Objective To study the effect of lipopolysaccharide ( LPS ) on the activity of primary cultured macrophages and the distribution of divalent metal transporter 1 ( DMT1 ) and ferroportin 1 ( FPN1 ) .Methods Primary cell culture , MTT chromotest , cytochemistry chromotest and cell immunofluorescence techniques were used in this work . Results DMT1 was mainly distributed in the cytoplasm , which illuminates that DMT1 mediates the macrophage intracellular transit of iron from phagolysosome to cytoplasm .FPN1 was distributed in the cytoplasm and membrane , and the cytoplasm was the main site of FPN 1 distribution in macrophages .Conclusion Iron liberation from heme inside the phagolysosome occurs after erythrophagocytosis and it is possible that FPN 1 mediates intracellular transit of iron released by heme catabolism .The study found that LPS promoted the cell growth and this effect was reached to the peak in the 10 -5μg/L LPS group, but the cell growth was blocked with the increase of LPS concentration .

OBJECTlVE To develop an LC-MS/MS method for simultaneous determination of pro-damine ( PDM) and its metabolite 2,4-dinitro-N3-propyl-6-trifluoromethyl-1,3-benzenediamine ( DTB) in rat plasma in order to study toxicokinetics of PDM in rats. METHODS SD male rats were administered a single dose of PDM ( ig: 100 and 1000 mg·kg-1; iv: 100 mg·kg-1 ) . LC-MS/MS method was used to determine PDM and DTB in rat plasma. Toxicokinetic parameters were fitted using DAS Ver2. 1. 1. RESULTS After ig administration of PDM 100 mg·kg-1 , the parameters of PDM and DTB were as fol-lows:AUC(0-t) was 2715±102 and (6845±316)μg·h·L-1, t1/2z was 9.0±1.4 and (7.1±1.3)h, Tmax was 7.0± 1.6 and (7.0±0.0)h, cmax was 146±51 and (473±103)μg·L-1. After ig administration of PDM 1000 mg·kg-1, the parameters of PDM and DTB were as follows:AUC(0-t) was 3401±242 and (10364± 573)μg·h·L-1, t1/2z was 8.8±2.1 and (6.0±1.8)h, Tmax was (7.0±1.6)h, cmax was 175±56 and (586± 152)μg·L-1 . The absolute bioavailability of PDM was 44.9%( 100 mg·kg-1 ) and 17.1%( 1000 mg·kg-1 ) . CONCLUSlON This method is suitable for the analysis of PDM and DTB in rat plasma. There is evidence that PDM and DTB display nonlinear toxicokinetic characteristics in the studied dose range.

OBJECTIVETo accomplish a kind of therapeutic gene for hemophilia A, and observe the expression of human factor VIII (hF VIII) in vivo.

METHODSHuman clotting factor VIII cDNA with B-domain deleted (Delta760aa approximately 1639aa) was inserted into vector pRC/RSV to form pRC/RSV-hF VIII BD, which conjugated with in vivo liposome transfection reagent (DOTAP-Cholesterol) to accomplish a kind of therapeutic gene, pRC/RSV-hF VIII BD-DOTAP-Cholesterol. Mice were injected with pRC/RSV-hF VIII BD-DOTAP-Cholesterol i.m. and sacrificed 48 hours, 10 days, 20 days, 30 days, 40 days and 50 days later, respectively. Tissues such as heart, liver, spleen, lung, kidney and muscle were harvested, the distribution and transcription as well as expression of hF VIII BD cDNA were detected by means of PCR, RT-PCR and immunohistochemistry techniques. In addition, the antigen and antibody of hF VIII in plasma were measured.

RESULTSThere was high expression of hF VIII in plasma and tissues at the 48(th) hour after injection. On day 10, antigen level of hF VIII in plasma reached its peak, 17.55 ng/ml, and gradually reduced later. The antibody of hF VIII in plasma emerged on day 10 after injection, and increased and gradually reached 37.06 U/ml on day 50 after injection. PCR, RT-PCR and immunohistochemistry showed that hF VIII BD cDNA and its transcription as well as expression existed in all kinds of tissues, and lasted longer in spleen, lungs and kidneys than in heart, liver and muscle.

