Polymorphisms of the coagulation factor VII gene and its plasma levels in relation to acute cerebral infarction differences in allelic frequencies between Chinese Han and European populations / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Coagulation factor VII (FVII) levels in plasma are usually related to ischemic heart disease (IHD) and cerebral infarction shares many of the risk factors related to IHD. Is there any relationship between factor VII and cerebral infarction? We investigated the relationship between FVII and acute cerebral infarction and reported genotype frequencies and allelic frequencies of FVII gene polymorphisms in the Chinese Han population.</p><p><b>METHODS</b>We recruited 62 patients with acute cerebral infarction confirmed by magnetic resonance imaging (MRI) from Ruijin Hospital, and 149 age-matched patients clinically free of vascular disease to act as controls. All of them were unrelated, and were from the Chinese Han population. FVII coagulant activity (FVIIc) was determined using an clotting assay, activated FVII (FVIIa) and FVII Ag were assayed using enzyme immunoassay kits. The FVII gene polymorphisms to be detected included-401G/T, -402G/A, 5'F7A1/A2, IVS7 and R353Q. 5'F7 and IVS7 were revealed by means of a PCR and direct agarose gel electrophoresis. The rest were examined by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).</p><p><b>RESULTS</b>The results showed that FVIIc, FVIIAg and FVIIa were higher in the acute cerebral infarction group than in the control group (P < 0.01, P < 0.05, P < 0.05, respectively). There were no significant differences in the genotype frequencies of FVII gene polymorphisms between the two groups. The allelic frequencies in the Chinese Han population were as follows: -401G/T (96.64/3.36), -402G/A (52.01/47.99), 5'F7A1/A2 (96.64/3.36), IVS7 H5/H6/H7/H8 (0.34/52.35/46.98/0.34) and R353Q (95.64/4.36). There were significant differences (P < 0.01, P < 0.001, P < 0.001, P < 0.001, P < 0.001, respectively) in these allelic frequencies between the Chinese Han and European populations.</p><p><b>CONCLUSIONS</b>The results indicate that increased plasma FVII levels may contribute to thrombosis in cerebral infarction. And there was no significant difference in genotype frequencies of these five FVII gene polymorphisms between the acute cerebral infarction and control groups. Moreover, these results showed that the frequencies of protective allele, including -401T, 5'F7 A2 and 353Q were lower, but that -402A, which was previously found to be associated with increased plasma FVII levels, is higher in Chinese Han population.</p>

In vivo transfection and expression of human coagulant factor VIII cDNA in mice / 中国实验血液学杂志

The aim is to observe the expression of human factor VIII gene in mice tranduced in vivo and ex vivo. The vector pLNC-FVIII BD was generated by cloning a B-domain-deleted (760aa-1639aa) FVIII cDNA (FVIIIBD cDNA) into retroviral vector pLNCX. 2 x 10(6) of mouse bone marrow stroma cells transduced by LNC-FVIII BD were infused into 4-week-old BALB/c mice by tail-vein injection. pLNC-FVIII BD was conjugated with PAMAM dendrimer to form complex PAMAM-pLNC-FVIII BD, with which C57BL/6J were injected by tail vein (200 micro l contained 15 micro g/mouse) and sacrificed at days 1, 2, 7, 14, 21 and 28, respectively after injection. Tissue such as liver, spleen, lung and kindney were harvested, with which the transcription were detected by means of RT-PCR. In addition, blood was collected to be measured human FVIII Ag, human FVIIIc and anti-FVIII of human inhibitors. The results showed that the highest level of human FVIII in the recipient BALB/c mice was 8.6 +/- 1.44 ng/ml detected on the first day post-injection; anti-FVIII antibodies were detected from the first week post-injection, and then the level of FVIII Ag decreased and cannot be measured on the fourth week. In the C57BL/6J mice physiological level of human FVIII was expressed in plasma at 48 hours after injection and the average human FVIIIc was 0.62 U/ml and the average human FVIII Ag was 115.5 ng/ml, and gradually reduced later. Anti-FVIII of human inhibitors was not revealed all the time. Syngene image scanning demonstrated that the transcription of the human FVIII BD cDNA occurred mainly in spleen and lung, and secondarily in liver and kidney. No side effects of PAMAM-pLNC-FVIII BD were observed in mice tissue by pathological examination at 4 weeks. In conclusion, retrovirus-transduced bone marrow stroma cells effectively produced human FVIII after ex vivo transduction, but the development of anti-FVIII antibodies in recipient mice influenced the expression level. The human FVIII gene can successfully be transduced in vivo through injecting PAMAM-pLNC-FVIII BD cDNA into mice intravenously. There was physiological level expression of human FVIII in plasma at 48 hours after injection and the average human FVIIIc is 0.62 U/ml and the peak in the six mice was 0.89 U/ml, and gradually reduced later.

