SMMC-7721肝癌细胞67kD层粘连蛋白受体的分离纯化

Isolation and Purification of 67 kD Laminin Receptor from SMMC-7721 Hepatocellular Carcinoma Cell

分离纯化肝癌细胞的 6 7kD层粘连蛋白受体 (6 7LR) ,以便进一步研究 6 7LR的结构、功能及其在肝癌浸润、转移过程中的作用 .以SMMC 772 1肝癌细胞和L 0 2正常肝细胞为材料 ,采用13 1I标记的层粘连蛋白测定其与细胞的结合能力 ;亲和层析法分离纯化层粘连蛋白受体 ,用SDS PAGE、放射自显影及体外竞争结合实验进行鉴定 .在相同条件下SMMC 772 1肝癌细胞与层粘连蛋白特异结合量为 17 5 4± 0 4 9ng 10 5细胞 ,而L 0 2正常肝细胞与层粘连蛋白的特异结合量为 8 36± 0 4 8ng 10 5细胞 .经过亲和层析 ,从SMMC 772 1肝癌细胞和L 0 2正常肝细胞均可获得纯化受体 ,SDS PAGE显示为单一条带 ,分子量为 6 7kD ,放射自显影及体外竞争结合实验表明其具有较强的与层粘连蛋白结合的活性 .体外竞争结合实验表明 ,SMMC 772 1肝癌细胞层粘连蛋白受体 (772 1LnR)的抑制率可达到 96 2 7± 2 2 9% ,而L 0 2正常肝细胞层粘连蛋白受体 (L 0 2LnR)的抑制率为 4 8 71± 3 79% ,这说明 772 1LnR与层粘连蛋白的亲和力明显高于L 0 2LnR(P <0 0 0 1) .结果表明 ,与L 0 2肝细胞比较 ,SMMC 772 1肝癌细胞具有与层粘连蛋白较强结合能力的特异受体 ,并从肝癌细胞膜上分离纯化到与层粘连蛋白有较强亲和力的 6

67kD laminin receptor(67LR) was isolated and purified from SMMC-7721 hepatocellular carcinoma(HCC) cell line and L-02 hepatic cell line so as to investigate its structure and function as well as its role in metastasis of hepatocellular carcinoma. The binding of laminin to cells was quantified by 131I-labeled laminin. Laminin receptor was isolated and purified by laminin fragment conjugated Sepharose affinity chromatography and the receptors were identified by SDS-PAGE, autoradiography and competitive binding assay. Under the same condition, the amount of radiolabelled laminin binding to SMMC-7721 HCC cells and L-02 hepatic cells was 17.54±0.49 ng per 10+5 cells and 8.36±0.48 ng per 10+5 cells respectively (P<0.001).After purification, these two receptors migrated as a single band of 67 kD on SDS-PAGE.The purified receptors retained a high binding affinity in autoradiography and competitive binding assay.The rate of competitive inhibition of the laminin receptor from SMMC-7721 HCC cells (7721LnR) was 96.27±2.29%,while the laminin receptor from L-02 hepatic cells (L-02LnR) was only 48.71±3.79%,suggesting that 7721LnR had higher binding affinity with laminin than L-02LnR.It has been shown that there is higher binding activity of laminin to SMMC-7721 HCC cells and the 67 kD laminin receptor isolated from SMMC-7721 HCC cells shows higher binding affinity with laminin,compared with that from L-02 hepatic cells.