北京生物医学工程

大鼠小梁细胞的体外培养及弹性模量的测量

Experimental method to determine the stiffness of rat trabecular meshwork cells

作者：于洋李林李姗姗刘志成

单位：首都医科大学生物医学工程学院（北京 100069）首都医科大学临床生物力学应用基础研究北京市重点实验室（北京 100069）

关键词：小梁细胞；原子力显微镜；弹性模量； SD大鼠；免疫组织化学

分类号：R318.01

出版年·卷·期（页码）：2019·38·2（145-150）

摘要：

目的小梁网是由小梁薄片和其上的小梁细胞构成的网状结构，它对眼压和房水流出具有重要的调节作用，同时小梁细胞的力学特性和生物学特性与房水流出阻力密切相关。因此本研究主要探讨体外培养的大鼠小梁细胞的生物学特性并应用原子力显微镜测量其弹性模量，为今后建立高眼压动物模型并探究原发性开角型青光眼的发病机制提供理论依据。方法取3只SD大鼠双眼小梁网组织，应用消化法对小梁细胞进行体外混合培养。倒置相差显微镜和免疫组化SABC染色的方法确定小梁细胞并观察其生物学特性。应用原子力显微镜压痕方法测量细胞的弹性模量。结果大鼠小梁细胞10 d左右达到融合，细胞形态多样，免疫组化检测结果显示层粘连蛋白、纤维连接蛋白和神经元特异性烯醇化酶染色阳性。小梁细胞弹性模量为1.02 kPa±0.66 kPa。结论消化法成功培养出大鼠小梁细胞，并利用原子力显微镜测得小梁细胞的弹性模量，为之后研究青光眼小梁细胞的特性奠定基础。

Objective Trabecular meshwork is a network composed of trabecular slices and trabecular meshwork cells (TMCs). It has an important effect on the regulation of intraocular pressure and aqueous humor outflow. Meanwhile, the mechanical and biological properties of TMCs are closely related with aqueous humor outflow resistance. In this study, the biological characteristics of rat TMCs cultured in vitro are investigated and their stiffness is measured by atomic force microscope (AFM). The study provides theoretical basis for the establishment of a high intraocular pressure animal model and the exploration of the pathogenesis of primary open angle glaucoma. Methods Trabecular meshwork tissues of three SD rats were removed and the TMCs were mixed-cultured in vitro by digestive method. Inverted phase contrast microscopy and immunohistochemistry SABC staining were used to determine TMCs and to observe their biological characteristics. The stiffness of cells was measured by AFM. Results The form of TMCs reached fusion in 10 days, and the morphology of the TMCs was diverse. The results of immunohistochemistry showed laminin (LN), fibronectin (FN) and neuron-specific enolase (NSE) staining positive. The stiffness of TMCs was 1.02 kPa±0.66 kPa. Conclusions The rat TMCs can be successfully cultured by digestion method and the stiffness of TMCs is measured by AFM, which lays a foundation for the study on the characteristics of glaucoma TMCs.

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