体外构建3D细胞压力培养模型研究压力对增生性瘢痕成纤维细胞的作用  

Study on the effects of pressure on the growth characteristics of hypertrophic scar fibroblasts by establishing in 3D model of cell pressure culture

作　　者：刘宁[1] 王鹏[1] 蔡瑞昭 李猛智 赵菁玲[1] 陈蕾[1] 舒斌[1] 刘旭盛[1] 祁少海[1] Liu Ning;Wang Peng;Cai Ruizhao;Li Mengzhi;Zhao Jingling;Chen Lei;Shu Bin;Liu Xusheng;Qi Shaohai(Department of Burns Surgery, First Affiliated Hospital, Sun Yat- sen University, Guangzhou 510080, China)

机构地区：[1]中山大学附属第一医院烧伤外科,广州510080

出　　处：《中华损伤与修复杂志（电子版）》2021年第2期132-139,共8页Chinese Journal of Injury Repair and Wound Healing(Electronic Edition)

基　　金：国家自然科学基金(81971833、81971884);广东省基础与应用基础研究基金项目(2019A1515110045、2020B1515020049)。

摘　　要：目的采用Flexcell-5000C压力系统和鼠尾I型胶原构建3D细胞压力培养模型,在体外细胞水平探讨压力对增生性瘢痕成纤维细胞(HSFb)的作用。方法胶原酶消化法分离培养原代HSFb,采用Flexcell-5000C压力系统和鼠尾I型胶原体外构建HSFb 3D培养模型。实验分为对照组(未施压)、10 mmHg压力组、20 mmHg压力组、30 mmHg压力组。通过免疫荧光观察不同压力下HSFb的表型变化;Ki-67/Edu免疫荧光染色检测HSFb增殖能力;Annexin-V-PI流式双染检测HSFb凋亡;PI流式单染检测HSFb细胞周期;划痕实验检测HSFb迁移能力。数据比较采用单因素方差分析和LSD-t检验。结果激光共聚焦显微镜下可见HSFb表型随着压力增大逐渐向正常皮肤成纤维细胞(FB)转变,且在30 mmHg压力下最显著;Ki-67/Edu免疫荧光染色显示:HSFb增殖能力与压力值成反比,20 mmHg、30 mmHg组较对照组HSFb增殖能力显著降低(F=10.61,P=0.0037),而其余各组组间HSFb增殖能力差异无统计学意义(P>0.05);Annexin-V-PI流式双染显示:相比于对照组、10 mmHg压力组、20 mmHg压力组及30 mmHg压力组HSFb早期凋亡率显著升高,差异有统计学意义(F=103.8,P<0.0001),而10、20 mmHg压力组HSFb早期凋亡率较对照组差异无统计学意义(P>0.05);PI流式单染结果显示:相比于对照组、10 mmHg压力组及20 mmHg压力组,30 mmHg压力组G0/G1期HSFb百分比显著升高(F=21.58,P=0.0003),S期及G2/M期HSFb百分比显著下降(F=93.89、54.11,P<0.001、=0.0066);相比于对照组,10 mmHg及20 mmHg G0/G1期HSFb百分比显著升高,S期及G2/M期HSFb百分比显著下降(P<0.05);而20 mmHg与10 mmHg相比,HSFb细胞周期差异无统计学意义(P>0.05);划痕实验结果显示,与对照组相比,30 mmHg压力组HSFb迁移能力明显下降(t=10.66,P=0.004)。结论基于Flexcell-5000C压力系统和鼠尾I型胶原构建的3D细胞压力培养模型便捷稳定,压力值实时可调可控,成功验证了压力能够抑制HSFb增殖、迁移,且促进其凋亡Objective To establish in vitro 3D model of cell culture and pressure with Flexcell-5000C pressure system and rat tail type I collagen to explore the in vitro effect of pressure on hypertrophic scar fibroblasts(HSFb)at cellular level.Methods The primary HSFbs were isolated and cultured by collagenase digestion.A 3D culture model of HSFb was constructed in vitro with Flexcell-5000C pressure system and rat tail type I collagen.All the HSFbs were randomly divided into control group(no pressure)and 10 mmHg,20 mmHg and 30 mmHg pressure groups.The cytomorphosis of HSFbs in different pressure was monitored by immunofluorescence.The cellular proliferation index of HSFbs was examined by ki67/EdU immunofluorescence staining of chromosome.The HSFbs apoptosis was detected by Annexin-V-PI double flow cytometry.The cell cycle was detected by PI single staining flow cytometry and cell migration rate,by scratch assay.Data were processed with one-way analysis of variance and the LSD-t test.Results Examined by confocal microscope,the morhpological changes happened in HSFbs under mechanical pressure.The HSFb phenotype was gradually transferred to normal fibroblast(FB)along with the increase of the pressure,especially under 30 mmHg pressure.It was indicated by Ki-67/Edu immunofluorescence staining that the proliferation capacity of HSFbs was inversely proportional to the pressure value.The proliferation value of HSFbs in 20 mmHg and 30 mmHg groups was evidently lower than that in control group,the difference was statistically significant(F=10.61,P=0.0037).But there was no significant difference of the proliferation value among all the other groups(P>0.05).It was indicated by Annexin-V-PI double flow cytometry that the early apoptosis rate in 30 mmHg group was much higher than that in the control,10 mmHg and 20 mmHg groups(F=103.8,P<0.0001).And there was no significant difference of apoptosis between 10 mmHg,20 mmHg and control groups(P>0.05).It was exhibited by PI single staining flow cytometry that the HSFb percentage in 30 mmHg gr

关 键 词：瘢痕 成纤维细胞 压力加载系统 3D细胞培养 

分 类 号：R622[医药卫生—整形外科]