摘要
目的建立小鼠视网膜神经节细胞的体外纯化培养方法。方法实验研究。采用Thy-1．2单克隆抗体免疫吸附法，将8～12只生后4～6d的C57BL／6小鼠视网膜消化后，制成视网膜神经细胞混合悬液，应用胶质细胞特异性抗体CDllb室温孵育后，去除混悬液中的胶质细胞，应用特异性抗体Thy．1．2吸附混合悬液中的视网膜神经节细胞，接种于预先应用多聚赖氨酸包被过的24孔培养板中，置于1cm×1cm的载玻片上。在温度37℃、体积分数5％的CO2培养箱中培养。观察视网膜神经节细胞的数目和轴突生长情况。结果细胞接种后24h即完全贴壁，视网膜神经节细胞的纯化率达90％以上，细胞生长良好，部分细胞伸出突起，随时问延长轴索逐渐延长，有些轴索的长度可达胞体的5倍以上，呈竹节状，近末端逐渐膨大。2周后可见到死亡细胞，3周时仍有少量存活细胞，但死亡的细胞占多数。结论采用Thy-1．2单克隆抗体免疫吸附法可成功纯化培养小鼠的视网膜神经节细胞。（中华腹科杂志，2011，47：129-133）
Objective To establish a cuhure system for purified mouse retinal ganglion cells （RGC） culture in order to lay a foundation for the in vitro study of RGC. Methods It was a experiment study. Eight to twelve C57BL/6 mice on postnatal day 4 to 6 were used. The retinas were dissected and dissociated enzymatically to make a suspension of single cells. The retinal suspension was incubated in rat anti-mouse-macrophage antiserum for 5 minutes and incubated on a 100 mm anti-rat IgG panning plate at room temperature for 30 min twice. The nonadherent cells were removed with the suspension and placed on the Thy-1.2 panning plate at room temperature. After 45 min, plates were washed 6-10 times with phosphate-buffered saline and swirled moderately to dislodge nonadherent cells. Trypsin （0. 125% solution） dissociation was used to remove adherent cells from the plate. Then cells were spun and resuspended in neurobasal medium containing some neurotrophic factors. The cell suspension was implanted in 24 well culture plates and cultured under 37℃ in an incubator with 5% C02. Results Most of the cells adhered to the plate after 24 hours in culture and showed dendrites at different lengths. The dendrites grew longer with time. Most of the RGC can survive more than two weeks. Conclusion Monoclonal antibody to the Thy-1.2 antigen can be used to purify the mice RGC. （Chin J Opbthalmol, 2011,47：129-133）
出处
《中华眼科杂志》
CAS
CSCD
北大核心
2011年第2期129-133,共5页
Chinese Journal of Ophthalmology
基金
国家自然科学基金（30772376）
作者简介
张绍敏，Email：ssz3@psu．edu
