Coagulation factor Ⅷ and antithrombin Ⅲ activity were detected in 15 health donors. It was found that antithrombin Ⅲ activity decreased obviously 12 h after blood drawing. It lost 56 % of the activity at the 3rd ...Coagulation factor Ⅷ and antithrombin Ⅲ activity were detected in 15 health donors. It was found that antithrombin Ⅲ activity decreased obviously 12 h after blood drawing. It lost 56 % of the activity at the 3rd day, and 70 % of the activity at the 7th day. FⅧ:c showed no obvious change after 24 h, until the 3rd day. It lost 40 %-60 % of the activity after 36 h and was reduced to the 30 % of the original activity at the 5th day. Our results suggested that at the 3rd day coagulation factor Ⅷ of bank stored blood can be used to replenish antithrombin Ⅲ, while bank stored blood in one day can be used to replenish FⅧ.展开更多
Objective Hemophilia A,an X-linked bleeding disorder,affecting 1 in 5 000 males is caused by heterogeneous mutations in factor Ⅷ gene.Inversion mutation in intron 22 of F8C gene remains its leading cause.The aim of t...Objective Hemophilia A,an X-linked bleeding disorder,affecting 1 in 5 000 males is caused by heterogeneous mutations in factor Ⅷ gene.Inversion mutation in intron 22 of F8C gene remains its leading cause.The aim of this study was to evaluate the frequency and distribution of the intron 22-inversion mutation in the patients and in the family members in the region.Methods 29 hemophilia A patients from Jammu and Kashmir(20 severe,8 moderate and 1 mild) were analyzed for intron 22-inversion mutation.Results 11(38%) were positive for the distal type of inversion mutation.The mutation was found in 9/20(45%) patients with severe factor Ⅷ deficiency and 2/8(25%) with moderate severity hemophilia A,whereas the patient with mild hemophilia A was found to be negative for inversion mutation.Evaluation of twenty-six female relatives from 11 families of inversion mutation positive patients identified one mother and one sister from one family to be the carrier,suggesting its origin in the mother. Conclusion The present study confirms the intron-22 inversion mutation in F8C gene as the major cause of hemophilia A in the population from Jammu and Kashmir with a higher frequency of inversion mutation in sporadic cases compared to the familial cases.展开更多
Abstract:Objective To develop a retroviral-mediated high efficient expression system of human coagulation factor Ⅷ. Methods The LNC-FⅧBD retroviral vector was generated by cloning a human B-domain-deleted (760aa～16...Abstract:Objective To develop a retroviral-mediated high efficient expression system of human coagulation factor Ⅷ. Methods The LNC-FⅧBD retroviral vector was generated by cloning a human B-domain-deleted (760aa～1639aa) Factor Ⅷ (FⅧ) cDNA (FⅧ cDNA BD) into the retroviral vector pLNCX. Several mammalian cell lines, including NIH3T3, CHO, Cos-7 and human hepatic cell line, L-02, were transduced with viral supernatant from the highest virus-producing PA317 clone. Antigen and coagulant activity of human FⅧ in cell culture medium were measured by ELISA and a one-stage method, respectively. RT-PCR was performed for the detection of FⅧBD mRNA. Results Human FⅧ was expressed in all four target cells, with the highest FⅧ expression observed in NIH3T3. The coagulant activity of secreted FⅧ was up to 1.6U/106 cells*24?hrs-1, and the FⅧ antigen was 500?ng/106 cells*24?hrs-1. FⅧ coagulant activity and antigen expressed by transduced CHO cells were 0.12?U/106 cells*24?hrs-1 and 62.4?ng/106 cells*24?hrs-1, respectively. Human FⅧ expression was relatively low in Cos-7 and L-02 cells. RT-PCR results demonstrated transcription of FⅧcDNA BD in the target cells.Conclusions The constructed retroviral vector was able to direct high level expression of human FⅧ in various mammalian cell lines. It has potential utility in the future gene therapy for Hemophilia A.展开更多
Background Hemophilia A （HA） is an X-linked inherited bleeding disorder caused by decreased activity of factor Ⅷ（FⅧ） due to heterogenous mutations in the FⅧ coding gene （F8）. The type of mutation plays an imp...Background Hemophilia A （HA） is an X-linked inherited bleeding disorder caused by decreased activity of factor Ⅷ（FⅧ） due to heterogenous mutations in the FⅧ coding gene （F8）. The type of mutation plays an important role in the FVIII inhibitor formation. To date, several studies on the spectra of F8 defects have been performed in Western populations, but similar studies in Asian races are scarce. Here, we reported the distribution of the F8 gene mutations in 18 unrelated Chinese patients with HA. Methods Intron 22 and intron 1 inversions in the F8 gene were screened in 158 unrelated patients with HA using a long-distance PCR and multiplex PCR method. Direct sequencing of the coding region of the F8 gene was used to identify the mutations responsible