绿色荧光蛋白标记牛乳腺上皮细胞生长特征和超微结构观察

为了研究绿色荧光蛋白标记对牛乳腺上皮细胞超微结构和细胞体外生长的影响,试验分离、培养和鉴定了牛乳腺上皮细胞,对细胞进行绿色荧光蛋白标记,制作超薄切片,用透射电镜观察细胞超微结构,并进行核型分析。结果发现,牛乳腺上皮细胞呈典型的"铺路石样"单层生长,经绿色荧光蛋白标记后,细胞发出绿色荧光,且维持转染前的基本形态,可以表达广谱细胞角蛋白。在超微结构上,转染前后的牛乳腺上皮细胞均有大而明显的核仁,核膜清晰,核内聚集有异染色质,表明遗传物质稳定。细胞表面微绒毛发达,显示物质、能量或信息的交流活动较强;胞质内线粒体、内质网丰富,胞质内均含有大量的脂滴,显示细胞营养物质代谢旺盛。对GFP标记乳腺上皮细胞进行染色体核型分析显示细胞内含有60条染色体,形态正常,呈二倍体核型。试验结果表明,绿色荧光蛋白可以用于标记牛乳腺上皮细胞,不会对细胞体外生长活性和超微结构等造成明显影响。

Observation of Growth Characteristics and Ultrastructure in GFP-labeled Bovine Mammary Epithelial Cells

The objective of this assay was to describe the effect of green fluorescent protein (GFP) on bovine mammary epithelial cell (MECs) growth morphology and ultrastructure. After bovine MECs were isolated, cultured and identificated, these cells were marked with GFP. The cellular growth morphology was observed after labeling,and the ultrathin sections were produced, cellular ultrastructure was observed by transmission electron microscopy. Subsequently the karyotype was analyzed. The growth morphology of typical "cobble-stone" monolayer was observed in bovine MECs labeled with GFP (named GFP-MECs) in cytoplasm, which similar to the MECs untransfected and both expressed the broad-spectrum keratin. On the ultrastructure, bovine GFP-MECs and MECs both had large and prominent nucleoli and clear nuclear membrane, and the huge amount of heterochromatin was concentrated in the nucleus, which represented the stability of the genetic material. Cellular surface microvilli developed strongly showed that energy or information exchange was active. Cytoplasm was rich in mitochondria, endoplasmic reticulum and lipid droplets suggested cell nutrients metabolism was strong. Karyotype analysis showed GFP-MECs were euploid karyotype. The results indicated that the green fluorescent protein could be used to label bovine MECs, and could not cause significant impact on the growth activity and ultrastructure.