Objective To study two new factor Ⅸ mutations Cys82Ser and Ile288Ser in vitro and research the molecular mechanism of haemophilia B. Methods PcDNA3. 1 ( - ) FⅨwt expression plasmid was prepared. The mutated FⅨcDNA expression plasmids, PcDNA3.1 ( - ) FⅨM1 (Cys82Ser) and PcDNA3. 1 ( - ) F Ⅸ M2 (Ile288Ser) were constructed by megaprimer method respectively. Transient expression experiments were performed using HEK293 cells transfected with the expression vectors containing the wild-type or the mutation recombinant cDNA. PcDNA3. 1 ( - ) was used as a blank control. The expression proteins were detected by ELISA, factor activity assay and flourescence stain. Results The results suggested that the two FⅨ gene mutations did not induce the reduction of the mutant FⅨ mRNA compared with the wild-type FⅨ mRNA. The FⅨ:Ag in culture media and cell lysate of wild type conduct were assigned as 100. 0%. The results of PcDNA3.1 ( - ) FⅨ M1 mutation protein were (27. 1 ± 5. 2)% and (99.4 ±4. 1)% respectively. For PcDNA3. 1( - )FⅨM2, the results were (5.3 ± 1.8)% and (31.7 ±2. 5)% respectively. The FⅨ: C in culture media of wild type conduct was also assigned as 100. 0%. Then the two types of mutant protein were ( 8. 5 ± 3.2 ) % and ＜ 1%, respectively. Immunofluorescence microscopy result suggested that the intensity of perinuclear spot was reduced in cells transfected with PcDNA3.1 ( - ) FⅨM2 while staining for PcDNA3. 1 ( - ) FⅨM1 was predominantely diffuse without perinnclear enhancement. Conclusions These results strongly suggest that the FⅨ Cys82Ser mutation protein is not been correctly folded, by any possibility. The mutation protein has secretion defect. The secretion dysfunction and the protein degradation intracellular are possiblely the molecular pathology of Ile288Ser mutant protein.

Objective To investigate the gene defects of a pedigree with inherited coagulation factor Ⅺ (FⅪ) deficiency by analyzing its phenotype and molecular genetic characteristics. Methods A pedigree with inherited FⅪ deficiency was enrolled in this study. The activated partial thromboplastin time (APTF), prothrombin time (PT), FⅪ activity (FⅪ: C) and FⅪ antigen (FⅪ: Ag) were determined for phenotype diagnosis. Fifteen exons and their flanks of F11 gene from the proband's genomic DNA were amplified by polymerase chain reaction (PCR), and the PCR products were directly sequenced to analyze the F11 gene mutation. The PCR products amplified from genomic DNA from the proband, her parents and 100 healthy donors were digested with restriction enzyme BssSI to exclude gene polymorphism and confirm the mutation site. The cleavage site in the signal peptide was predicted by the SignalP software. Results The values of APTT, PT, FⅪ: C and FⅪ: Ag of the proband were 69.5 s, 12.3 s, 2.6% and 2.5%, respectively, indicating that this case was cross-reacting material (CRM) negative. The same values of healthy controls were 35 s, 13 s, 100% and 100%, respectively. As compared with Genbank AY191837 sequence, four variants in F11 exons were found. G3733C heterozygous mutation in exon 2 causod Gly to Arg substitution at-1 amino acid position in signal peptide (G-1R). The G3733C mutation in exon 2 introduced a new BssSI enzyme digestion site. Further analysis of the 100 randomly collected DNA samples from the normal population excluded the possibility of G3733C as a polymorphism. CI6642T heterozygous mutation in exon 8 introduced a premature stop codon at 263 amino acid position (Q263Term). Conclusions G-1R mutation and Q263Term compound heterozygous mutation in F11 gene are the mechanism of FⅪ deficiency for the proband. G-1R mutation is a novel F11 gene mutation causing inherited FⅪ deficiency.

