Construction of IL-2 gene-modified human hepatocyte and its cultivation with microcarrier.

AIM

To construct interleukin-2 gene-modified human hepatocyte line (L-02/IL-2) and investigate the changes of the function of liver cells and IL-2 secretion in culture with microcarrier,laying the foundation for further experimentation on hepatocyte transplantation.

METHODS

hIL-2 gene was transduced into L-02 hepatocytes by recombinant retroviral vector pLNCIL-2, and the changes of morphology and clonogenicity rate of the transduced cells were observed, the secretion levels of hIL-2 in cultural supernatant were detected by ELISA and NeoR gene was amplified by PCR. The growth of L-02/IL-2, the special biochemistry items and the levels of IL-2 were detected after cultivation with microcarrier.

RESULTS

The clonogenicity rate of the L-02/IL-2 cells was lower than that of L-02/Neo cells and L-02 cells. The levels of hIL-2 could reach 32,000 pg/10(6) cells per day and kept secreting for more than ten weeks. NeoR gene segment was respectively obtained by PCR from both L-02/IL-2 and L-02/Neo cell's genomic DNA. At the 6(th) day in culture with microcarrier, the matrix-induced liver cell aggregates were formed, the number of alive L-02/IL-2 cell were 16.8+/-0.53 x 10(6)/flask and the levels of ALB and UREA were 52.54+/-1.28 mg/L and 5.29+/-0.17 mmol/L, respectively. These data had not significantly changed as compared with those of L-02 cells (P>0.05); However, the levels of IL-2 in IL-2/L-02 cells remarkably exceeded that in L-02 cells in the whole culture process (P<0.001).

CONCLUSION

The IL-2 gene-modified hepatocyte line has been successfully constructed. The L-02/IL-2 cellular aggregates cultured with microcarrier have a high capacity of IL-2 production as well as protein synthesis and amino acid metabolism.