This paper describes the first human gene therapy trial for hemophilia B. Retroviruses were used to introduce human factor Ⅸ into autologous, primary human skin fibroblasts from the patients. Recombinant retroviral v...This paper describes the first human gene therapy trial for hemophilia B. Retroviruses were used to introduce human factor Ⅸ into autologous, primary human skin fibroblasts from the patients. Recombinant retroviral vector containing human FIX cDNA driven by viral LTR promoter (XL-Ⅸ) and double-copy retroviral vector driven by human cytomegalovirus enhancer-promoter (N2CMV-Ⅸ)were constructed. After the safety assessment, including soft-agar test, cell morphology observation, analysis of endotoxin, chromosome karyotype, allergic reaction test, nude mice test, routine pathological test, electromicroscopic analysis, and virus detection by PCR, etc., the engineered cells were pooled and embedded in collagen mixture, autologously injected into the patients respectively. The concentration of human FIX protein of Patient 1 increased from 71 ng/ml to 220 ng/ml, witha maximum level of 245 ng/ml. The expression of FIX has lasted for 6 months at the time of writing. The clotting activity also increased from 2.9% to 6.3%, his clinical symptoms have been alleviated obviously. The secretion rate of FIX for Patient 2 increased from 130 to 250 ng/ml, maintained at the level of 220 ng/ml for 5.5 months at the time of writing, but the clotting activity has not been increased steadily. There is no deleterious effect to be found in the two patients since the ex-vivo cells were implanted. The two patients are now under follow-up investigation. We suggested that retrovirus-mediated transfer of genes into skin fibroblasts, to be embedded in collagen and subcutaneously injected into patients, is a simple and effective approach for the gene therapy for hemophilia B.展开更多
This research was to examine if rice (Oryza sativa L.), a monocotyledon of angiosperm, was able to synthesize chlorophyll (Chl) in complete darkness. Five-cm-tall etiolated seedlings of rice were used as starting mate...This research was to examine if rice (Oryza sativa L.), a monocotyledon of angiosperm, was able to synthesize chlorophyll (Chl) in complete darkness. Five-cm-tall etiolated seedlings of rice were used as starting materials and treated with or without various concentrations of glucose and/or δ-aminolevulinic acid (ALA) in the dark. Leaves harvested at the indicated time were determined for their contents of Chl, protoporphyrin Ⅸ (Proto), Mg-protoporphyrin Ⅸ (Mg-Proto) and protochlorophyllide (Pchlide). The mole percentage of porphyrin was calculated. The Chl content in the etiolated rice seedlings slightly increased from about 2.5 μg/g to 7.5 μg/g within 12 d in the dark, but the total Chl of dark-grown rice increased from 0.36 μg/g to 3.6 μg/g. While the mole percentages of Proto, Mg-Proto and Pchlide in the dark-grown seedlings without any treatment were about 65%, 27.5% and 7.5% at the beginning, respectively, those in the light-grown seedlings were about 42.5%, 35% and 22.5%, respectively. The mole percentage of porphyrin of etiolated seedlings resumed its normal ratio within 2 d after treatment with glucose. While the Chl content of etiolated seedlings grown in culture solution with 3% and 6% glucose increased 2.5 and 4.0 folds, respectively, those with 3% and 6% glucose and 1 mmol/L ALA increased 22 and 24 folds, respectively. It is concluded that angiosperm might be able to synthesize a small amount of Chl in complete darkness, that either glucose or ALA could stimulate dark Chl synthesis in angiosperm, and that a combination of glucose and ALA exhibited an additional effect. It is still unknown and remains to be further explored what is the mechanism of the effect of glucose and ALA on the Chl synthesis of rice in the dark.展开更多
The iron chelators can be utilized in target cells to improve 5-aminolaevulinic acid （ALA）-based photodynamic therapy （PDT）. The purpose of this study is to compare the effect of two kinds of iron chelators, desfe...The iron chelators can be utilized in target cells to improve 5-aminolaevulinic acid （ALA）-based photodynamic therapy （PDT）. The purpose of this study is to compare the effect of two kinds of iron chelators, desferrioxamine （DFO） and ethylenediaminetetraacetic acid （EDTA） on the enhancement of ALA-PDT. HaCat cells were cultured in medium containing 2.0 mmol/L of ALA and 0.5 mmol/L of DFO or EDTA. After 3-h incubation in the dark, the concentration of cellular pro-toporphyrin Ⅸ （PpⅨ） was detected by high performance liquid chromatography （HPLC）, and the fluorescence of PpⅨ was observed at 630 nm emission