Objective To compare the effect of different positive end-expiratory pressures (PEEPs) on the efficacy of volume therapy guided by global end-diastolic volume index (GEDVI) and central venous pressure (CVP) in patients with septic shock.Methods Twenty-five patients with septic shock complicated with respiratory failure,of both sexes,aged 18-64 yr,were enrolled in the study.Their APACHE [[scores were 13-31.The patients were endotracheally intubated and underwent volume-controlled ventilation,PEEP was 5-15 cmH2O,and pulse oxygen saturation was maintained ＞ 90 ％.The patients were divided into low PEEP (5-10 cmH2 O) group and high PEEP (11-15 cmH2 O) group depending on the different PEEP levels.6 ％ hydroxyethyl starch (200/0.5)6 ml/kg was infused over 30 min for volume therapy.Right internal jugular vein or subclavian vein was cannulated for CVP monitoring,and GEDVI was continuously monitored by pulse indicator continuous output monitoring (PiCCO) technology.CVP and GEDVI were recorded before and after volume therapy and the changing rate was calculated.Results Compared with CVP and GEDVI before volume therapy,CVP and GEDVI were significantly increased after volume therapy in low PEEP group (P ＜ 0.05),and GEDVI was increased after volume therapy (P ＜ 0.05) and no significant change was found in CVP after volume therapy in high PEEP group (P ＞ 0.05).Compared with low PEEP group,the changing rate of CVP was significantly decreased (P ＜ 0.05),and no significant change was found in the changing rate of GEDVI in high PEEP group (P ＞ 0.05).Conclusion High PEEP can decrease the efficacy of volume therapy guided by CVP,while exerts no effect on the efficacy of volume therapy guided by GEDVI in patients with septic shock.

OBJECTIVE To find the infla mmation bio markers induced by coke oven e missions (COE),we investigated the changes of T helper 17 (Th17 )cytokines in hu man bronchial epithelial (16HBE)cells.METHODS 16HBE cells were exposed to organic extracts of COE collected fro m co-king plant at the concentrations of 5,10 and 20 mg·L -1 for 24 h or 5 d to establish short-term and long-term cell models,respectively.Cell viability was measured by MTT assay and infla mmatory da mage was assessed by lactate dehydrogenase assay (LDH).The cytokines in culture supernatant sa mples was detected by co mmercial hu man Th17 cytokine panel kit.RESULTS COE Can induce infla mmation in COE 20 mg·L -1 group and no expression on IL-17 F and IL-1 β.The concentration of IL-10 was 1 .25 ± 0.54,1 .39 ±0.13 and (1 .90 ±0.73)pg·mL -1 in COE 5,10 and 20 mg·L -1 group showing good con-centration-effect relationship (r=0.98,P <0.05 ).IL-23 expression was found only higher at 10 and 20 mg·L -1 and the concentrations were 3.38 ±3.90 and (1 .74 ±2.00 )pg·mL -1 ,respectively.In 16HBE cells treated by COE for 5 d,elevated expression of IL-17A was found in COE 5 and 10 mg·L -1 group,and there was statistically sigificant difference between COE 10 mg·L -1 and DMSO group (P<0.05).Elevated concentration of IL-17F of 10.2 ±1 1 .78 and (6.79 ±7.84)pg·mL -1 was found in COE 5 and 10 mg·L -1 group.The concentration of IL-10 was 1 .71 ±0.02,1 .49 ±0.25 and (2.82 ± 0.33)pg·mL -1 in COE 5,10 and 20 mg·L -1 group,respectively.We found increased IL-1 βexpression with concentration of 2.72 ±0.62,2.25 ±0.33 and (0.93 ±0.21 )pg·mL -1 in COE 5,10 and 20 mg·L -1 group with negative dose-response relationship.We also found more elevated TNF-αlevels in the 5 d than in the 24 h model with no COE specific relationship.CONCLUSION COE induces expression changes of Th17 cytokines profile in 16HBE cells,including IL-23 and IL-1 βfor early and long-term infla mmation,respectively.IL-10 may be a candidate marker for population study on COE induced infla mmatory injury.

objective To make genetic diagnosis in two haemophilia families with recombination. Methods For hemophilia A(HA)family,screening of the F Ⅷ intron 22 and intron 1 inversion mutations was employed to identify the mutation. Linkage analysis with 8 polymorphic markers was adopted in the pedigree. For hemophilia B(HB)family,DNA sequencing of all coding regions of FⅨ gene Was used to detect the mutation directly. The muhifluorescent PCR method employing six FⅨ related STR was adopted in linkage analysis.Results In the HA family,the proband was positive in inversion 1 detection and the relative female was inversion 1 carrier. But linkage analysis with polymorphic markers showed contrary resuhs. Some markers certified that the female inherited the disease chromosome of the family while the others showed contrary results.In the HB family,it was unsuccessful in sequencing the exon 7 of the F Ⅸ gene in the proband and there was no mutation found in the other parts. The relative female and her amniocyte DNA were successful in sequencing the whole F Ⅸ gene and no mutation was detected.The linkage analysis of the family showed contrary results. Recombination occured in these two families. Conclusions Although the linkage analysis iS convenient and effective in carrier and prenatal diagnosis of hemophilia families. The recombination risk shouldn't be neglected especially when the polymorphic markers give inconsistent information for linkage analysis. It is necessary to find some high inforrnative markers intragenic or on the telomeric side to the gene in order to prevent the risk of recombination.