CONCLUSIONTherapeutic gene, pRC/RSV-hF VIII BD-DOTAP-Cholesterol, produced by combination of pRC/RSV-hF VIII BD and DOTAP-Cholesterol liposome can express human F VIII successfully in vivo, which lays an experimental foundation for curing hemophilia A by gene-drug in clinic.

Animals ; DNA, Complementary ; Disease Models, Animal ; Factor VIII ; biosynthesis ; genetics ; therapeutic use ; Gene Expression ; Genetic Therapy ; Genetic Vectors ; Hemophilia A ; therapy ; Humans ; Liposomes ; Mice ; Mice, Inbred BALB C ; Tissue Distribution ; Transfection

OBJECTIVETo identify the mutation of coagulation factor VII (F VII) gene in two pedigrees with hereditary F VII deficiency.

METHODSF VII gene mutations were analysed in two propositi and their family members by direct DNA sequencing. Allele specific PCR and PCR combined with restricted enzyme digestion were used to confirm the detected mutations.

RESULTSTwo gene mutations were detected in the propositus of pedigree A: G to C transition at position 6390 resulting in Trp40Cys and G to A at 11496 resulting in Arg353Gln, both are heterozygotes. The heterozygosity for polymorphism Arg353Gln was confirmed with the restriction enzyme Msp I digestion in his mother. In the propositus of pedigree B, there was a T to G transition at position 11482 resulting in His348Gln, heterozygosity of which was confirmed with Nsp I digestion in the propositus and his daughter. G to T transition at position 11514 resulting in Thr359Met was also found in the propositus of pedigree B, and the heterozygosity for Thr359Met was confirmed with allele specific PCR in the propositus and his son.

CONCLUSIONThree missense mutations were found in two pedigrees with hereditary F VII deficiency. A novel Trp40Cys mutation was reported for the first time.

Factor VII ; genetics ; Factor VII Deficiency ; genetics ; Female ; Heterozygote ; Humans ; Male ; Mutation, Missense ; Pedigree ; Point Mutation ; Polymerase Chain Reaction ; methods

OBJECTIVETo explore gene defect of hereditary coagulation factor XIII deficiency.

METHODSPCR and gene sequencing or ARMS-PCR were used to detect the FXIIIA gene of peripheral white blood cell (PBC) from two Chinese hereditary coagulation factor XIII deficiency family members and 60 normal subjects respectively. The level of FXIIIA gene mRNA was tested by RT-PCR.

RESULTS(1) Nucleotide sequence analysis of the two probands' and their family members' DNA revealed that all of the three patients had homozygous missense mutation in FXIII A subunit gene. Proband 1 had a C to G transition at nucleotide (nt) 1 241 in exon 10 and proband 2 and his sister a C to T transition at nt 232 in exon 3 of FXIII A gene, which resulted in the substitution of Ser413 with Trp and Arg 77 with Cys, respectively. Family study showed that the two mutations were inherited from the parents who were correspondingly heterozygotes at nt 1 241 or nt 232. (2) The two mutations were not found in the normal subjects. (3) The FXIIIA gene mRNA level in the two probands was a little decreasing.

CONCLUSIONIt is the two novel mutations that results in FXIIIA deficiency. The two mutations of FXIIIA gene may affect its function or alter protein folding. The defective FXIII which is unstable and degraded rapidly in cytoplasm may be the main cause of FXIII deficiency.

Blood Coagulation Disorders, Inherited ; genetics ; Child ; Exons ; genetics ; Factor XIII ; genetics ; Factor XIII Deficiency ; genetics ; Female ; Heterozygote ; Humans ; Pedigree ; Point Mutation ; Polymerase Chain Reaction ; methods

OBJECTIVETo explore the effect and mechanism of sodium butyrate on expression of human clotting factor VIII in vitro.

