大鼠视网膜神经细胞体外培养

In vitro culture of retinal neuron cells of rat

目的 建立视网膜神经层分散细胞的培养方法 ,以进一步对视网膜神经元细胞 (尤其是视网膜神经节细胞 )和神经胶质细胞进行体外实验研究。方法 取出生后 1～ 3d的SD乳鼠的视网膜 ,用胰酶消化法制备分散细胞悬液后 ,接种于置有包被poly D lysine玻片的 2 4孔培养板中培养 ,倒置相差显微镜观察细胞生长规律。培养第 7d ,用抗神经元特异性核 (NeuN)抗体和抗神经丝 (Neurofilament 2 0 0 ,NF 2 0 0 )抗体分别标记视网膜神经元细胞和神经节细胞 (RGCs) ,并计算每 10个高倍镜下的细胞数以及RGCs所占百分数。结果 体外培养的视网膜神经元细胞经历了贴壁、重聚、迁移和相互接触的过程 ,有突起样生长 ,其中RGCs占神经元的 5 5 %左右。结论 视网膜神经层分散细胞体外培养成功 ,并有较好的RGCs生长率 ,为进一步进行视网膜疾病的研究创造了条件。

ObjectiveTo establish a culture system of retinal neuron cells for further research on RGCs,other neurons and glial cells.MethodsRetina of postnatal 1-3-day SD rats was dissected into cell suspension by using trypsin digestion.The cell suspension was planted coverslips covered with Poly-D-lysine in 24 well plates.The growth regularity of cells in vitro was observed under phase-contrast microscope.All neuron cells were identified using anti-rat NeuN antibody and RGCs using anti-rat NF-200 antibody after 7 days.The numbers of the all positive staining cells were counted in each 10-field high power view(HP,200×) under the light microscope.The percentage of NF-positive cells was measured.ResultsAll cultured cells underwent adherence,migration,reunion and contact with each other,and neurites were well developed with the RGCs about 49%.ConclusionSuccessful culture of retinal neuron cells and well developed RGCs in vitro is helpful in the research of retinal diseases.