Objective To investigate the phenotype, genotype and molecular mechanisms in four Chinese pedigrees with venous thrombosis caused by hereditary PC deficiency. Methods The plasma activity of PC: A, TPS: A and FPS: A of the probands and their family members were detected with chromogenic and coagulation assay. The antigen of PC and FPS were identified with ELISA. Thrombin generation tests were applied to indicate the coagulation status. All of the nine exons and intron-exon boundaries of PC gene and PS gene were amplified by PCR and directly sequenced for mutaiton investigation. Results Compound heterozygous mutations of L-34P, K150del and A209V with 36% of PC: A and 57% of PC: Ag were identified in proband 1. PC: A was 46% , PC: Ag was 64. 4% while TPS: A, FPS: A and FPS: Ag were 36% , 19.5% and 20.9% respectively in proband 2. Two independent heterozygous mutations of R147W in PC gene inherited from his mother and T519stop in PS gene inherited from his father were identified. The anticoagulant activity of Proband 2 and his parents were declined in thrombin generation assay. In proband with PS defeciency and his father, the inhibition of thrombin generation capacity was decreased with exogenous APC, while his mother did not have significant difference. In Proband 3, PC: A was 32% while PC: Ag was 48.42% . Two independent mutations of R147W and R178W in Exon 7 were detected. Compound heterozygous mutations of R178W and D255H,with 21% of PC : A and 18. 36% of PC: Ag were identified in the Proband 4. Conclusions Hereditary PC deficiency or combined PC and PS deficiency result in venous thrombosis in four Chinese families. Mutants of L-34P, A209V, R178W, R147W and D255H might be the molecular mechanisms of PC deficiency.

Objective To investigate the clinical phenotype and genotype in three probands with antithmmbin(AT)deficiency and their families,and to identify the molecular mechanism of AT deficiency.Methods Chromogenic substrate method and immunoturbidimetry assay was used to detect the plasma levels of AT:A and AT:Ag,respectively.Genomic DNA was extracted from the peripheral blood.All 7 exons and the flanking sequences were amplified by PCR.and the abnormal mutant genes were analyzed by direct sequencing.Western blot was used to detect the AT levels and thrombin generation tests were used to detect coagulation status.Results The plasma levels of AT:A and AT:Ag of the three probands declined by 50%.G7386C(Trp225Cys)mutation in exon 4,C2591G(Ser36stop)in exon 2 and C9819T(Arg359stop)in exon 5 were characterized in the three prebands and they could result in W(Trp)225C(Cys)missense mutation,S(Set)36X(stop)nonsense mutation and R(Arg)359X(stop)nonsense mutation respectively,The testing results of phenotype and genotype from some of their family members showed consistent with results from the probands.Western blot results indicated that the Icyels of PC:Ag were lower compared with the normal pooled plasma.The hypercoagulative status was present in the probands using thrombin generation tests.Conclusions Type Ⅰ hereditary AT deficiency was found in these three families.The 3 heterozygous mutations.W225C,S36X and R359X are genetic defects of hereditary AT deficiency.W225C and S36X have not been described before.

0bjective To make genetic and prenatal diagnosis of a female with Haemophilia A.Methotis The FⅧ:C.BT and VWF were detected to make phenotypic diagnosis.LD-PCR was adopted for screening the intron 22 inversion and PCR was adopted for the screening the intron 1 inversion.The coding and boundary sequences of FⅧgene were analyzed by PCR and DNA equencing.Eight combined polymorphie markers(Amelo,F8IVS13,CA22,DXS15,DXS9901,G6PD,DXS1073 and DXS1108)were applied for linkage analysis of the family by multiplex fluorescent PCR.The polymorphism of DXS52 (ST14)was analyzed by PCR and electrophoresis. Assessment of X inactivation was performed using an Hpa II-polymerase chain eaction (PCR)assay for the X-inked human androgen receptor gene(HUMARA). Results The female HA patient showed severe FW deficiency(FⅧ:C 2.1%)and other phenotypie tests were normal.Her family members showed normal in all tests.The female proposita was found to be a carrier of FW gene intron 22 inversion.But her family members as well as her etus showed negative results.Except this inversion,no other mutation Wag found then.The female inherited two X chromosomes from both her parents' and her fetus inherited the maternally derived X chromosome from the female proposita according to the linkage analysis.Furthermore.X-inactivation paRern of the female was unbalanced and her aternally derived X chromosome Wag inaetived mostly while the majority of her paternal derived one kept active.Conclusions The severe haemophilia A in the proposita resulted from the de novo Ⅷ intron 22 inversion which most probably arose in the paternal germ line.Associated with a skewing pattern of inactivation of the maternally derived X chromosome.Her etus is normal female.

