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中华眼科医学杂志(电子版) ›› 2025, Vol. 15 ›› Issue (02) : 71 -77. doi: 10.3877/cma.j.issn.2095-2007.2025.02.002

论著

聚偏二氟乙烯对人角膜上皮细胞迁移和增殖作用与机制的实验研究
陈嘉玮1,2, 王培红2, 刘一昀2, 李泽凯1,2, 王策1,2, 蓝倩倩3, 齐虹1,2,   
  1. 1. 100191 北京大学医学部医学技术研究院
    2. 100191 北京大学第三医院眼科
    3. 530000 南宁,广西壮族自治区人民医院眼科
  • 收稿日期:2025-02-03 出版日期:2025-04-28
  • 通信作者: 齐虹
  • 基金资助:
    国家自然科学基金面上项目(82171022,82371026)北京大学第三医院创新转化基金项目(BYSYZHKC2023113)南宁市青秀区科学技术局重点研发计划项目(2020036)

Effects and mechanisms of polyvinylidene fluoride on migration and proliferation of human corneal epithelial cells

Jiawei Chen1,2, Peihong Wang2, Yiyun Liu2, Zekai Li1,2, Ce Wang1,2, Qianqian Lan3, Hong Qi1,2,   

  1. 1. Institute of Medical Technology,Peking University Health Science Center,Beijing 100191,China
    2. Department of Ophthalmology,Peking University Third Hospital,Beijing 100191,China
    3. Department of Ophthalmology, The People′s Hospital of Guangxi Zhuang Autonomous Region,Naning 530000,China
  • Received:2025-02-03 Published:2025-04-28
  • Corresponding author: Hong Qi
引用本文:

陈嘉玮, 王培红, 刘一昀, 李泽凯, 王策, 蓝倩倩, 齐虹. 聚偏二氟乙烯对人角膜上皮细胞迁移和增殖作用与机制的实验研究[J/OL]. 中华眼科医学杂志(电子版), 2025, 15(02): 71-77.

Jiawei Chen, Peihong Wang, Yiyun Liu, Zekai Li, Ce Wang, Qianqian Lan, Hong Qi. Effects and mechanisms of polyvinylidene fluoride on migration and proliferation of human corneal epithelial cells[J/OL]. Chinese Journal of Ophthalmologic Medicine(Electronic Edition), 2025, 15(02): 71-77.

目的

探讨聚偏二氟乙烯(PVDF)对人角膜上皮细胞(HCEC)迁移和增殖的作用与机制。

方法

通过溶液流延法和静电纺丝法制备PVDF薄膜,使用扫描电子显微镜、X射线衍射仪、傅里叶红外光谱仪、差示扫描量热仪及紫外-可见光分光光度仪从表面形貌、结晶行为以及可见光透过率等方面对两种材料制备工艺进行比较,筛选最佳工艺用于后续细胞实验。根据是否电晕极化将PVDF分为未极化处理组和极化处理组,使用细胞计数试剂盒-8评估PVDF的生物相容性;通过划痕实验、Transwell迁移实验及EdU增殖实验评估PVDF对细胞迁移与增殖能力的影响;通过免疫荧光法和高内涵细胞纹理分析模块评估PVDF对细胞骨架的作用。各组细胞迁移率、迁移细胞数、细胞增殖率、细胞骨架长度及宽度均满足方差齐性和正态分布,以±s表示,组间比较采用单因素方差分析及Bonferroni事后检验。

结果

溶液流延法制备的PVDF薄膜在理化特性中优于静电纺丝法,并选定用于后续细胞实验。在划痕实验中,对照组、PVDF未极化组及极化组迁移率分别为(61.43±2.86)%、(70.36±5.87)%及(96.12±0.48)%,组间差异有统计学意义(F=75.91,P<0.05);Transwell实验中,对照组、PVDF未极化组及极化组迁移细胞数分别为(1643.11±51.17)个、(1827.44±51.81)个及(2392.89±57.99)个,组间差异有统计学意义(F=105.69,P<0.05)。对照组、PVDF未极化组及极化组细胞增殖率分别为(27.83±1.62)%、(30.58±1.46)%及(41.20±1.89)%,组间差异有统计学意义(F=115.13,P<0.05)。对照组、PVDF未极化组及极化组HCEC微管长度和宽度分别为(1.41±0.06)μm和(0.41±0.01)μm、(1.52±0.02)μm和(0.44±0.01)μm及(1.72±0.03)μm和(0.44±0.01)μm,组间差异有统计学意义(F=29.76,9.67;P<0.05);对照组、PVDF未极化组及极化组HCEC细胞微丝的长度和宽度分别为(1.24±0.01)μm和(0.38±0.01)μm、(1.31±0.01)μm和(0.40±0.01)μm及(1.40±0.03)μm和(0.40±0.01)μm,组间差异有统计学意义(F=42.70,62.70;P<0.05)。

结论

溶液流延法所制备的PVDF材料具有优异的理化特性与生物相容性,可通过自发极化产生的电微环境调控细胞骨架重组,从而促进HCEC迁移与增殖,且电晕极化处理能显著增强此特性,这能为角膜再生医学提供新策略。

Objective

The aim of this study is to investigate the effects and mechanisms of polyvinylidene fluoride(PVDF)on the migration and proliferation of human corneal epithelial cell(HCEC).