for HA in 18 unrelated Chinese HA patients who were negative for intron 22 and intron 1 inversions; sequences were compared with the HAMSTERS database. A clotting method was used to assay the FⅧ activity level and the Bethesda assay was used to detect the FⅧ inhibitor. Results A total of 18 different HA F8 mutations were identified, seven of which were described for the first time. These novel mutations included five small deletions, one point mutation and one small insertion. One novel mutation （4382-3 AC deletion） was associated with inhibitor development. Conclusion These data extend our insight into the mechanisms by which novel amino acid mutations may lead to HA and how the HA patient genotypes influence the risk of FⅧ inhibitor.展开更多
Milk fat globule epithelial growth factor VIII(MFG-E8) is a novel adhesion protein mainly produced by macrophages and dendritic cells; it is expressed in most of the human tissues and functions to prompt cancer progre...Milk fat globule epithelial growth factor VIII(MFG-E8) is a novel adhesion protein mainly produced by macrophages and dendritic cells; it is expressed in most of the human tissues and functions to prompt cancer progression and survival. MFG-E8 contains a signal sequence for secretion, two epidermal growth factor(EGF)-like domains at the NH2 terminus and two discoidin domains with blood-clotting factor V/factor Ⅷ(C1 and C2) at the COOH terminus. The second EGF domain contains an arginine-glycine-aspartic(RGD) integrin-binding motif that engages α_vβ_5 integrins to facilitate cell adhesion and induce integrinmediated signal transduction. Integrin α_vβ_3 associates with VEGF receptor 2, engagement of integrins can promote angiogenesis, which plays key roles in growth, proliferation, and survival of cancer cells. VEGF stimulates the expression of α_vβ_3 and α_vβ_5 integrins on angiogenic vasculature, thereby potentiating effects of VEGF receptor engagement. Mice expressing a mutant form of α_vβ_3 integrin are unable to undergo tyrosine phosphorylation, confirming the important role that this integrin plays in pathological angiogenesis and providing important mechanistic insights. The C-terminus discoidin-like domains promote binding to membrane phospholipids, functioning close to VEGF like angiogenesis. MFG-E8 is an opsonin for apoptotic cells, and it acts as a bridging protein between apoptotic cells and phagocytes. It also influences cell immunities by altering CD4^+ and/or CD8^+ cells. Antibody or small peptide works with MFG-E8 at different functional sites or interacts with EGF-like domains and/or discoidin-like domains may play an important role in anti-angiogenesis or immune restoration. Altering the structures and/or functions of MFG-E8 and/or its domains is promising for development of novel anti-cancer strategies.展开更多
文摘Coagulation factor Ⅷ and antithrombin Ⅲ activity were detected in 15 health donors. It was found that antithrombin Ⅲ activity decreased obviously 12 h after blood drawing. It lost 56 % of the activity at the 3rd day, and 70 % of the activity at the 7th day. FⅧ:c showed no obvious change after 24 h, until the 3rd day. It lost 40 %-60 % of the activity after 36 h and was reduced to the 30 % of the original activity at the 5th day. Our results suggested that at the 3rd day coagulation factor Ⅷ of bank stored blood can be used to replenish antithrombin Ⅲ, while bank stored blood in one day can be used to replenish FⅧ.
文摘Objective Hemophilia A,an X-linked bleeding disorder,affecting 1 in 5 000 males is caused by heterogeneous mutations in factor Ⅷ gene.Inversion mutation in intron 22 of F8C gene remains its leading cause.The aim of this study was to evaluate the frequency and distribution of the intron 22-inversion mutation in the patients and in the family members in the region.Methods 29 hemophilia A patients from Jammu and Kashmir(20 severe,8 moderate and 1 mild) were analyzed for intron 22-inversion mutation.Results 11(38%) were positive for the distal type of inversion mutation.The mutation was found in 9/20(45%) patients with severe factor Ⅷ deficiency and 2/8(25%) with moderate severity hemophilia A,whereas the patient with mild hemophilia A was found to be negative for inversion mutation.Evaluation of twenty-six female relatives from 11 families of inversion mutation positive patients identified one mother and one sister from one family to be the carrier,suggesting its origin in the mother. Conclusion The present study confirms the intron-22 inversion mutation in F8C gene as the major cause of hemophilia A in the population from Jammu and Kashmir with a higher frequency of inversion mutation in sporadic cases compared to the familial cases.
基金ThisworkwassupportedbytheDrug developmentFoundationof ShanghaiScienceandTechnicalCommittee (No .975 4190 0 1)andDr .ClydeWuFoundationofShanghaiInstituteofHematology .