Objective To investigate the genetic diagnosis and molecular pathogenesis of four patients with combined deficiency of coagulation factor Ⅴ and Ⅷ and their family members. Methods The APPT, FT, FⅤ: C, FⅧ: C were detected for phenotypic diagnosis. Thrombin generation assay was applied to determine the generation condition of thrombin in patients and healthy controls. Cenomic DNA was extracted from peripheral blood using the TianGen RelaxCene Blood DNA System;amniotic fluid DNA was extracted with phenol-ethyl ether method. The LMAN1 and MCFD2 genes were analyzed by PCR. Gene mutations were detected with nucleotid sequences by using end-labeling dideoxy method. Results The APTT of Proband 1 was significantly prolonged to 88. 2s and her PT was prolonged to 19. 6 s. The combined deficiency was identified with FⅧ (FⅧ: C 24. 2% ) and FV(FⅤ: C 9. 1% ). Proband 2 and 3 were sisters. The coagulation studies revealed that both of them had prolonged APTT (71.6 s and 74.6 s respectively) and PT (22. 1 s and 18. 3 s respectively). The combined deficiency of FⅤ (FⅤ: C 7. 6% and 14. 5% respectively) and FⅧ( FⅧ: C 25% and 19.6% respectively) were identified. Proband 4 was detected to have the prolonged APTT (70.3 s),PT (18.2 s) and the deficiency of FⅤ(FⅤ: C 9. 4% ) and FⅧ (15. 7% ). The remaining phenotype indicators test of the 4 probands were normal. The diagnosis for the 4 probands was combined deficiency of factor Ⅴ and Ⅷ. The proband 1 was detected to have compound heterozygous mutations in LMAN1 gene while having the LMAN1 and MCFD2 direct gene sequencing. One mutation was a small insertion located on exon 8 [ nt912insA (X71661. 1)] that resulted in p. 305frameshiftX20 and her mother was detected to have the same heterozygous mutation on the the locus. The other mutation was located on exon 11: nt1366C > CT ( X71661. 1 ) , p. 456Arg > Stop which was inherited from her father. Amniocyte DNA was detected to have only one heterozygous mutaion [nt1366C > CT (X71661. 1) , 456Arg > Stop] inherited from the father. No mutation in MCFD2 gene was found in proband 1 and her parents. The analysis of the MCFD2 gene in proband 2 and 3 revealed a novel homozygous single base substitution (nt411T>C) in exon 4, which results in the exchange of the amino acid isoleucine by the amino acid threonine at amino acid position 136 (p. Ile136Thr). Sequencing of the whole LMAN1 gene showed that the proband 4 had one homozygous nonsence mutation in the exon 5 of the LMAN1 ( nt615C >T,p. 202 Arg> Stop). All of the 4 probands with combined deficiency of FⅤ and FⅧ showed declined endogenous thrombin potential in the thrombin generation tests. Conclusion The combined deficiency of FⅤ and FⅧ in the proband 1 results from the compound heterozygous mutations ( nt1366C > CT and nt912insA) in LMAN1 gene, which are inherited from her parents respectively. The prenatal genetic investigation for the patient mother with preganency indicates that the fetus is a female carrier with one mutation (nt1366C > CT) inherited from the father. The homozygous missence mutation ( nt411T > C, p. Ile136Thr) in the MCFD2 gene accounts for the proband 2 and 3. The daughter of the proband 2 is a carrier with a heterozygous mutation inherited from her mother. The homozygous nonsence mutation in the LMAN1 gene of the proband 4 results in the deficency of F Ⅴ and FⅧ.

Objective To apply color Doppler flow imaging (CDFI) and transcranial Doppler (TCD) to the follow-up observation of the changes of cervical vessel,intracranial hemodynamics and cerebrovascular reserve capacity (CVR) of the patients after carotid artery stent implantation.Methods Totally 96 patients with carotid artery stent implantation underwent CDFI and TCD examinations,and the changes of hemodynamics were compared before and 1 month,6 months,1 a and 2 a after implantation.Results The values of peak systolic velocity (PSV) and resistance index (RI) at the areas of carotid stenosis were lower significantly than those before implantation,while the values of PSV,pulsatility index (PI) and CVR of the middle cerebral artery were obviously higher than those before implantation (P＜0.05).There were no significant differences between the cervical and intracranial hemodynamics indexes 1 month,6 months,1 a and 2 a after treatment.Two-year follow-up found 4 cases of restenoses after implantation,and the rate for restenosis was 4.2％.Conclusion Carotid artery stent implantation improves significantly cervical and intracranial blood supply as well as CVR of the carotid stenosis patient,and CDFI combined with TCD can be used for the accurate evaluation of the efficacy and postoperative follow-up of carotid artery stent implantation.