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The purpose of this study was to purify and characterize hepatic stimulatory substance (HSS), a protein found in the liver that has been reported to stimulate hepatic DNA synthesis and hepatocyte replication and that was previously identified by La-Brecque et al1–3 and Francavilla et al in the rat liver,4,5 as well as by Starzl et al in the canine liver.6,7
HSS has been partially purified by successive steps, involving ethanol precipitation, ultrafiltration, and fast protein lipid chromatography (FPLC). The activity of this factor has been tested in 40% hepatectomized rats.
Male Fischer (F344) rats (180 to 200 g) and weanling Fischer (F344) rats (60 to 90 g), were purchased from Hilltop Lab Animals, Scottsdale, PA, and were kept in temperature and light controlled rooms. They received food and water ad libitum.
Forty percent partial hepatectomies (PH) were performed in rats according to Higgins and Anderson.8 Control animals underwent a sham operation consisting of laparotomy and manual manipulation of the liver.
The activity of various fractions was determined in vivo using 40% hepatectomized rats. Experiments were carried out according to LaBrecque and Pesch.1 [3H]-thymidine incorporation, labeling and mitotic indexes were determined as previously described.13
Statistical analysis of groups was carried out by one-way analysis of variance using SPSS/PC statistical software (SPSS, Inc, Chicago) on an IBM-AT microcomputer.
Table 2 describes the purification steps of HSS and the physico-chemical characteristics of active fractions named F150 on the basis of its elution from the FPLC columns with MaCl at 150 mmol/L.
HSS seems to be a protein with a molecular weight (MW) between 50 and 14 kd, which is resistant to neuraminidase, is destroyed by trypsin, and is resistant to heating at 95°C for ten minutes. The purification of F I50 results in a 38,000-fold increase of activity over the original cytosol. As previously described,14 each preparation of F150 obtained from FPLC chromatography was completely free of recognizable hormones such as insulin, glucagon, vasopressin, and EGF.14 The activity of each fraction during the various purification steps was tested in 40% hepatectomized rats. All fractions demonstrated significantly more activity than PBS when injected into rats.
The activity of F150 was dose-dependent over a range of 1.76 μg to 6.8 μg per 100 g body weight of the recipient rat. Thymidine incorporation results were confirmed by labeling and mitotic index results, as shown in Fig 1.
It has been clearly demonstrated that F150 is organ, but not species specific.14 This fraction, as it presently exists, contains a few proteins, as indicated by SDS page with a MW ranging between 14 and 60 kd. Further studies are in process to obtain the final purification of HSS. Completely purified HSS would represent an important step in the knowledge of hepatocyte proliferation as well as in clinical therapy.
The use of growth factor therapy for acute liver failure as well as in acute rejection after liver transplantation in animals and in humans is, in fact, the main objective of this study. In fact, we have already shown that this type of therapy, using fractions obtained during the HSS purification, significantly improves the survival rate of rats intoxicated with the selective hepatotoxin D-Galactosamine.13
Supported by Research grants from the Veterans Administration and Project Grant No. AM-29961 from the National Institutes of Health, Bethesda, MD, and by Grant No. 885/02 16544 from the Consiglio Nazionale delle Ricerche, Italy.
We are grateful to John Prelich for technical assistance.