A Conditional Immortalized Mouse Muller Glial Cell Line Expressing Glial and Retinal Stem Cell Genes
A Conditional Immortalized Mouse Muller Glial Cell Line Expressing Glial and Retinal Stem Cell Genes
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DOI：
10.1167/iovs.10-5395
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发表时间：
2010-11-01
影响因子：
4.4
通讯作者：
Phillips, M. Joseph
中科院分区：
文献类型：
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作者：
Otteson, Deborah C.;Phillips, M. Joseph
目的。米勒胶质细胞在视网膜中具有多种功能，包括神经营养因子的合成、神经递质的摄取和代谢、离子的空间缓冲、血视网膜屏障的维持以及对损伤的反应。米勒胶质细胞群体在体内和体外都具有一些干细胞样特征。本研究的目的是从出生后的小鼠视网膜中产生新的 Muller 胶质细胞系并对其进行表征。方法。木瓜蛋白酶解离后，从出生后第 10 天（P）双杂合转基因（H-2K（b）-tsA58/+；HRhoGFP/+）或 C57BL/6 小鼠培养细胞。通过免疫组织化学和蛋白质印迹分析测定了 SV40 T 抗原 (TAg) 的干扰素 γ (IFN γ) 诱导。通过 BrdU 摄取和钙黄绿素 AM/溴化乙锭染色细胞的细胞计数来测定增殖。通过RT-PCR和免疫组织化学分析基因表达。结果。通过对层粘连蛋白的不同粘附来选择条件永生化（ImM10 [Immortmouse Muller P10]）和自发永生化（C57M10 [C57BL/6 Muller P10]）Muller 胶质细胞系；两者均由贴壁扁平细胞组成，具有大的、弥漫性染色的细胞核和上皮形态。在来自 H-2K(b)-tsA58/+ 的混合视网膜培养物中，TAg 诱导刺激 Muller 胶质细胞摄取 BrdU； HRhoGFP/+ 小鼠并增加了 ImM10 细胞的增殖。 ImM10 和 C57M10 细胞表达米勒神经胶质细胞特征的基因，但不表达分化视网膜神经元特征的基因。 ImM10 细胞还表达视网膜干细胞基因。结论。 ImM10 细胞系是一种从 P10 小鼠视网膜中分离出来的新型条件永生化 Muller 胶质细胞系，表达 Muller 胶质细胞和视网膜干细胞特征的基因。 （投资眼科可见科学。2010；51：5991-6000）DOI：10.1167/iovs.10-5395
PURPOSE. Muller glia have multiple functions in the retina, including synthesis of neurotrophic factors, uptake and metabolism of neurotransmitters, spatial buffering of ions, maintenance of the blood-retinal barrier, and response to injury. A population of Muller glia has some stem cell-like characteristics both in vivo and in vitro. The purpose of this study was to generate and characterize novel Muller glial cell lines from the postnatal mouse retina.METHODS. Cells were cultured from postnatal day (P) 10 double heterozygous transgenic (H-2K(b)-tsA58/+; HRhoGFP/+) or C57BL/6 mice after papain dissociation. Interferon gamma (IFN gamma) induction of the SV40 T-antigen (TAg) was assayed by immunohistochemistry and Western blot analysis. Proliferation was assayed by BrdU uptake and cell counts of calcein AM/ethidium bromide-stained cells. Gene expression was analyzed by RT-PCR and immunohistochemistry.RESULTS. Conditionally immortalized (ImM10 [Immortmouse Muller P10]) and spontaneously immortalized (C57M10 [C57BL/6 Muller P10]) Muller glial cell lines were selected by differential adherence to laminin; both consisted of adherent flat cells with large, diffusely staining nuclei and an epithelial morphology. TAg induction stimulated BrdU uptake by Muller glia in mixed retinal cultures from H-2K(b)-tsA58/+; HRhoGFP/+ mice and increased the proliferation of ImM10 cells. ImM10 and C57M10 cells expressed genes characteristic of Muller glia but not genes characteristic of differentiated retinal neurons. ImM10 cells also expressed retinal stem cell genes.CONCLUSIONS. The ImM10 cell line is a novel, conditionally immortalized Muller glial cell line isolated from the P10 mouse retina that expresses genes characteristic of Muller glial and retinal stem cells. (Invest Ophthalmol Vis Sci. 2010;51:5991-6000) DOI:10.1167/iovs.10-5395