To investigate the non-viral vector mediating human coagulation factor VIII gene expression in mouse 32D cell line, a recombinant plasmid vector, pRC/RSV-hFVIIIBDcDNA, was constructed by cloning B-domain-deleted (Delta760aa-1639aa) human factor VIII cDNA (hFVIIIBDcDNA) into plasmid vector, pRC/RSV. The plasmid RC/RSV-hFVIIIBDcDNA was then transfected by means of SuperFect Transfection Reagent into mouse 32D cell line. After screening with G418, the procoagulant activity (hFVIII:C) and antigen (hFVIII:Ag) of human factor VIII in the culture medium were detected using one-stage method and ELISA, respectively. Furthermore, RT-PCR was performed to observe the transcription of hFVIIIBDcDNA. The results showed that human coagulation factor VIII protein existed in culture medium with hFVIII:C up to 2.01 U/(10(6) cell x 24 hours) and hFVIII:Ag to 450.08 ng/(10(6) cell x 24 hours). RT-PCR displayed mRNA of hFVIIIBDcDNA in 32D cells. It is concluded that the recombinant plasmid RC/RSV-hFVIIIBDcDNA can successfully express human FVIII in mouse 32D cell line, and hFVIII expressed in vitro presents the similar coagulant activity to the native hFVIII existing in normal human plasma.
Animals ; Cell Line ; DNA, Complementary ; genetics ; Enzyme-Linked Immunosorbent Assay ; Factor VIII ; genetics ; metabolism ; Gene Expression Regulation ; Humans ; Mice ; Plasmids ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVETo investigate the gene mutations in a pedigree with inherited prothrombin (FII) deficiency.

METHODSThe activated partial thromboplastin time (APTT), prothrombin time (PT), FII activity (FII:C) and FII antigen (FII:Ag) test were used for phenotype diagnosis. The genomic DNA was extracted from the peripheral blood of the propositus. All the 14 exons, intron/exon boundaries and the 5' and 3' untranslated regions (UTR) of the prothrombin gene were amplified by polymerase chain reaction (PCR). The PCR products were screened by direct sequencing and the mutations detected were further confirmed by restricted enzyme digestion. One hundred and three healthy blood donors were used as controls.

RESULTSThe phenotype of the propositus was prothrombin deficiency (type I). With reference to the prothrombin nucleotide sequence published by Degen & Dacie, three variations were found in the FII gene of the propositus. Among them, the novel mutation was a homozygous A601G subtitution in exon 2.

CONCLUSIONThe prothrombin deficiency of the propositus is caused by a homozygous Glu29 to Gly mutation in the prothrombin gene.

Blood Coagulation ; Child ; Female ; Humans ; Hypoprothrombinemias ; blood ; genetics ; Point Mutation ; Prothrombin ; genetics

OBJECTIVETo explore the gene mutation type of an inherited coagulation factor VII deficiency pedigree.

METHODSFVII: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.

RESULTSHomozygosity 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.

CONCLUSIONSHomozygous 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.

Adolescent ; Adult ; Aged ; DNA Mutational Analysis ; Factor VII ; genetics ; Factor VII Deficiency ; genetics ; Female ; Homozygote ; Humans ; Male ; Middle Aged ; Mutation, Missense ; Pedigree ; Polymerase Chain Reaction

Objective:To investigate the protective effect of ferulic acid on PC12 cells injured by H₂O₂ and the molecular mechanisms. Method:The oxidative stress model was established by treating PC12 cells with H₂O₂, and then different dosages of ferulic acid (1, 10, 100 μmol·L^-1) were used for intervention. Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the cell viability,lactate dehydrogenase (LDH) and malondialdelyde (MDA) in cell supernatant, and superoxidedismutase (SOD) in cells was tested by biochemical method respectively. Insulin-like growth factors-1 (IGF-1) mRNA and protein expressions were detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) and Western blot. Result:The 24 h intervention with different dosages of ferulic acid (1, 10, 100 μmol·L^-1) could significantly improve the oxidative damage of PC12 cells induced by H₂O₂, compared with the model group, ferulic acid at 1,10,100 μmol·L^-1 significantly increased PC12 cells viability,significantly decreased LDH and MDA content in cell supernatant (P<0.05, P<0.01), and enhanced SOD activity(P<0.01). Real-time PCR and Western blot showed that compared with control group,IGF-1 mRNA and protein expressions in model group decreased significantly (P<0.01), compared with model group, IGF-1 mRNA and protein expressions in ferulic acid group increased significantly (P<0.01). Conclusion:Ferulic acid exerts a protective effect on H₂O₂-inducing PC12 cells injury,which might be related to insulin signaling pathways.