under confocal laser scanning microscope. For PDT, HaCat cells were irradiated using 632.8 nm laser, and the fractions of apoptotic and necrotic cells were flow cytometrically assayed. Related differences in morphology and ultrastructure of Ha-Cat cells were observed using optical microscope or transmission electron microscope. Compared to incubation with ALA alone, the addition of DFO or EDTA increased the concentration of cellular PpⅨ and the fluorescent density of PpⅨ, and also increased cell death ratio after PDT. PDT using ALA plus DFO produced the highest cellular PpⅨ level, greatest cell death ratio and most severe structural damage to the cells. It was concluded that both DFO and EDTA could enhance ALA-based PpⅨ production and PDT. Compared to the non-specific iron chelator of EDTA, the specific chelator, DFO, showed more potential for the enhancement.展开更多
THE mammary gland bioreactor system of lactating animal, established by germ-linemicroinjection and embryo transplantation, has been one of the most exciting projects in thefield of biotechnology. However, this system...THE mammary gland bioreactor system of lactating animal, established by germ-linemicroinjection and embryo transplantation, has been one of the most exciting projects in thefield of biotechnology. However, this system is costly, laborious and tim-cosuming. In 1994,Johanna et al. reported that secretion of a foreign protein, human growth hormone (hGH),in milk had been achieved after direct introduction of the cDNA into the mammary gland ofgoats by replication-defective retroviral vector. In that work, the in vivo expression of展开更多
Mouse myoblast C2C12 cell was used as target cell for gene transfer study of human clotting factor IX (hFIX) cDNA. In addition to the previously constructed retroviral vectors XLIX, LNCIX and GINaCIX, GlNaMCIX with hF...Mouse myoblast C2C12 cell was used as target cell for gene transfer study of human clotting factor IX (hFIX) cDNA. In addition to the previously constructed retroviral vectors XLIX, LNCIX and GINaCIX, GlNaMCIX with hFIX driven by muscle creatine kinase (MCK) enhancer and human cytomegalovirus (CMV) was constructed, based on the retroviral vector GINa. These four retroviral vectors were used to transduce mouse my-oblasts C2C12. With ELISA assays, it has been found that the expression levels of human clotting factor IX detected in those transduced C2C12 cells are GlNaMCIX>GlNaCIX> LNCIX>XLIX. Mixed colonal cells transduced with GlNaMCIX expressed hFIX protein at the level of 640 ng/106 cell every 24 h. The modified C2C12 cells transduced with GlNaMCIX were implanted into skeletal muscle of the hindlegs of C3H mice; a stable expression of hFIX was detected and lasted for 35 d, with a maximum level of 206 ng/mL plasma. The regulation of hFIX cDNA expression in myoblasts was discussed and it was strongly suggested that a myoblast-mediated gene delivery system had the poten-tial to be optimized as a safe and effective therapeutic modality for hemophilia B.展开更多
To study the possibility of somatic gene therapy for hemophilia B via gene transfer to primary factor Ⅸ-deficient skin fibroblasts, we constructed four retroviral vectors containing factor Ⅸ cDNA driven by retrovira...To study the possibility of somatic gene therapy for hemophilia B via gene transfer to primary factor Ⅸ-deficient skin fibroblasts, we constructed four retroviral vectors containing factor Ⅸ cDNA driven by retroviral LTR promoter, SV40 early promoter and mouse MT-I promoter, respectively. These retroviral vectors were transfected into an amphotropic packaging cell line, PA317 cells, by electroporation, and a human iibrosarcoma cell line, HT1080 cells, was used to assay the factor Ⅸ-virus titers of these four virus-producing PA317 cells, which ranged from 2×10~4 to 5×10~5 cfu/ml. The factor Ⅸ proteins produced by bulk population of four virus-producing PA317 cells were determined by ELISA. Results showed that LTR promoter directed the highest production of factor Ⅸ at the rate of 584 rig/10~6 cells/24h, while SV40 early promoter and MT promoter directed about 10 and 20 times less production of factor Ⅸ than LTR promoter. The highest expressed retroviral vector XL-Ⅸ was used to infect a line of factor Ⅸ-deficie human primary skin fibroblasts, FDⅨ cells. The factor Ⅸ secretion rate of the infected FDⅨ cells was about 549 ng/10~6 cells/24h and over 75% of secreted factor Ⅸ was biologically active. We are convinced that this factor Ⅸ-deficient human primary skin fibroblast had been cured, or genetically corrected, by retroviral-mediated gene therapy in vitro.展开更多
