心室成纤维细胞分泌心肌营养活性蛋白的分离提纯及其作用

PURIFICATION AND EFFECTION OF CARDIOTROPHIC PROTEINS SECRETED BY VENTRICULAR FIBROBLASTS

收集FCGM,用0.2μm微孔滤膜除去细胞碎片,再经Diaflo-YM-10滤膜超滤浓缩,截留分子量大于10 KD的FCGM,行Sephadex-G75凝胶层析,得到Ⅰ、Ⅱ两个洗脱峰。经生物学鉴定,证明Ⅰ峰洗脱液具有明显的心肌营养活性。将凝胶层析Ⅰ峰洗脱液透析、浓缩,行PAK200SW高效液相色谱分析,结果得到五个不同的洗脱峰。经生物学鉴定,第Ⅰ峰具有生物活性。将已行半制备高效液相色谱分析Ⅰ峰洗脱液透析、浓缩,再行高效液相色谱分析,结果获单一峰。用该单一峰物质行SDS-PAGE电泳,以标准蛋白质作对照,可知该单一峰中含有数种物质,分子量在25-35 KD。

Fibroblasts conditioned growth medium( FOGM) was collected after culture of three days. It was removed debris of cells using 0. 2 μm filters,and then ulterafiltrated and concentrated by Diafio YM-10,so the fraction of molecular weight(MW) > 10 KD from FCGM was obtained. FCGM of MW> 10 KD was seperated by gel filtration through Sephadex G75 column at 4℃ . Two fractions of peak Ⅰ and Ⅱ were collected and bioassayed respectively. The fraction I was identified to possessed cardiotrophic activity. Above mentioned biologically active fraction I was dialysed, concentrated, and then further purified by high performance liquid chromatography( HPLC). The five elutions of Peak were obtained and the first fraction was identified to be of biological activity. The elution of peak I .which was dialysed and concentrated, was applied to the high perfermance liquid chromtography column again,Protein peak was detected and gained only one elution peak. Elution protein of peak I of HPLC was analysed by SDS-PAGE. Contrast to the standard proteins,several substances were found in the one elution peak and their MWs were between 25 - 35 KD.