Isolation and distribution of rabbit keratocyte precursors
Isolation and distribution of rabbit keratocyte precursors
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发表时间：
2008-01
期刊：
影响因子：
2.2
通讯作者：
T. Mimura;S. Amano;Seiichi Yokoo;S. Uchida;T. Usui;S. Yamagami
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文献类型：
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作者：
T. Mimura;S. Amano;Seiichi Yokoo;S. Uchida;T. Usui;S. Yamagami
目的从兔角膜基质中分离多能性前体细胞，比较角膜基质中心区和周边区前体细胞的分布和增殖能力。方法将兔角膜基质分为周边区（直径6.0- 10.0mm）和中心区（直径6.0mm）。然后进行成球测定以从每个区域的基质中分离前体。为了促进分化，将分离的球形集落铺在含有胎牛血清的培养基的威尔斯孔中。使用免疫细胞化学和/或逆转录聚合酶链反应（RT-PCR）检测球集落及其后代的各种标志物的表达。结果外周间质细胞的原代球形成率（51.4±10.1/10，000细胞）显著高于中央间质细胞（35.9±3.0/10，000细胞; p=0.00021）。外周基质中的次级球体形成率（45.6±6.4/10，000个细胞）显著高于中央基质（33.4±2.1/10，000个细胞; p=0.00002）。来自球体的细胞对CD 34和巢蛋白呈阳性。他们的后代表现出角化细胞样梭形，表达波形蛋白、α-平滑肌肌动蛋白和两种神经分化标记物（微管相关蛋白-2和神经元特异性烯醇化酶）。RT-PCR证实巢蛋白和波形蛋白的表达。结论角膜基质周边区和中央区均含有大量的前体细胞，但周边区前体细胞较多，增殖能力较中央区强。
Purpose To isolate multipotent precursors from the rabbit corneal stroma and to compare the distribution and proliferative capacity of keratocyte precursors obtained from the central and peripheral regions of the corneal stroma. Methods The rabbit corneal stroma was divided into a peripheral region (6.0–10.0 mm in diameter) and a central region (6.0 mm in diameter). A sphere-forming assay was then performed to isolate precursors from the stroma of each region. To promote differentiation, isolated sphere colonies were plated in wells with a medium containing fetal bovine serum. Expression of various markers by the sphere colonies and their progeny was examined using immunocytochemistry and/or reverse-transcription polymerase chain reaction (RT–PCR). Results The rate of primary sphere formation by cells from the peripheral stroma (51.4±10.1/10,000 cells) was significantly higher than by cells from the central stroma (35.9±3.0/10,000 cells; p=0.00021). Secondary sphere formation rate was significantly higher in the peripheral stroma (45.6±6.4/10,000 cells) than in the central stroma (33.4±2.1/10,000 cells; p=0.00002). Cells from the spheres were positive for CD34 and nestin. Their progeny showed a keratocyte-like spindle shape and expressed vimentin, α-smooth muscle actin, and two neural differentiation markers (microtubule-associated protein-2 and neuron-specific enolase). Expression of nestin and vimentin was confirmed by RT–PCR. Conclusions Our findings demonstrate that both the peripheral and central regions of the corneal stroma contain a significant number of precursors, but the peripheral stroma has more precursors with a stronger proliferative capacity than that of cells from the central stroma.