Double-copy retroviral vector containing human factor Ⅸ cDNA driven by human cytomegalovirus enhancer-promoter was constructed. The vector was introduced into the amphotropic packaging cell line PA317. The recombinan...Double-copy retroviral vector containing human factor Ⅸ cDNA driven by human cytomegalovirus enhancer-promoter was constructed. The vector was introduced into the amphotropic packaging cell line PA317. The recombinant virus produced in PA317 was used to transduce skin fibroblasts from a hemophilia B patient. The infected cells produced high levels of biologically active human factor Ⅸ at a rate of 3420 ng/10~6 cells/24 h. These cells were embedded in a collagen matrix and implanted into the peritoneal cavity or subcutaneous space of mice. It was demonstrated that human factor Ⅸ was produced by the implants for at least 12 days in vivo, reaching a peak of 105 ng/ml plasma. Over 90% of the protein was functionally active. This technique has the potential to be developed into a new approach for gene therapy for hemophilia B.展开更多
Objective: To investigate the role of human host heme-oxygenase-1(HO-1) in pathogenesis of cerebral malaria in the in vitro model,Methods: The effect of human host HO-1 [human brain microvascular endothelial cell(HBME...Objective: To investigate the role of human host heme-oxygenase-1(HO-1) in pathogenesis of cerebral malaria in the in vitro model,Methods: The effect of human host HO-1 [human brain microvascular endothelial cell(HBMEC)] on hemoglobin degradation in the co-culture model of HBMEC and ITG Plasmodium falciparum-infected red cells(i RBC) through measurement of the enzymatic products iron and bilirubin,Results: Following exposure to the HO-1 inducer Co PPIX at all concentrations,the HBMEC cells apoptosis occurred,which could be prominently observed at 15 μM of 3 h exposure,In contrast,there was no significant change in the morphology in the non-exposed i RBC at all concentrations and exposure time,This observation was in agreement with the levels of the enzymatic degradation products iron and bilirubin,of which the highest levels(106.03 and 1 753.54% of baseline level,respectively) were observed at 15 μM vs,20 μM at 3 h vs,24 h exposure,For the effect of the HO-1 inhibitor Zn PPIX,HBMEC cell morphology was mostly unchanged,but significant inhibitory effect on cell apoptosis was seen at 10 μM for the exposure period of 3 h(37.17% of baseline level),The degree of the inhibitory effect as reflected by the level of iron produced was not clearly observed(highest effect at 10 μM and 3 h exposure),Conclusions: Results provide at least in part,insight into the contribution of HO-1 on CM pathogenesis and need to be confirmed in animal model.展开更多
Clotting factor Ⅸ is an essential member in the intrinsic dotting pathway and its defect may cause an inherited X-chromosome linked bleeding disorder——hemophilia B, or Christmas disease. Factor Ⅸ is synthesized in...Clotting factor Ⅸ is an essential member in the intrinsic dotting pathway and its defect may cause an inherited X-chromosome linked bleeding disorder——hemophilia B, or Christmas disease. Factor Ⅸ is synthesized in liver hepatocytes where it undergoes complex vitamin K-dependent post-translational modifications which are essential for the activation of factor Ⅸ. Recently, several groups have cloned factor Ⅸ cDNA and reported that, in the presence of vitamin K, active factor Ⅸ could be secreted in human and mouse fibroblasts,展开更多
Hemophilia B is an inherited bleeding disorder due to a deficiency of factor Ⅸ.Currently, conventional therapy depends on the infusion of human plasma or factor Ⅸ concentrate. The therapeutic effect is temporary and...Hemophilia B is an inherited bleeding disorder due to a deficiency of factor Ⅸ.Currently, conventional therapy depends on the infusion of human plasma or factor Ⅸ concentrate. The therapeutic effect is temporary and periodic infusions are required. This approach of treatment is expensive, and is associated with a significant risk of AIDS and hepatitis. Recent advances in gene-transfer techniques have provided a new approach of therapy for genetic diseases, including factor Ⅸ deficiency. Previously, we constructed sev-展开更多
