An eukaryotic expressing vector that expresses CH50, a recombinant polypeptide of human fibronectin, in mice was constructed, and its chemotactic and anti-tumor function by in vivo gene transfection was investigated. The plasmid was constructed by recombination techniques. The cDNA fragment coding CH50 polypeptide from a prokaryotic expressing vector of CH50 was ligated with 5'-terminal noncoding region and coding region of signal peptide of mouse IFN-gamma cDNA at 5' side and 3'-terminal noncoden region of human FN cDNA at 3' side. The recombinant cDNA was inserted into plasmid pREP8. The resulted expressing plasmid was designated as pCH503. The macrophages transfected with pCH503 in vivo and cultured in vitro could produce CH50. The expressed product was identified by heparin-affinity chromatography and SDS-PAGE. By counting and Giemsa-staining of coeliac cells and histotomy and staining of muscle tissue, the chemotaxis on immune cells was observed after transfection of pCH503 either in peritoneal cavity or in muscle. The inhibition of gene transfection of pCH503 on melanoma was observed in mice. The number of melanoma nodes in mice was reduced by 50%-60% after coeliac transfection with pCH503. The pCH503, an eukaryotic expressing vector of CH50, can express in vivo in mice. The transfection of pCH503 in vivo has the chemotaxis on immune cells and can inhibit the formation of tumor nodes, suggesting that plasmid pCH503 is potentially useful in combined treatment of tumor.
The inhibition of CH50, a recombinant polypeptide of human fibronectin, on the formation of tumor metastases in vivo was studied by inoculation of melanoma B16/F1 cells by hypodermic or intraperitoneal injection, and the size and amount of tumor nodes after therapy were measured. In the treatment of hypodermic tumor, local injection of CH50 produced much better efficacy than distance injection of CH50 did. The inhibitory effect of CH50 on the growth of tumor reached 50%. In the treatment of peritoneal metastasis, the inhibition of CH50 on metastases smaller than 1 mm was above 80%, and above 50% on metastases larger than 1 mm. A better efficacy was achieved if CH50 was used in combination with hydroxycamptothecine (HCPT), a chemotherapeutic agent. CH50 displayed a strong inhibitory effect on the formation of small metastasis and growth of larger nodes of tumor, suggesting that CH50 plays a very important role in combined treatment of tumor therapy.
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