范宇晨, 郭涛. 人眼组织小梁网细胞的体外培养及鉴定的新方法[J]. 蚌埠医学院学报, 2020, 45(2): 163-166. DOI: 10.13898/j.cnki.issn.1000-2200.2020.02.007
    引用本文: 范宇晨, 郭涛. 人眼组织小梁网细胞的体外培养及鉴定的新方法[J]. 蚌埠医学院学报, 2020, 45(2): 163-166. DOI: 10.13898/j.cnki.issn.1000-2200.2020.02.007
    FAN Yu-chen, GUO Tao. A new method of culture and identification of human eye trabecular meshwork cells in vitro[J]. Journal of Bengbu Medical College, 2020, 45(2): 163-166. DOI: 10.13898/j.cnki.issn.1000-2200.2020.02.007
    Citation: FAN Yu-chen, GUO Tao. A new method of culture and identification of human eye trabecular meshwork cells in vitro[J]. Journal of Bengbu Medical College, 2020, 45(2): 163-166. DOI: 10.13898/j.cnki.issn.1000-2200.2020.02.007

    人眼组织小梁网细胞的体外培养及鉴定的新方法

    A new method of culture and identification of human eye trabecular meshwork cells in vitro

    • 摘要:
      目的探讨人原代小梁网细胞的体外培养,以及利用其特性建立一种鉴定小梁网细胞的新方法。
      方法从眼球破裂伤病人的眼球以及角膜移植后剩余的角膜环分离出小梁网组织,利用组织块贴壁法以及消化法对人原代小梁网细胞进行体外培养。倒置显微镜下观察细胞生长状态并利用CCK8法检测其生长速率。利用细胞免疫荧光技术对所培养的细胞进行纤维连接蛋白、Ⅳ型胶原蛋白、层黏连蛋白、水通道蛋白-1等蛋白的染色鉴定。并利用100 nmol/L地塞米松对所培养的细胞诱导10 d,通过荧光定量PCR和western blotting方法检测myocilin的表达水平以确定所培养的细胞是否为小梁网细胞。
      结果小梁网组织块贴壁培养1~2周后,开始有细胞从组织块旁向外长出,并逐渐增多。传代后小梁网细胞在第1~4天生长较快,第5~7天生长速度有所减慢,但依然显著高于第4天(P < 0.01)。所培养的细胞纤维连接蛋白、Ⅳ型胶原蛋白、层黏连蛋白、水通道蛋白-1的免疫荧光染色均呈阳性。地塞米松诱导后,与对照组相比,小梁网细胞中myocilin mRNA和蛋白表达水平均明显上升(P < 0.01)。
      结论本实验中所培养的细胞通过对其特点进行检测,确定所培养的细胞为人原代小梁网细胞。

       

      Abstract:
      ObjectiveTo explore the culture of human eye trabecular meshwork cells in vitro, and establish a new method of identifying cells according to its characters.
      MethodsThe trabecular meshwork tissue was isolated from the eyeballs of patients with ocular rupture injury, and corneal ring left after corneal transplantation, and the human primary trabecular meshwork cells were cultured in vitro using tissue block adherence and digestion methods.The cell growth was observed under an inverted microscope, and the growth rate was measured using CCK8.The fiber connection protein, type Ⅳ collagen protein, layer adhesion protein and water channel protein-1 were identified using cell immunofluorescence staining.The cultured cells were induced using 100 nml/L of dexamethasone for 10 d, and the mRNA and protein expression levels of myocilin were detected using fluorescence quantitative PCR and western blotting, respectively.
      ResultsAfter culturing for 1 to 2 weeks, the cells began to grow out from the side of the trabecular meshwork, and gradually increased.After passaging, the trabecular meshwork cells grew faster on 1-4 days, and slower on 5-7 days, which still significantly higher than that on day 4(P < 0.01).The staining of fibronectin, collagen-Ⅳ, laminin and aquaporin 1 in cultured cells were positive using immunofluorescence.After dexamethasone inducing, the myocilin mRNA and protein expression levels in trabecular meshwork cells significantly increased compared with the control group(P < 0.01).
      ConclusionsThe primary trabecular meshwork cells can be identified by detecting their characteristics.

       

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