In this study, rabbits were used as a model for gene therapy for hemophilia B, Human factor Ⅸ cDNA was transferred to cultured normal rabbit skin fibroblasts (RSF) by a recombinant plasmid (pCMVIX) or retrovirus(XL-I...In this study, rabbits were used as a model for gene therapy for hemophilia B, Human factor Ⅸ cDNA was transferred to cultured normal rabbit skin fibroblasts (RSF) by a recombinant plasmid (pCMVIX) or retrovirus(XL-IX or N2CMVIX) constructed in our laboratoy. Infected fibroblasts capable of synthesizing and secreting high levels of biologically active human factor Ⅸ protein were selected and embedded in a collagen matrix. The latter was surgically implanted into rabbits as autografts or allografts. Human factor Ⅸ protein was detected in the plasma of all the grafted rabbits, and its expression has been maintained for more than 10 months at the time of writing. In addition, we have improved and simplified the method of implantation from surgically grafting the tissue-like matrix to the injection of the infected cell-collagen mixture subcutaneously. Using the latter method, human factor Ⅸ in rabbits injected with RSF-N2CMVIX reached a peak of 480 ng/ml plasma, and its expression has continued for more than 3 months at the time of writing. We suggest that the simplified method of transplantation by subcutaneous injection would offer an effective and acceptable approach to somatic cell gene therapy and may be practical for human trials.展开更多
Ⅰ. INTRODUCTION Clotting factor Ⅸ is an essential member in the intrinsic clotting pathway, and the deficiency of factor Ⅸ may cause hemophilia B. An important subject on gene therapy for hemophilia B is to increas...Ⅰ. INTRODUCTION Clotting factor Ⅸ is an essential member in the intrinsic clotting pathway, and the deficiency of factor Ⅸ may cause hemophilia B. An important subject on gene therapy for hemophilia B is to increase the protein products of factor Ⅸ in human cells. In our earlier studies, we have constructed several recombinant retroviral vectors with factor展开更多
Polymerase chain reaction (PCR) is a recently devoloped molecular biological technique, using specific oligonucleotide primers and DNA polymerase to amplify the specific fragment of DNA in vitro. This technique has be...Polymerase chain reaction (PCR) is a recently devoloped molecular biological technique, using specific oligonucleotide primers and DNA polymerase to amplify the specific fragment of DNA in vitro. This technique has been successfully introduced in gene diagnosis, mutation detection, polymorphism analysis and nucleotide sequencing. In the study of gene transfer and gene therapy, it is necessary to analyse DNA in the展开更多
Factor IX is an important component in human clotting system.Its deficiency ordysfunction leads to hemophilia B.Recently,with the advance of efficient gene transfertechniques,great progress has been made in the gene t...Factor IX is an important component in human clotting system.Its deficiency ordysfunction leads to hemophilia B.Recently,with the advance of efficient gene transfertechniques,great progress has been made in the gene therapy of hemophilia B.It hasbeen moved from labs to clinics and positive results have been reported.In this study,a double-copy retroviral vector(N2CMVIX)and a recombinant plasmid(pCMVIX),bothcontaining human factor IX cDNA,were constructed.The vectors were transferred into cul-tured rabbit skin fibroblast(RSF).The cells capable of secreting high level of human fac-tor IX protein were embedded in collagen matrix and implanted in rabbits allograft展开更多
文摘This paper describes the first human gene therapy trial for hemophilia B. Retroviruses were used to introduce human factor Ⅸ into autologous, primary human skin fibroblasts from the patients. Recombinant retroviral vector containing human FIX cDNA driven by viral LTR promoter (XL-Ⅸ) and double-copy retroviral vector driven by human cytomegalovirus enhancer-promoter (N2CMV-Ⅸ)were constructed. After the safety assessment, including soft-agar test, cell morphology observation, analysis of endotoxin, chromosome karyotype, allergic reaction test, nude mice test, routine pathological test, electromicroscopic analysis, and virus detection by PCR, etc., the engineered cells were pooled and embedded in collagen mixture, autologously injected into the patients respectively. The concentration of human FIX protein of Patient 1 increased from 71 ng/ml to 220 ng/ml, witha maximum level of 245 ng/ml. The expression of FIX has lasted for 6 months at the time of writing. The clotting activity also increased from 2.9% to 6.3%, his clinical symptoms have been alleviated obviously. The secretion rate of FIX for Patient 2 increased from 130 to 250 ng/ml, maintained at the level of 220 ng/ml for 5.5 months at the time of writing, but the clotting activity has not been increased steadily. There is no deleterious effect to be found in the two patients since the ex-vivo cells were implanted. The two patients are now under follow-up investigation. We suggested that retrovirus-mediated transfer of genes into skin fibroblasts, to be embedded in collagen and subcutaneously injected into patients, is a simple and effective approach for the gene therapy for hemophilia B.
