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分离培养原代树鼩角膜基质细胞
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(昆明医科大学第四附属医院眼科,云南省第二人民医院眼科,云南省眼科研究所,云南省眼科疾病研究重点实验室,昆明650021)

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R-33


Isolation and culture of tree shrew primary corneal stromal cells
Author:
  • ZHANG Lu

    ZHANG Lu

    Department of Ophthalmology, the Fourth Affiliated Hospital of Kunming Medical University; Department of Ophthalmology,Yunnan Second People’s Hospital; Yunnan Ophthalmology Research Institute; Yunnan Key Laboratory of Ophthalmological Research, Kunming 650021, China
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  • LI Yan

    LI Yan

    Department of Ophthalmology, the Fourth Affiliated Hospital of Kunming Medical University; Department of Ophthalmology,Yunnan Second People’s Hospital; Yunnan Ophthalmology Research Institute; Yunnan Key Laboratory of Ophthalmological Research, Kunming 650021, China
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  • SUN Ziwen

    SUN Ziwen

    Department of Ophthalmology, the Fourth Affiliated Hospital of Kunming Medical University; Department of Ophthalmology,Yunnan Second People’s Hospital; Yunnan Ophthalmology Research Institute; Yunnan Key Laboratory of Ophthalmological Research, Kunming 650021, China
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  • LI Nanyu

    LI Nanyu

    Department of Ophthalmology, the Fourth Affiliated Hospital of Kunming Medical University; Department of Ophthalmology,Yunnan Second People’s Hospital; Yunnan Ophthalmology Research Institute; Yunnan Key Laboratory of Ophthalmological Research, Kunming 650021, China
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  • HU Zhulin

    HU Zhulin

    Department of Ophthalmology, the Fourth Affiliated Hospital of Kunming Medical University; Department of Ophthalmology,Yunnan Second People’s Hospital; Yunnan Ophthalmology Research Institute; Yunnan Key Laboratory of Ophthalmological Research, Kunming 650021, China
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Affiliation:

(Department of Ophthalmology, the Fourth Affiliated Hospital of Kunming Medical University; Department of Ophthalmology,Yunnan Second People’s Hospital; Yunnan Ophthalmology Research Institute; Yunnan Key Laboratory of Ophthalmological Research, Kunming 650021, China)

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    摘要:

    目的 探索一种简单?易行的体外快速?分离培养树鼩角膜基质细胞(corneal stromal cells, CSCs)的方法?方法 实验分为实验组A 和实验组B,取树鼩角膜基质层,实验组A 剪碎,用1% 的Ⅱ型胶原酶消化60 min,离心后重悬接种;实验组B 用Ⅱ型胶原酶联合中性蛋白酶消化?比较记录原代及传代细胞生长情况?传代时间,细胞行波形蛋白免疫组化及免疫荧光鉴定?结果 胶原酶消化法可成功的获取原代细胞, 9 d 后即可进行传代,细胞形态好;传代细胞生长较快,2~3 d 即可传一代;双酶消化法获得的CSCs 较少, 10 d 才可见少量散落细胞,15~20 d 可传代;波形蛋白免疫组化及免疫荧光表达阳性?结论 两种方法均可成功培养出CSCs,但胶原酶消化法能更容易高效分离培养出大量树鼩CSCs?

    Abstract:

    Objective To explore a simple and easy method for the rapid isdation and culture of corneal stromalcells (CSCs). Methods Two experimental groups A and B were designated. The corneal stromal layer was removed fromtree horny cornea. In the group A, it was cut, digested with 1% type II collagenase for 60 min, and resuspended aftercentrifugation. In the group B, type II collagenase was used first,and then treated with neutral protease digestion. Thegrowth and passage time of primary and passage cells were recorded and compared. Vimentin was detected byimmunohistochemistry and immunofluorescence assays. Results The collagenase digestion method which was used in thegroup A successfully isolated primary cells. After 9 days, the cells were passaged and showed good cell morphology. Thepassage cells grew fast and were passaged for 2 to 3 days. The double enzyme digestion method used in the group B isolatedfewer CSCs by day 10. A small number of scattered cells were observed, which were passaged from 15 to 20 days.Immunohistochemistry and immunofluorescence assays showed positive vimentin expression of the corneal stromal cells inboth groups. Conclusions Both methods can successfully isolated CSCs for culture, but the digestion method with only collagenase is easier and more efficiently to isolat a large number of tree shrew CSCs.

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张璐,李妍,孙子雯,李楠钰,胡竹林.分离培养原代树鼩角膜基质细胞[J].中国比较医学杂志,2019,29(5):109~115.

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  • 收稿日期:2018-10-15
  • 在线发布日期: 2019-06-05
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