Detection methods of non-Gal xenoantibody in human serum / 器官移植

Objective To investigate the optimal condition for the detection of anti-non-galactose (Gal) xenoantigen and antibody in human serum.Mehtods Peripheral blood mononuclear cell (PBMC) obtained from Wuzhishan miniature pig models with α-1,3-galactosyltransferase gene knockout (GTKO) were used as target cells,mixed and incubated with healthy human serum of different concentrations (4.8％,16.7％ and 100％) for 0.5,1.0,2.0,3.0 and 6.0 h,respectively.The abilities of PBMC to bind with IgM and IgG were detected by flow cytometry.Results At the serum concentration of 16.7％,the ability ofnon-Gal IgM to bind with PBMC was significantly enhanced from 0.5 h to 3.0 h incubation (P＜0.01),whereas no statistical significance was noted in terms of IgG (P＞0.05).Increasing serum concentration could also enhance the ability of non-Gal IgM to bind with PBMC.At the serum concentration of 100％ and incubation for 3 h,the ability of IgM to bind with PBMC was the highest among all groups (P＜0.01).At the serum concentration of 100％ and incubation for 6 h,the ability of IgG to bind with PBMC was significantly enhanced (P＜0.05).Prolonging incubation time and increasing serum concentration did not affect the activity of PBMC.Conclusions The optimal condition for detection of anti-non-Gal xenoantigen and antibody is determined.A quantity of 1×105 PBMC from pig should be incubated with 100％ human serum for 3 h for detection of IgM level,or incubated with 100％ human serum for 6 h for measurement of IgG level.This optimized condition contributes to screening the donor pigs which lowly express non-Gal antigen.

Generation of factor VIII gene knockout mouse by tetraploid embryo complementation technology / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>Factor VIII( FVIII) gene knockout mouse model was established for further study on the treatment of hemophilia A.</p><p><b>METHODS</b>Exons 16-19 of the mouse FVIII gene were knocked out by ET clone, ES homologous recombination and tetraploid embryo compensation technology. PCR, reverse transcriptase-PCR(RT-PCR) and immunohistochemistry were used to detect the transcription and translation pattern of FVIII. The phenotype of the knockout mice was analyzed by examining the activated partial thromboplastin time (APTT) and FVIII activity (FVIII:C).</p><p><b>RESULTS</b>PCR, RT-PCR and immunohistochemistry confirmed that FVIII was deficient in the FVIII gene knockout mouse. The APTT results showed that FVIII-deficient mouse plasma had a prolonged clotting time compared to normal mouse plasma. The FVIII:C in heterozygous, hemizygous and homozygous mice was 80%, 8% and 10% of that in normal mice, respectively.</p><p><b>CONCLUSION</b>The phenotype of the FVIII gene knockout mouse appears grossly similar to that of human with hemophilia A. Establishment of this model may promote the development of new technologies of treatment to hemophilia A.</p>