Methods

PVDF films were prepared using solution casting and electrospinning methods.Scanning electron microscopy,X-ray diffraction,fourier-transform infrared spectroscopy,differential scanning calorimetry,and UV-Vis spectrophotometry were employed to select the optimal process from the aspect of the surface morphology,crystallinity,and visible light transmittance for two fabrication methods,and used for subsequent cell experiments.PVDF was divided into non-polarized and corona-poled groups with or without spontaneous polarization.Biocompatibility was assessed using cell counting kit-8.Scratch assay,transwell migration assay,and EdU proliferation assay were used to evaluate the effects of PVDF on cell migration and proliferation.Immunofluorescence and high-content cellular texture analysis were applied to assess cytoskeletal changes.Migration rates,migrated cell counts,proliferation rates,cytoskeletal length,and width met homogeneity of variance and normal distribution,were expressed as ±s and compared by one-way ANOVA with Bonferroni post hoc tests for intergroup.

Results

Solution-cast PVDF films exhibited superior physicochemical properties compared to electrospun films and were selected for cell experiments.PVDF significantly promoted HCEC migration and proliferation.In the scratch assay,the migration rates of the control group,non-polarized PVDF group,and polarized PVDF group were(61.43±2.86)%,(70.36±5.87)%,and(96.12±0.48)%,respectively,with statistically significant differences(F=75.91,P<0.05).In the Transwell assay,the numbers of migrating cells in the control group,non-polarized PVDF group,and polarized PVDF group were(1643.11±51.17),(1827.44±51.81),and(2392.89±57.99),respectively,with statistically significant intergroup differences(F=105.69,P<0.05).The cell proliferation rates in the control group,non-polarized PVDF group,and polarized PVDF group were(27.83±1.62)%,(30.58±1.46)%,and(41.20±1.89)%,respectively,with statistically significant intergroup differences(F=115.13,P<0.05).The microtubule lengths and widths of HCECin the control group,non-polarized PVDF group,and polarized PVDF group were(1.41±0.06)μm,(0.41±0.01)μm,(1.52±0.02)μm,(0.44±0.01)μm,(1.72±0.03)μm,and(0.44±0.01)μm,respectively.Statistically significant differences were observed among the three groups in both microtubule length and width(F=29.76,9.67;P<0.05).The microfilament lengths and widths were(1.24±0.01)μm,(0.38±0.01)μm,(1.31±0.01)μm,(0.40±0.01)μm,(1.40±0.03)μm,and(0.40±0.01)μm,respectively,with statistically significant intergroup differences in both microfilament length and width(F=42.70,62.70;P<0.05).

Conclusions

Solution-cast PVDF demonstrates excellent physicochemical properties and biocompatibility.It promotes HCECmigration and proliferation by regulating cytoskeletal reorganization via an electrical microenvironment generated by spontaneous polarization,with corona poling significantly enhancing this effect,which providing a novel strategy for corneal regenerative medicine.

图1 扫描电子显微镜下制备聚偏二氟乙烯的表面形貌(×2000) 图1A示溶液流延法制备聚偏二氟乙烯薄膜的表面形貌;图1B示静电纺丝法制备聚偏二氟乙烯薄膜的表面形貌 图2 溶液流延法与静电纺丝法所制备聚偏二氟乙烯的X射线衍射图谱 图3 溶液流延法与静电纺丝法制备聚偏二氟乙烯的傅里叶红外光谱 图4 溶液流延法与静电纺丝法制备聚偏二氟乙烯差示扫描量热仪升温的结晶图 图5 溶液流延法与静电纺丝法制备聚偏二氟乙烯在280~780 nm范围内的透过率
图6 倒置显微镜下聚偏二氟乙烯对单层人角膜上皮细胞划痕迁移的细胞状态(×40) 图6A~图6C分别示对照组、未极化组与极化组共培养人角膜上皮细胞在0 h时的初始划痕状态;图6D~图6F分别示对照组、未极化组与极化组共培养人角膜上皮细胞在12 h时的细胞迁移状态
图7 倒置显微镜下聚偏二氟乙烯对单层人角膜上皮细胞培养12 h时的显微结构图(×40) 图7A ~图7C分别示对照组、未极化组及极化组人角膜上皮细胞共培养12 h时的迁移细胞数;图7D示Transwell实验中12 h时细胞迁移数的柱状图
图8 倒置荧光显微镜下聚偏二氟乙烯对单层人角膜上皮细胞增殖率影响的显微结构图(×40)图8A ~图8C分别示对照组、未极化组及极化组共培养人角膜上皮细胞的EdU荧光染色复合图;图8D示聚偏二氟乙烯对细胞增殖率影响的柱状图
图9 倒置荧光显微镜下聚偏二氟乙烯对单层人角膜上皮细胞骨架影响的显微结构图(×200) 图9A ~图9C分别示对照组、未极化组及极化组共培养人角膜上皮细胞的微管与微丝荧光染色复合图;图9D示聚偏二氟乙烯对细胞骨架影响的柱状图
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