Analysis of an inherited FVII deficiency pedigree caused by homozygosity of Thr359Met / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the gene mutation type of an inherited coagulation factor VII deficiency pedigree.</p><p><b>METHODS</b>FVII:Ag, FVII:C, FVIIa were detected to classify deficiency type. FVII gene mutations were analysed in the proband and her family members by DNA directly sequencing. Biostructural pathology of the identified mutation was analysed by molecular modeling.</p><p><b>RESULTS</b>Homozygosity of C-->T transition at position 11514 in exon 8 resulting in Thr359Met was identified in the proband, and heterozygosity for Thr359Met was confirmed in her parents, her son and some other family members. Thr359Met induces CRM-deficiency. It is found by computer simulated molecular model that the replacement of Thr by Met which has a larger and longer side chain might cause steric hindrance, and change the number of H-bonds.</p><p><b>CONCLUSIONS</b>Homozygous missense mutation Thr359Met was found in a pedigree of hereditary FVII deficiency. This mutation might change the configuration of protein molecule and result in severe FVII deficiency.</p>

Effective and stable in vitro expression of human coagulation factor VIII by retrovirus-based plasmid vector coupled with polyamidoamine dendrimer / 中华血液学杂志

<p><b>OBJECTIVE</b>To demonstrate the effectiveness of a retrovirus-based plasmid vector coupled with nanometer material-polyamidoamine (PAMAM) dendrimer in stable gene expression of FVIII in vitro and to study the cytotoxicity of PAMAM.</p><p><b>METHODS</b>The retrovirus-based plasmid vector pLNC-FVIII BD was generated by cloning a B-domain-deleted (760aa - 1639aa) FVIII cDNA (FVIIIBD cDNA) into retroviral vector pLNCX. The complex that contained PAMAM and pLNC-FVIII BD transfer FVIII BD cDNA into NIH3T3 cell line. In day 2, 5, 10, 15, 30 after transferring, the antigen and procoagulant activity of human FVIII in the cell culture medium were measured by ELISA assay and one-stage method, respectively. RT-PCR was performed for the detection of FVIII BD mRNA. Inhibitory percentage of cell vitality was used for cytotoxicity of PAMAM.</p><p><b>RESULTS</b>Human FVIII was expressed for 30 days by transfected cells. The mean procoagulant activity of secreted FVIII in these 30 days was 0.929 U/ml, and the FVIII antigen was 0.188 micro g/ml by 10(6) cells in 24 hours, respectively. The level of FVIII didn't significantly decreased during these days. Inhibitory percent of cell vitality was only 5.32%.</p><p><b>CONCLUSION</b>PAMAM could effectively transfer pLNC-FVIII BD into NIH3T3 cells and FVIII could be stably and effectively expressed by the transfected cells. Cytotoxicity of PAMAM was low.</p>

Sperm-mediated expression of human clotting factor VIII gene in transgenic offspring of mice / 中国实验血液学杂志

The expression of human clotting factor VIII gene was observed in transgenic off spring of mice through artificial insemination with sperm as carriers. Female mice were impregnated through artificial insemination by introducing sperm carrying pRC/RSV-hF VIII BD, which contained human F VIII BD (B-domain deleted) cDNA (hF VIII BD c DNA), into the uteri. During the fourth week after the birth of new-born mice, PCR was used to screen hF VIII BD cDNA positive transgenic mice, then blood of which was collected for detecting the antigen and Anti-hF VIII inhibitors, simultaneously, the transcription and expression of hF VIII BD cDNA were investigated by Northern blot and Western blot. The results showed that 7 became pregnant of 20 inseminated mice, and 11 new-born mice came into the world, out of which 9 survived at last. Three hF VIII BD cDNA-positive-transgenic mice had been screened out by PC R, in which the antigen of human F VIII in plasma was 8.65 ng/ml, 7.84 ng/ml and 8.44 ng/ml, respectively, the Anti-hF VIII inhibitors were all negative. Northern blot and Western blot showed that the transcription and expression of hF VIII BD cDNA existed in tissues such as spleen, liver, lung and kidney of 3 transgenic mice. It was concluded that transgenic mice carrying human F VIII gene can be generated by sperm-carrier techniques and express human F VIII protein. This experiment provides important data for manufacturing transgenic animal carrying human F VIII gene, which can work as a biological reactor to produce human F VIII protein, through sperm-carrier techniques.