文摘Abstract:Objective To develop a retroviral-mediated high efficient expression system of human coagulation factor Ⅷ. Methods The LNC-FⅧBD retroviral vector was generated by cloning a human B-domain-deleted (760aa～1639aa) Factor Ⅷ (FⅧ) cDNA (FⅧ cDNA BD) into the retroviral vector pLNCX. Several mammalian cell lines, including NIH3T3, CHO, Cos-7 and human hepatic cell line, L-02, were transduced with viral supernatant from the highest virus-producing PA317 clone. Antigen and coagulant activity of human FⅧ in cell culture medium were measured by ELISA and a one-stage method, respectively. RT-PCR was performed for the detection of FⅧBD mRNA. Results Human FⅧ was expressed in all four target cells, with the highest FⅧ expression observed in NIH3T3. The coagulant activity of secreted FⅧ was up to 1.6U/106 cells*24?hrs-1, and the FⅧ antigen was 500?ng/106 cells*24?hrs-1. FⅧ coagulant activity and antigen expressed by transduced CHO cells were 0.12?U/106 cells*24?hrs-1 and 62.4?ng/106 cells*24?hrs-1, respectively. Human FⅧ expression was relatively low in Cos-7 and L-02 cells. RT-PCR results demonstrated transcription of FⅧcDNA BD in the target cells.Conclusions The constructed retroviral vector was able to direct high level expression of human FⅧ in various mammalian cell lines. It has potential utility in the future gene therapy for Hemophilia A.
文摘Background Hemophilia A （HA） is an X-linked inherited bleeding disorder caused by decreased activity of factor Ⅷ（FⅧ） due to heterogenous mutations in the FⅧ coding gene （F8）. The type of mutation plays an important role in the FVIII inhibitor formation. To date, several studies on the spectra of F8 defects have been performed in Western populations, but similar studies in Asian races are scarce. Here, we reported the distribution of the F8 gene mutations in 18 unrelated Chinese patients with HA. Methods Intron 22 and intron 1 inversions in the F8 gene were screened in 158 unrelated patients with HA using a long-distance PCR and multiplex PCR method. Direct sequencing of the coding region of the F8 gene was used to identify the mutations responsible for HA in 18 unrelated Chinese HA patients who were negative for intron 22 and intron 1 inversions; sequences were compared with the HAMSTERS database. A clotting method was used to assay the FⅧ activity level and the Bethesda assay was used to detect the FⅧ inhibitor. Results A total of 18 different HA F8 mutations were identified, seven of which were described for the first time. These novel mutations included five small deletions, one point mutation and one small insertion. One novel mutation （4382-3 AC deletion） was associated with inhibitor development. Conclusion These data extend our insight into the mechanisms by which novel amino acid mutations may lead to HA and how the HA patient genotypes influence the risk of FⅧ inhibitor.
基金Supported by a grant from Medical Technology Research Center for Health Development of China National Health and Family Planning Commission(No.W2012FZ007)
文摘Milk fat globule epithelial growth factor VIII(MFG-E8) is a novel adhesion protein mainly produced by macrophages and dendritic cells; it is expressed in most of the human tissues and functions to prompt cancer progression and survival. MFG-E8 contains a signal sequence for secretion, two epidermal growth factor(EGF)-like domains at the NH2 terminus and two discoidin domains with blood-clotting factor V/factor Ⅷ(C1 and C2) at the COOH terminus. The second EGF domain contains an arginine-glycine-aspartic(RGD) integrin-binding motif that engages α_vβ_5 integrins to facilitate cell adhesion and induce integrinmediated signal transduction. Integrin α_vβ_3 associates with VEGF receptor 2, engagement of integrins can promote angiogenesis, which plays key roles in growth, proliferation, and survival of cancer cells. VEGF stimulates the expression of α_vβ_3 and α_vβ_5 integrins on angiogenic vasculature, thereby potentiating effects of VEGF receptor engagement. Mice expressing a mutant form of α_vβ_3 integrin are unable to undergo tyrosine phosphorylation, confirming the important role that this integrin plays in pathological angiogenesis and providing important mechanistic insights. The C-terminus discoidin-like domains promote binding to membrane phospholipids, functioning close to VEGF like angiogenesis. MFG-E8 is an opsonin for apoptotic cells, and it acts as a bridging protein between apoptotic cells and phagocytes. It also influences cell immunities by altering CD4^+ and/or CD8^+ cells. Antibody or small peptide works with MFG-E8 at different functional sites or interacts with EGF-like domains and/or discoidin-like domains may play an important role in anti-angiogenesis or immune restoration. Altering the structures and/or functions of MFG-E8 and/or its domains is promising for development of novel anti-cancer strategies.
