hG-CSF cDNA Cloning and the Construction and Applicaltion in Gene Therapy of Its Retroviral Vector / 中国肿瘤生物治疗杂志

The hG -CSF cDNA was cloned by RT-PCR and confirmed by sequencing, which contains the full length of hG-CSF encoding region and parts of 5 '、3' non - coding region. Then the hG - CSF retroviral vector pLGSN was constructed by orientationally inserting the hG-CSF cDNA into the EcoRI/XhoI cloning sites of pLXSN vector. Packaged with CRE and CRIP packaging cell lines which are considered to be unlikely to produce helper viruses, the final pLGSN retrovirion titer reached 1. 1 ?106 CFU/ml. During constitutive passaging, the CRIP - LGSN cell clone produced relatively stable tilers of pLGSN retrovirion, ranging from 6. 8?105CFU/ml to 1. 1?106CFU/ml. By infecting the murine fibroblast cell line NIH3T3 with pLGSN retrovirion, a cell clone designated as NIH3T3 -G -CSF was obstained, secreting 168U/ml G-CSF . The integration and expression of hG-CSF gene in this cell clone were confirmed by Southern and Northern blotting analyses. Western blotting has also detected specifically the hG-CSF protein in the condensed supernalants from NIH3T3-G-CSF cells . After packaging the hG-CSF-secreting fibroblasts with collagen and implanting them into synergenic mice peritoneally , we detected a certain levels of G-CSF in the sera of mice, which suggested the implanted NIH3T3-G-CSF fibroblast cells could constitutively express and release hG-CSF in vivo. Our data showed the constructed hG-CSF retroviral vector could be used to further investigate the fibroblasl-mediated hG-CSF gene therapy .

STUDY ON OPTIMAL CONDITIONS OF LEUKOREGULIN INDUCTION BY USING ORTHOGONAL EXPERIMENTAL DESIGN / 中国免疫学杂志

Leukoregulin is an immunologic hormone whose anticancer action includethe inhibition of cellular proliferation and cytolysis of tumor cells eitherdirectly or indirectly by enhancing the tumor cell's susceptibility to dest-ruction mediated by natural killer cells.The optimal condition for the ind-uction of leukoregulin Were studied by using orthogonal experimental design.The optimal concentration is 1x10~7/ml for human spleen cells;3.1?g/mlfor phytohemagglutinin(PHA);12.5ng/ml for 12-0-tetradecanoyl-phorbol-13-acetate;15% for boyin serum.Leukoregulin activity prepared by 70 hincubation is maximum,the experiment also confirmed that the inducingleukoregulin effect of PHA-PⅢis significantly high.