by MTT method and EdU method.The effect of FG-3019 on HTFs migration was evaluated by wound healing experiment.The expression of CTGF,p38 MAPK,p-p38 MAPK,α-SMA,FN and collagen I was detected by qRT-PCR or western blotting.Results:Immunofluorescence staining showed that vimentin was positive(98.17%)and cytokeratin was negative(1.83%)in HTFs.Compared with Control group,the cell viability of HTFs in 200,300,400,500μg/m L groups decreased significantly(P<0.05).Compared with TGF-β1 group,the OD_(490nm) of TGF-β1+FG-3019 group decreased by 19.64%,the proliferation index decreased by 57.87%,and the wound healing rate decreased by 56.46%(P<0.05).Compared with TGF-β1 group,the relative expression ofα-SMA,FN and collagen I protein in TGF-β1+FG-3019 group decreased by 70.78%,70.99%and 70.04%,respectively,the relative expression of p-p38 MAPK protein decreased by 75.83%and 60.73%,respectively,and the relative expression of p-p38 MAPK protein decreased by70.22%(P<0.05).Compared with the TGF-β1+FG-3019 group,the TGF-β1+FG-3019+anisomycin group had increased proliferation,migration,phenotypic transformation,relative expression of CTGF m RNA and protein,and relative expression of p-p38 MAPK protein(P<0.05).Conclusion:FG-3019 partially inhibits the proliferation,migration and phenotypic transformation of HTFs induced by TGF-β1by inhibiting p38 MAPK signal pathway.
作者
范雅稚
何娜
王建明
冯海晓
邢瑶
FAN Ya-zhi;HE Na;WANG Jian-ming;FENG Hai-xiao;XING Yao(Department of Ophthalmology,the Second Affiliated Hospital of Xi’an Jiaotong University,Xi'an,Shaanxi,710004,China)
出处
《现代生物医学进展》
CAS
2022年第12期2237-2244,共8页
Progress in Modern Biomedicine
基金
陕西省重点研发项目(S2019-YF-YBSF-0813)。