METHODSMouse NIH/3T3 cell line was transfected with recombinant plasmid vector pRC/RSV-BDD-hFVIII, which enclosed B-domain deleted (760aa approximately 1 639aa) human factor VIII cDNA (BDD-hFVIII cDNA). Then cells were incubated in Dulbecco's modification of Eagle's medium (DMEM) containing sodium butyrate for 24 hours, hFVIII: C and hFVIII: Ag in the cell culture medium were measured by ELISA assay and one-stage method, respectively. In addition, the effect of sodium butyrate on transcription of cDNA encoding the whole hFVIII, heavy and light chain of hFVIII was also investigated by means of run-on assay.

RESULTSAfter stimulation of sodium butyrate, the levels of hFVIII: C and hFVIII: Ag increased 70% than those of control. Run-on assay showed that sodium butyrate enhanced the transcription of cDNA which encoded heavy chain of hFVIII.

CONCLUSIONSodium butyrate can improve the expression of hFVIII through enhancing the transcription of hFVIII heavy chain encoding cDNA. It demonstrated that sodium butyrate had potential utility in inducing the expression of hFVIII in vitro.

3T3 Cells ; Animals ; Butyrates ; pharmacology ; Factor VIII ; genetics ; Gene Expression Regulation ; drug effects ; Humans ; Mice

OBJECTIVETo investigate the plasma levels of coagulation factor VII (FVII) and polymorphisms of FVII gene in patients with coronary heart disease (CHD), and evaluate the effect of plasma FVII levels and FVII gene polymorphisms on CHD.

METHODSPlasma FVIIa, FVII: Ag and FVIIc were measured and polymorphisms of FVII gene were analyzed in 149 control cases and 60 CHD cases, including 33 acute myocardial infarction (AMI) cases by a combination of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and agarose gel electrophoresis.

RESULTSFVIIa, FVIIc in AMI group were significantly higher than that in control group, but FVII: Ag wasn't. There were no significant difference in plasma FVIIa, FVII: Ag and FVIIc between CHD and control group. The IVS7 genotypic frequency in AMI group was significantly different from that in control group. There was no significant difference in genotypic frequencies and allelic frequencies in other polymphism sites. FVII: Ag was significantly higher in -402A homozygote than that in -402G homozygote.

CONCLUSIONSIncreased FVII levels, especially FVIIa and FVIIc in plasma, may contribute to coronary artery thrombosis. There was significant difference in IVS7 genotype frequency between control and AMI groups, but the rest weren't significantly different. FVII: Ag was significantly higher in -402A homozygote than that in -402G homozygote. Polymorphism of -402 G/A may play an indirect role in AMI by regulating plasma FVII levels.

Coronary Disease ; blood ; genetics ; Factor VII ; analysis ; genetics ; Female ; Genotype ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length

Objective:To investigate the application and effect of blended teaching model in clinical skill training.Methods:According to the practical teaching syllabus for undergraduate medical students in Shanghai Jiao Tong University School of Medicine and the national practical operation examination for medical practitioners, 20 basic skills of internal medicine, surgery, gynecology, pediatrics, and emergency were selected. A total of 120 junior medical students in the experimental group received the practice course of basic clinical skill operations using the online and offline blended teaching model in the academic years of 2020-2021, and 120 medical students in the control group received this course using the offline teaching model alone during the same period of time. The two groups were compared in terms of process completeness, skill proficiency, aseptic concept, communication and care, and clinical thinking, and a questionnaire survey was conducted for the students in the experimental group to investigate course design, adaptability, interest, satisfaction, and learning effect. SPSS 21.0 was used to perform the Wilcoxon rank-sum test and the chi-square test.Results:There were no significant differences between the two groups in general information such as sex and major. The experimental group had significantly better scores of the above practical abilities than the control group [88 (86,89) vs. 75 (72,77), 57 (56,58) vs. 52 (50,54), 7 (6,8) vs. 5 (4,6), 8 (7,8) vs. 5 (4,6), 9 (8,9) vs. 6(6,7), 8 (7,9) vs. 7 (6,7), P<0.001], and the questionnaire survey showed that the students in the experimental group gave a relatively high overall evaluation of the course (4.0-4.8 points). Conclusions:The blended teaching model is beneficial to the cultivation of clinical skills and practical ability in undergraduate medical students and can help to enhance their self-learning and operational abilities and improve classroom efficiency and teaching effectiveness.