| 范雅稚,何 娜,王建明,冯海晓,邢 瑶.Pamrevlumab（FG-3019）通过p38 MAPK信号通路调控人Tenon's囊成纤维细胞的增殖、移行和表型转化[J].,2022,(12):2237-2244 |
| Pamrevlumab（FG-3019）通过p38 MAPK信号通路调控人Tenon's囊成纤维细胞的增殖、移行和表型转化 |
| Pamrevlumab (FG-3019) Regulates the Proliferation, Migration and Phenotypic Transformation of Human Tenon's Cystic Fibroblasts through p38 MAPK Signal Pathway |
| 投稿时间：2021-12-29 修订日期：2022-01-25 |
| DOI：10.13241/j.cnki.pmb.2022.12.008 |
| 中文关键词: 青光眼 Pamrevlumab（FG-3019） 人Tenon's囊成纤维细胞 增殖 移行 表型转化 p38 MAPK信号通路 |
| 英文关键词: Glaucoma Pamrevlumab (FG-3019) Human Tenon's cystic fibroblasts Proliferation Migration Phenotypic transformation p38 MAPK signal pathway |
| 基金项目:陕西省重点研发项目（S2019-YF-YBSF-0813） |
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| 中文摘要: |
| 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、500 μg/mL，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100 μg/mL FG-3019培养48 h）、TGF-β1+FG-3019+anisomycin组（10 ng/mL的TGF-β1、100 μg/mL FG-3019和10 μg/mL 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500 μg/mL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OD490nm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 mRNA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 mRNA和蛋白相对表达量、p-p38 MAPK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。 |
| 英文摘要: |
| ABSTRACT 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, 500 μg/mL, 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 100 μg/mL FG-3019 for 48 h), TGF-β1+FG-3019+anisomycin group (cultured with 10 ng/mL TGF-β1, 100 μg/mL FG-3019 and 10 μg/mL 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, 500 μg/mL groups decreased significantly (P<0.05). Compared with TGF-β1 group, the OD490nm 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 of α-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 by 70.22% (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 mRNA 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-β1 by inhibiting p38 MAPK signal pathway. |
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