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刚度可调丝素蛋白-胶原复合水凝胶影响人乳腺上皮细胞生长与表型

张一宁, 焦慧, 郭丽娜, 马馨雨, 盖高成, 张津滔, 阳文青, 王秀丽^*

大连医科大学基础医学院，大连 116044

摘要

细胞外基质(extracellular matrix, ECM)的刚度特性与乳腺组织的生理及病理状态密切相关。本研究旨在探讨刚度可调的丝素蛋白-胶原复合水凝胶培养基质对正常乳腺上皮细胞生长与表型的影响及其作用机制。利用辣根过氧化物酶(horseradish peroxidase, HRP)-H2O2酶促交联反应制备刚度可调的丝素蛋白-胶原复合水凝胶，用旋转流变仪检测其弹性模量。接种人乳腺上皮细胞(MCF-10A细胞)至不同刚度(19.10~4 932.36 Pa)复合水凝胶并维持三维(three dimensional, 3D)培养，用CCK-8试剂盒检测MCF-10A细胞增殖能力，并利用whole-mount洋红染色和苏木素-伊红(HE)染色评价细胞的形态与分布。结果显示，随着基质刚度增加，MAC-10A细胞增殖能力减弱，并且其形成的腺泡样上皮结构逐渐减少、分支样结构增多。同时，基质刚度的增加显著阻遏MCF-10A细胞生理极性的建立。实时定量PCR结果显示，伴随培养基质刚度的增加，ECM重塑相关基因mmp-2、mmp-3、mmp-9表达水平显著上调，而上皮-间质转化(epithelial-mesenchymal transition, EMT)相关基因E-cadherin表达水平显著下调，Vimentin基因和Wnt通路中Snail、Wnt5b、Integrin β1基因表达水平均显著上调。上述结果提示，刚度可调的丝素蛋白-胶原复合水凝胶可调控MCF-10A细胞的增殖、表型和功能基因的表达，该调控作用可能与乳腺上皮细胞极性的改变、ECM重塑以及EMT的发生密切相关。

关键词： 基质刚度; 三维培养; 微环境; 上皮-间质转化

Silk fibroin/collagen composite hydrogels with different matrix stiffness influence the growth and phenotype of human mammary epithelial cells

ZHANG Yi-Ning, JIAO Hui, GUO Li-Na, MA Xin-Yu, GAI Gao-Cheng, ZHANG Jin-Tao, YANG Wen-Qing, WANG Xiu-Li^*

College of Basic Medical Sciences, Dalian Medical University, Dalian 116044, China

Abstract

Extracellular matrix (ECM) stiffness is closely related to the physiological and pathological states of breast tissue. The current study was aimed to investigate the effect of silk fibroin/collagen composite hydrogels with adjustable matrix stiffness on the growth and phenotype of normal breast epithelial cells. In this study, the enzymatic reaction of horseradish peroxidase (HRP) with hydrogen peroxide (H2O2) was used to change the degree of cross-linking of the silk fibroin solution. The rotational rheometer was used to characterize the composite hydrogel’s biomechanical properties. Human normal mammary epithelial cell line MCF-10A were inoculated into composite hydrogels with various stiffness (19.10–4 932.36 Pa) to construct a three dimensional (3D) culture system of mammary epithelial cells. The CCK-8 assay was applied to detect the cell proliferation rate and active states in each group. Hematoxylin-Eosin (HE) staining and whole-mount magenta staining were used for histological evaluation of cell morphology and distribution. The results showed that with the increase of matrix stiffness, MCF-10A cells exhibited inhibited proliferation rate, decreased formation of acinus structures and increased branching structures. Meanwhile, with the increase of matrix stiffness, the polarity of MCF-10A cells was impeded. And the increase of matrix stiffness up-regulated the expression levels of mmp-2, mmp-3, and mmp-9 in MCF-10A cells. Among the genes related to epithelial-mesenchymal transition (EMT), the expression level of the epithelial marker gene E-cadherin was significantly down-regulated, while the interstitial cell marker gene Vimentin was up-regulated, and the expression levels of Snail, Wnt5b and Integrin β1 in the Wnt pathway were up-regulated. These results suggest that the silk fibroin/collagen composite hydrogels with adjustable matrix stiffness regulates the proliferation and the phenotype of MCF-10A cells. The effects of increased matrix stiffness may be closely related to the changes of the polar structures and function of MCF-10A cells, as well as the occurrence of ECM-remodeling and EMT. 

Key words: matrix stiffness; three dimensional culture; microenvironment; epithelial-mesenchymal transition

收稿日期：　　录用日期：

通讯作者：王秀丽　　E-mail: panpan1210@dicp.ac.cn

DOI: 10.13294/j.aps.2022.0043

引用本文：

张一宁, 焦慧, 郭丽娜, 马馨雨, 盖高成, 张津滔, 阳文青, 王秀丽. 刚度可调丝素蛋白-胶原复合水凝胶影响人乳腺上皮细胞生长与表型[J]. 生理学报 2022; 74 (3): 381-391.

ZHANG Yi-Ning, JIAO Hui, GUO Li-Na, MA Xin-Yu, GAI Gao-Cheng, ZHANG Jin-Tao, YANG Wen-Qing, WANG Xiu-Li. Silk fibroin/collagen composite hydrogels with different matrix stiffness influence the growth and phenotype of human mammary epithelial cells. Acta Physiol Sin 2022; 74 (3): 381-391 (in Chinese with English abstract).