Objective:To investigate the protective effect and molecular mechanism of Angelicae Sinensis Radix-Chuanxiong Rhizoma medicated serum （ASRCRS） against oxidative damage of PC12 cells induced by H₂O₂. Method:Oxidative damage of PC12 cells was induced by H₂O₂ in vitro， and intervention was performed in the low-， medium-， and high-dose ASRCRS groups with a final volume fraction of 15%. The cell viability was determined by methyl thiazolyl tetrazolium （MTT） assay. Cell morphology was observed by an inverted fluorescence microscope. The content of lactate dehydrogenase （LDH） and malondialdehyde （MDA）， the activity of superoxide dismutase （SOD）， and the distribution of reactive oxygen species （ROS） in the cell supernatant were detected by the kits. Cell apoptosis was detected by Annexin V-FITC/PI double staining. The protein expression levels of nuclear factor E₂-related factor 2 （Nrf2）， Kelch-like epichlorohydrin associated protein-1 （Keap1）， heme oxygenase-1 （HO-1）， and SOD1 were detected by Western blot. Result:Oxidative damage was induced by 300 μmol·L^-1 H₂O₂ for 24 hours. Compared with the normal group， the model group showed abnormal cell morphology， reduced cell viability （P<0.01）， increased LDH and MDA （P<0.01）， blunted SOD activity， elevated intracellular distribution of ROS， down-regulated protein expression of Nrf2， HO-1， and SOD1 （P<0.05， P<0.05）， and up-regulated protein expression of Keap1 （P<0.01）. Compared with the model group， ASRCRS groups displayed improved cell morphology， increased cell viability， inhibited cell apoptosis， potentiated SOD activity （P<0.01）， suppressed release of LDH （P<0.01） and generation of ROS， decreased content of MDA （P<0.01）， up-regulated protein expression of Nrf2， HO-1 and SOD1 （P<0.05， P<0.01）， and down-regulated protein expression of Keap1 （P<0.01）. Conclusion:ASRCRS could protect PC12 cells from oxidative damage induced by H₂O₂ by up-regulating the expression of Nrf2 to activate the Nrf2/antioxidant response element （ARE） signaling pathway， enhancing the ability to resist oxidative damage， and inhibiting cell apoptosis.

Objective: To investigate the azole susceptibility of Candida albicans ( C. albicans) from vulvovaginal candidosis patients and to analyze the relationship between ERG11 gene mutations in these isolates and azole resistance. Methods: Three hundred and two clinical isolates of Candida species were collected. Azole susceptibility was tested in vitro in microdilution studies. The ERG11 genes of 17 isolates of C. albicans (2 susceptibles, 5 dose-dependent resistants and 10 resistants) were amplified and sequenced. Results: Of the 302 isolates collected, 70.2% were C. albicans, of which 8.5%, 3.8% and 4.2% were resistant to fluconazole, itraconazole and voriconazole, respectively. In total, 27 missense mutations were detected in ERG11 genes from resistant/susceptible dose-dependent isolates. Among them, Y132H, A114S, and Y257H substitutions were most prevalent and were known to cause fluconazole resistance. G464S and F72S also have been proved to cause fluconazole resistance. Two novel substitutions (T285A, S457P) in hotspot regions were identified. Conclusions: Twenty seven mutations in the ERG11 gene were identified in azole-resistant C. albicans isolates, which indicated a possible relation with the increase in resistance to azole drugs and the recurrence of vulvovaginal candidosis. The relationship of two novel substitutions (T285A, S457P) with fluconazole resistance needs to be further verified by site-directed mutagenesis.

Objective: To determine the characteristics and progress of the visual acuity and refractive state of schoolchildren in Huangzhong District, Xining City, Qinghai Province in China. Methods: Cohort study. Department of Ophthalmology, Beijing Children's Hospital carried out a cohort study by collecting the visual acuity and refractive state of Grade 1-5 schoolchildren among 16 primary schools in Huangzhong District, Xining City, Qinghai Province in September 2020 and July 2021. Cycloplegic retinoscopy with eye drop which contained tropicamide (0.5%) and phenylephrine hydrochloride (0.5%) was performed in children with low vision(<1.0). Myopia was defined as the spherical equivalent (SE) ≤-0.5 D after cycloplegic retinoscopy. Measurement data was analyzed by t-test and enumeration data was analyzed by χ² test. Multiple linear regression was used to analyze the influencing factors. Results: The 2 489 individuals with repeated tests in two years were included in the follow-up study, among whom the prevalence of myopia was 26.24%(653/2 489) in 2020, while 32.94% (820/2 489)respectively in 2021. The incidence of myopia in one school year from grades 1 to 5 was 11.19%(47/420), 5.44%(21/386), 6.39%(25/391), 11.52%(44/382) and 11.67%(30/257). The average SE of children in all grades in 2021 increased negatively from the previous year (Grade 1 to Grade 5 increased respectively: 0.40 D, 0.69 D, 0.62 D, 0.52 D and 0.37 D). Conclusions: The prevalence of myopia among schoolchildren in Huangzhong District, Xining City, Qinghai Province was relatively high. There were two peaks of myopia incidence in the first, fourth and fifth grades. Female, age, and the baseline of SE were the related influencing factors for myopia progression.
Child ; Cohort Studies ; Female ; Follow-Up Studies ; Humans ; Mydriatics ; Myopia/epidemiology* ; Ophthalmic Solutions ; Phenylephrine ; Prevalence ; Prospective Studies ; Tropicamide