Application of long distance-PCR to hemophilia A carrier detection and prenatal diagnosis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish an effective laboratory examination system for carrier detection and prenatal diagnosis of haemophilia A (HA).</p><p><b>METHODS</b>Twenty-five carriers of severe HA were directly detected by long-distance PCR (LD-PCR) in search of the factor FVIII (FVIII) gene inversion. Prenatal diagnosis was carried out using pregnant woman's venous blood sample, husband's venous blood sample and fetal navel venous sample at 20-24 weeks of gestation. The plasma coagulation factor VIII activity (FVIII:C) was detected by one-stage method. The concentration of von Willbrand factor (Vwf) was assayed by ELISA. Prenatal diagnosis was finally made by LD-PCR. The results of LD-PCR were proved by DNA sequencing.</p><p><b>RESULTS</b>Eight out of 25 cases were diagnosed as having FVIII geneinversion. Four of these 8 carriers underwent the LD-PCR for prenatal diagnosis, and 2 of them had to terminate pregnancy because their fetuses were diagnosed as having HA. The other two carriers were finally diagnosed to have normal fetuses by combined use of LD-PCR with plasma FVIII:C, vWF in pregnant woman's venous blood, husband's venous blood and fetal navel venous blood, and the one-year follow-up study demonstrated that the babies were normal and living well.</p><p><b>CONCLUSION</b>LD-PCR technique was adopted in this study to detect the factor VIII gene inversion; it could accurately and rapidly diagnose the severe cases of HA and could be used for the HA carriers in need of pregnant diagnosis.</p>

Inhibition of HBV DNA replication and expression in 2.2.15 hepatoma cells infected with AFP-mediated HBX antisense RNA / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the specific expression of the antisense RNA against hepatitis B virus X (HBX) gene in hepatoblastoma cell line and its anti -HBV activity.</p><p><b>METHODS</b>HBX gene (nt.1370-1827) was amplified by PCR, then cloned into EB virus vector pEBAF which contained human alpha-fetoprotein promoter and enhancer. After transfected into 2.2.15 hepatoma cells and ECV304 human endothelial cells by lipofectin, northern blot, ELISA and real-time qualitative PCR were carried out to assay the expression of HBX mRNA, HBV antigens and HBV DNA level, respectively.</p><p><b>RESULTS</b>The HBX antisense RNA expression vector pEBAF-as-HBX which could be expressed specifically in 2.2.15 hepatoblastoma cells was successfully constructed. Both HBV DNA level and the expressions of hepatitis B virus surface antigen (HBsAg) and e antigen (HBeAg) in 2.2.15 hepatoblastoma cells were inhibited by pEBAF-as-HBX. Compared with those in sense control (pEBAF-s-HBX), the inhibitory rates of HBsAg, HBeAg, and HBV DNA were 37.9%, 36.8%, and 25%, respectively.</p><p><b>CONCLUSIONS</b>The pEBAF-as-HBX expression vector may lead to targeted-expression of HBX antisense RNA in hepatoma cells and shows great inhibition effect on HBV.</p>