北京生物醫(yī)學(xué)工程

大鼠小梁細(xì)胞的體外培養(yǎng)及彈性模量的測(cè)量

Experimental method to determine the stiffness of rat trabecular meshwork cells

作者：于洋李林李?yuàn)檴?/a> 劉志成

單位：首都醫(yī)科大學(xué)生物醫(yī)學(xué)工程學(xué)院（北京 100069）首都醫(yī)科大學(xué)臨床生物力學(xué)應(yīng)用基礎(chǔ)研究北京市重點(diǎn)實(shí)驗(yàn)室（北京 100069）

關(guān)鍵詞：小梁細(xì)胞；原子力顯微鏡；彈性模量； SD大鼠；免疫組織化學(xué)

分類號(hào)：R318.01

出版年·卷·期（頁(yè)碼）：2019·38·2（145-150）

摘要：

目的小梁網(wǎng)是由小梁薄片和其上的小梁細(xì)胞構(gòu)成的網(wǎng)狀結(jié)構(gòu)，它對(duì)眼壓和房水流出具有重要的調(diào)節(jié)作用，同時(shí)小梁細(xì)胞的力學(xué)特性和生物學(xué)特性與房水流出阻力密切相關(guān)。因此本研究主要探討體外培養(yǎng)的大鼠小梁細(xì)胞的生物學(xué)特性并應(yīng)用原子力顯微鏡測(cè)量其彈性模量，為今后建立高眼壓動(dòng)物模型并探究原發(fā)性開角型青光眼的發(fā)病機(jī)制提供理論依據(jù)。方法取3只SD大鼠雙眼小梁網(wǎng)組織，應(yīng)用消化法對(duì)小梁細(xì)胞進(jìn)行體外混合培養(yǎng)。倒置相差顯微鏡和免疫組化SABC染色的方法確定小梁細(xì)胞并觀察其生物學(xué)特性。應(yīng)用原子力顯微鏡壓痕方法測(cè)量細(xì)胞的彈性模量。結(jié)果大鼠小梁細(xì)胞10 d左右達(dá)到融合，細(xì)胞形態(tài)多樣，免疫組化檢測(cè)結(jié)果顯示層粘連蛋白、纖維連接蛋白和神經(jīng)元特異性烯醇化酶染色陽(yáng)性。小梁細(xì)胞彈性模量為1.02 kPa±0.66 kPa。結(jié)論消化法成功培養(yǎng)出大鼠小梁細(xì)胞，并利用原子力顯微鏡測(cè)得小梁細(xì)胞的彈性模量，為之后研究青光眼小梁細(xì)胞的特性奠定基礎(chǔ)。

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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