[Expression of augmenter of liver regeneration in hepatic tumor cells and its clinical significance].

OBJECTIVE: To investigate the effects of augmenter of liver regeneration (ALR) on the proliferation of hepatocytes and hepatic tumor cells and the expression of ALR in herpatocellular carcinoma (HCC). METHODS: Primary rat hepatocytes, QGY and HepG2 cells were cultured separately with ALR from different species. Cell proliferation was detected by their 3H-TdR uptake. The expression of ALR was examined in 9 normal hepatic tissues and 21 HCC cases using immunohistochemistry method. RESULTS: Different ALRs could stimulate the proliferation of HepG2 and QGY cells in a dose-dependent way in vitro, but all ALR had no influence in the proliferation of primary rat hepatocytes. The expression of ALR was absent in normal hepatic tissues, but present in all HCC hepatic tissues. However, the expression of ALR had no relationship with the differentiation and size of the carcinomas. CONCLUSION: ALR might play an important role in the occurrence and development of HCC.

Expression of human augmenter of liver regeneration in pichia pastoris yeast and its bioactivity in vitro.

AIM: To construct a yeast expression system of human augmenter of liver regeneration (hALR) and to examine its bioactivity in vitro. METHODS: With PCR and gene recombination techniques, cDNA of open reading frame of hALR was obtained from recombinant plasmid pcDNA3.1-hALR and inserted into plasmid pPIC9. The cDNA of hALR from recombinant plasmid pPIC9-hALR demonstrated by sequencing was subcloned into plasmid pPIC9K. The recombinant plasmid pPIC9K-hALR was transformed into GS115 with electroporation. hALR was expressed by GS115 under the induction of 5 mL/L methanol and purified with ultrafiltration after it was analyzed by 15% SDS-PAGE and Western blot. The effects of hALR on in vitro proliferation of QGY and HepG(2) cells were evaluated by (3)H-TdR methods. RESULTS: The correctness and integrity of recombinant plasmids pPIC9-hALR and pPIC9K-hALR were identified by restriction digestion, PCR and sequencing methods, respectively. hALR as a secretive protein was successfully expressed by GS115. Its molecular weight was about 15 ku and the target protein was about 60% of the total protein in the supernatant from GS115 with plasmid pPIC9K-hALR. The results of Western blot of hALR showed the specific band. The high qualitative hALR was obtained through ultrafiltration. hALR could stimulate in vitro proliferation of QGY and HepG(2) cells in a dose-dependent manner, but there was a difference in reactivity to hALR between QGY and HepG(2). CONCLUSION: The hALR as a secretive protein can be successfully expressed by GS115. It may stimulate in vitro proliferation of QGY and HepG(2) cells at a dose-dependent manner. But QGY and HepG(2) cells have different reactivities to hALR.

[Expression of rat augmenter of liver regeneration in pichia pastoris and evaluation of its bioactivity in vitro].

OBJECTIVES: To construct yeast expression plasmid of rat augmenter of liver regeneration (rALR), express it in GS115, and examine its bioactivity in vitro. METHODS: With PCR and genetic recombination techniques, the gene fragment of rALR was amplified from recombinant plasmid pcDNA3.1-rALR, and the recombinant plasmid pPIC9K-rALR was constructed. The recombinant plasmid pPIC9K-rALR was transducted into GS115 by electroporation after identified with endonuclease digestion and PCR amplification. The target protein was expressed by GS115 under the induction of 0.5% methanol. The recombinant rALR (rrALR) was purified with ultrafiltration after demonstrated by 15% SDS-PAGE and western blot. The effects of rrALR on the proliferation of QGY, HepG2 cells and primary rat hepatocytes in vitro were evaluated by 3H-TdR intake method. RESULTS: Recombinant plasmid pPIC9K-rALR was identified by restriction digestion and PCR methods. The rrALR as a secretive protein was successfully expressed in GS115. Its molecular weight, 1.5x10(4), was in correspondence with theoretic value. The rrALR could bind with the polyclonal antibody against human ALR in western blot, which demonstrated that there was cross-antigenicity between human and rat ALR. The high qualitative rrALR was obtained through ultrafiltration. Among the experimental concentrations, the rrALR could stimulate the proliferations of QGY and HepG2 cells in a dose-dependent manner in vitro, but had little effect on the proliferation of primary rat hepatocytes in vitro. CONCLUSIONS: The rrALR as a secretive protein is expressed in GS115 efficiently. The rrALR can stimulate the proliferations of QGY and HepG2 cells in a dose-dependent manner in vitro, but had no effect on primary rat hepatocytes, which demonstrates that there exists different receptors on the normal hepatocytes and hepatocarcinoma cells. There are cross-bioactivity and cross-antigenicity between human and rat ALR.