人胎肝中肝细胞生长因子的纯化及某些生物学特性的研究

Studies on the purification and biological peculiarities of hepatocyte growth factors of fetal liver origin

人胎肝细胞裂解后,胞液分别经分子量6万Da、3万Da,1万Da的滤膜超滤,在分子量1—3万Da组分中可检测出肝细胞生长因子(hHGF)活性。此样品再经DEAE离子交换层析,FPLCmonoQ色谱及HPLC分子排阻色谱,已将hHGF纯化为单体,相对活性提高5万倍。在HPLC-TSK2000SW柱上hHGF蛋白峰的分子量为11,100Da,纯hHGF在SDS-PAGE上为一条分子量14,900Da的条带。纯hHGF的生物活性、细胞特异性及理化性质与hHGF粗品一致,也与再生或断乳动物肝中初步纯化的肝生长刺激物相似,而与体液来源的肝生长因子不同。这两种肝细胞生长因子可能共同参与肝细胞生长调控。

Human hepatocyte growth factor (hHGF) was isolated from hepatocyte cytosol of human fetal livers and seperated by ultrafiltration with membrane filters of graded molecular size. hHGF activity was found mainly in the subfractione of molecular weight ranging from 10000 to 30000 daltons. This crude ultrafiltrate was further purified successively by using techniques of DEAE cellulose chromatography, ion exchange fast protein liquid chromatography and size exclusion high performance liquid chromatography. A single band was displayed on sodium dodecyl sulfate polyacrylamide gel electrophoresis, which corresponds to molecular weight of 14900 daltons. The specific activity of purified hHGF was increased about 50000 fold in comparing with the crude subfraction after ultrafiltration. Experimental results indicate that the biological peculiarities of purified hHGF is consistant with the essential nature of the semi-purified hepatic stimulating substances from regenerating livers or wealing animal livers reported, but it is differing from the serum-derived hepatoproteins. However, it is speculated that both HGFs join the growth resulation of hepatocytes in vivo.