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 .

Objective: To investigate the change of hepatitis B surface antibody (HBsAb) titer and its long-term protection and infection rates between 1 and 3-year-old children whose mothers were chronic hepatitis B pregnant woman with HBeAg positive and high viral load after successful blocking of mother-to-child transmission. Methods: One-year-old children whose mothers were hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) positive, with HBV DNA≥10⁶IU/ml were enrolled, then were followed up till 3 years old, and tested the five serological markers of hepatitis B and biochemical parameters at the age of one and three years respectively, and analyzed HBsAb titer, positive rate, negative rate and infection rate of 1 to 3-year-old children without enhanced vaccination; meanwhile, data of HBsAb titers at the age of 7 months were collected HBsAb titer, positive rate, and negative rate were analyzed. Results: Totally 264 1-year-old children were enrolled into the study, including 178 children without enhanced vaccination between seven months and 1 year of age, and 114 children without enhanced vaccination between 1 year and 3 years of age. Our result showed that there were no infected children at the age between 1 and 3 years. HBsAb titer decreased from 7 months to 1 year old and dropped from 1 000 IU/L to 509.43 IU/L (P<0.05), and the antibody was still protective. From 1 year to 3 years old, HBsAb titer dropped from 466.72 IU/L to 67.3 IU/L (P< 0.05); at the age of 3 years, 60.52 % children were either weakly positive or negative, but still protective, but significantly less than those who had the reinforced vaccination. As a result , the children without the enhanced vaccination between 1 and 3 years of age were still at high risk. Conclusions If the antibody was protective at 7 months, children were not easily infected between 1 year and 3 years of age. At the age of 3, the antibody dropped to low or no responsive levels, and the children were still at high risk. It is necessary to take protective measures and supplement the vaccine.