肝细胞生长因子和肝细胞生长抑制物质的分离

ISOLATION OF HEPATOCYTE GROWTH INHIBITORY SUBSTANCE AND HEPATOCYTE GROWTH FACTOR

①目的用改良亲和层析法从人胎盘中分离肝细胞生长因子和肝细胞生长抑制物质。②方法取３０ｇ人胎盘加４倍体积（Ｗ／Ｖ）的缓冲溶液，匀浆，４℃，离心（１１０００ｒ／ｍｉｎ）３０ｍｉｎ，收集上清液；用４５％饱和度硫酸铵进行盐析。沉淀复溶后对洗脱缓冲溶液透析２０ｈ，再进行Ｓｅｐｈａｒｏｓｅ４Ｂ凝胶过滤柱层析。最后，将层析第２峰过肝素－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ亲和层析柱，用改良的洗脱方法进行亲和层析。③结果在分离肝细胞生长因子的同时，还分离得到一种肝细胞生长抑制物质。肝细胞生长因子可明显刺激原代培养的大鼠肝细胞ＤＮＡ的合成（ｔ＝６４８．３９，Ｐ＜０．０１），而肝细胞生长因子则抑制原代培养的大鼠肝细胞的ＤＮＡ合成（ｔ＝－１０．４４，Ｐ＜０．０１）。④结论改良亲和层析法在分离效率方面比传统方法有明显提高，而用该法分离到的肝细胞生长抑制物质可能参与了肝细胞的发育调节。

Objective To isolate hepatocyte growth inhibitory substance and hepatocyte growth factor from human placenta by modified affinity chromatogrophy. Methods Thirty grams of human placenta was homogenized in 4 volume of buffer(V/W), and centifuged at 11 000 r/min for 30 minutes at 4℃. The supernatant was pooled and salinedout with ammonium sulfate (45% saturation). After dialized against buffer at 4℃ for 20 hours, the dissolved sediment ran through column filled with Sepharose 4B for gel filtration. Finally, peak 2 ran through column filled with heparinSepharose CL6B for a modified affinity chromatography. Results Together with heptocyte growth factor (HGF), a kind of hepatocyte growth inhibitory substance was isolated. Compared with the control group, HGF apparently stimulated DNA synthesis of rat hepatocyte in primary culture (t=648.39, P<0.01 ), while the hepatocyte growth inhibitory substance inhibited it (t=-10.44, P<0.01) . Conclusion The modified affinity chromatography was more efficient in isolation of hepatocyte growth factor than the classical way. The hepatocyte growth inhibitory substance thus obtained might participate in the proliferation regulation of fetal liver cells.