Isolation and characterization of porcine Müller cells. Myofibroblastic dedifferentiation in culture.
Isolation and characterization of porcine Müller cells. Myofibroblastic dedifferentiation in culture.
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发表时间：
1996-04
影响因子：
4.4
通讯作者：
C. Guidry
中科院分区：
文献类型：
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作者：
C. Guidry
目的研究分离的<s:1> ller细胞在扩展培养过程中的表型和抗原变化。方法采用木瓜蛋白酶和dna酶序酶切、培养、密度梯度离心等方法从猪视网膜中分离出<s:1> ller细胞。通过免疫检测碳酸酐酶II （CA-II）、细胞视黄醛结合蛋白（CRALBP）、胶质纤维酸性蛋白（GFAP）、波形蛋白和δ平滑肌肌动蛋白（alpha SMA）来确认分离细胞的身份。在培养中建立的连续增殖细胞检测这些抗原表达的变化。结果纯化的m<s:1> ller细胞原代培养在常规培养条件下增殖，2周内失去免疫检测的CRALBP。CA-II的表达也随时间减少，但明显低于CRALBP。GFAP表达的丧失更为缓慢，并在第5代完成。一些细胞在培养12天内可检测到α - SMA的从头表达，所有细胞在传代5时均可检测到。在此期间，vimentin的表达质量保持不变。结论：分离的猪Muller细胞在培养过程中经历表型去分化为成纤维细胞样细胞，包括失去可检测的CRALBP、CA-II和GFAP，并获得肌样标记α - SMA的表达。
PURPOSE To characterize phenotypic and antigenic changes in isolated Müller cells during proliferation in extended culture. METHODS Müller cells were isolated from porcine retina by sequential papain and DNase digestion, trituration, and density gradient centrifugation. The identity of the isolated cells was confirmed by immunodetection of carbonic anhydrase II (CA-II), cellular retinaldehyde-binding protein (CRALBP), glial fibrillary acidic protein (GFAP), vimentin, and delta smooth muscle actin (alpha SMA). Continuously proliferating cells established in culture were examined for changes in the expression of these antigens. RESULTS Primary cultures of purified Müller cells, incubated under routine culture conditions, were proliferative and lost immunodetectable CRALBP within 2 weeks. The expression of CA-II also diminished with time, but at an apparently lower rate than that of CRALBP. Loss of GFAP expression was even more gradual and was complete by passage 5. De novo expression of alpha SMA was detectable in some cells within 12 days in culture and by all cells by passage 5. During this period, vimentin expression remained qualitatively unchanged. CONCLUSIONS Isolated porcine Muller cells in culture undergo a phenotypic dedifferentiation to a fibroblast-like cell, which includes loss of detectable CRALBP, CA-II, and GFAP, and they acquire expression of the myoid marker alpha SMA.