| From the moment of fertilization, embryonic development at all levels is a direct or indirect result of synthetic activities within cell. One of the most important aspects of embryonic development is the nature of the regulatory mechanisms that restrict or permit the synthesis of specific proteins and other macromolecules. To hunt and identify the specific proteins, localize the tissue and time of their appearance will contribute greatly to our understanding of developmental process and tissue regeneration. Liver development comprises multiple stages. Liver develops from the precursors to differentiated organ on 12-week postfertilization. We supposed that liver in this stage is a endocrine-like organ with fuction of secreting developmental signal, and play a vital role in the specification of embryonic cells and histogenesis. In this study, we collected 30 embryo to assay the activity of liver supernate. The results showed that an substance with biological activity of stimulating hepatoma cells proliferation existed in embryonic liver (less 12-week postfertilization). We purified this substance and characterized its physicochemical characteristics and biological activity. It had been confirmed as a novel factor with activity of stimulating hepatoma cells proliferation. We called it human embryo liver-derived factor(hELF). 1. Purification and characterization of hELFAssay the biological activity of 30 tissue homogenate from fetal livers, the activity of stimulating HepG2 and SSMC-7721 hepatoma cell line proliferation was shown in the homogenate from embryo less 12w. First, SA was fractionated with acetone (45%～60%).Then,ultrafiltration up to M.W. 10 000 was performed to confirm the M.W. of the active substance. The result showed that the active substance M.W. was beyond 10 000. The active supernate was applied to Mono S column previously equilibrated with 50 mM acetate buffer (pH 4.0), and material was eluted with the same solution containing 2 M NaCl at a flow rate of 1ml/min. The final step of purification of hELF was achieved by applying the active fractions to Mono S again with the same method. The fractions were collected and assayed the proliferation activity. The overall recovery survival activity in the 4 steps was 9.3%, with 11387-fold increase in specific activity that in the homogenate of liver. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, hELF showed band M. W. 30 000. After the various treatment of the active material, the factor seemed to be a heat-stable protein, it retained the activity in a wide range of pH. Its activity was not influenced by urea treatment. From the results, we can conclude that we have purified the hELF and obtained hELF 200～400ng from tissue homogenate with 30～50mg protein from fetal livers.2. Assay of biology activity of hELFEffect of hELF on different target cells proliferation was assayed. Cultured HepG2 and SSMC-7721 hepatoma cells showed reaction to hELF. These cell lines were used to characterized the growth stimulatory action of hELF purification. The number of viable cells was measured routinely by MTT method.HepG2 cells cultured proliferated slowly in 2% new born calf serum. hELF accelerated the proliferation significantly. Even in the medium without NBCS, HepG2 with hELF survived and proliferated for over 5 days. After cultured 2 days, compared to hELF-free control conditions, HepG2 have proliferated obviously with clones began to form. At Day 7 of culture, clones of HepG2 with hELF have occupied almost the whole dish, and joined together. At the same time, the cell number of control group increased hardly any. SSMC-7721 was also responsive to hELF. The growth-promoting activity of the active substance was dose dependent, and showed direct relation in a range.In order to define futher the range of biological targets of hELF, we examined its action on cultured normal rat hepatocytes, on human fetal hepatocytes, on mouse breast cancer cell line D2F2 and on human uterine cervix cancer cell line Hela.hELF could not...
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