文摘This research was to examine if rice (Oryza sativa L.), a monocotyledon of angiosperm, was able to synthesize chlorophyll (Chl) in complete darkness. Five-cm-tall etiolated seedlings of rice were used as starting materials and treated with or without various concentrations of glucose and/or δ-aminolevulinic acid (ALA) in the dark. Leaves harvested at the indicated time were determined for their contents of Chl, protoporphyrin Ⅸ (Proto), Mg-protoporphyrin Ⅸ (Mg-Proto) and protochlorophyllide (Pchlide). The mole percentage of porphyrin was calculated. The Chl content in the etiolated rice seedlings slightly increased from about 2.5 μg/g to 7.5 μg/g within 12 d in the dark, but the total Chl of dark-grown rice increased from 0.36 μg/g to 3.6 μg/g. While the mole percentages of Proto, Mg-Proto and Pchlide in the dark-grown seedlings without any treatment were about 65%, 27.5% and 7.5% at the beginning, respectively, those in the light-grown seedlings were about 42.5%, 35% and 22.5%, respectively. The mole percentage of porphyrin of etiolated seedlings resumed its normal ratio within 2 d after treatment with glucose. While the Chl content of etiolated seedlings grown in culture solution with 3% and 6% glucose increased 2.5 and 4.0 folds, respectively, those with 3% and 6% glucose and 1 mmol/L ALA increased 22 and 24 folds, respectively. It is concluded that angiosperm might be able to synthesize a small amount of Chl in complete darkness, that either glucose or ALA could stimulate dark Chl synthesis in angiosperm, and that a combination of glucose and ALA exhibited an additional effect. It is still unknown and remains to be further explored what is the mechanism of the effect of glucose and ALA on the Chl synthesis of rice in the dark.
文摘The iron chelators can be utilized in target cells to improve 5-aminolaevulinic acid （ALA）-based photodynamic therapy （PDT）. The purpose of this study is to compare the effect of two kinds of iron chelators, desferrioxamine （DFO） and ethylenediaminetetraacetic acid （EDTA） on the enhancement of ALA-PDT. HaCat cells were cultured in medium containing 2.0 mmol/L of ALA and 0.5 mmol/L of DFO or EDTA. After 3-h incubation in the dark, the concentration of cellular pro-toporphyrin Ⅸ （PpⅨ） was detected by high performance liquid chromatography （HPLC）, and the fluorescence of PpⅨ was observed at 630 nm emission under confocal laser scanning microscope. For PDT, HaCat cells were irradiated using 632.8 nm laser, and the fractions of apoptotic and necrotic cells were flow cytometrically assayed. Related differences in morphology and ultrastructure of Ha-Cat cells were observed using optical microscope or transmission electron microscope. Compared to incubation with ALA alone, the addition of DFO or EDTA increased the concentration of cellular PpⅨ and the fluorescent density of PpⅨ, and also increased cell death ratio after PDT. PDT using ALA plus DFO produced the highest cellular PpⅨ level, greatest cell death ratio and most severe structural damage to the cells. It was concluded that both DFO and EDTA could enhance ALA-based PpⅨ production and PDT. Compared to the non-specific iron chelator of EDTA, the specific chelator, DFO, showed more potential for the enhancement.
文摘THE mammary gland bioreactor system of lactating animal, established by germ-linemicroinjection and embryo transplantation, has been one of the most exciting projects in thefield of biotechnology. However, this system is costly, laborious and tim-cosuming. In 1994,Johanna et al. reported that secretion of a foreign protein, human growth hormone (hGH),in milk had been achieved after direct introduction of the cDNA into the mammary gland ofgoats by replication-defective retroviral vector. In that work, the in vivo expression of
基金Project supported by the State High Technology Development Program and Technology and Development Program of Shanghai.
文摘Mouse myoblast C2C12 cell was used as target cell for gene transfer study of human clotting factor IX (hFIX) cDNA. In addition to the previously constructed retroviral vectors XLIX, LNCIX and GINaCIX, GlNaMCIX with hFIX driven by muscle creatine kinase (MCK) enhancer and human cytomegalovirus (CMV) was constructed, based on the retroviral vector GINa. These four retroviral vectors were used to transduce mouse my-oblasts C2C12. With ELISA assays, it has been found that the expression levels of human clotting factor IX detected in those transduced C2C12 cells are GlNaMCIX>GlNaCIX> LNCIX>XLIX. Mixed colonal cells transduced with GlNaMCIX expressed hFIX protein at the level of 640 ng/106 cell every 24 h. The modified C2C12 cells transduced with GlNaMCIX were implanted into skeletal muscle of the hindlegs of C3H mice; a stable expression of hFIX was detected and lasted for 35 d, with a maximum level of 206 ng/mL plasma. The regulation of hFIX cDNA expression in myoblasts was discussed and it was strongly suggested that a myoblast-mediated gene delivery system had the poten-tial to be optimized as a safe and effective therapeutic modality for hemophilia B.
基金This work was supported by a grant from the State High Technology Development Program 863-102-17-40
文摘To study the possibility of somatic gene therapy for hemophilia B via gene transfer to primary factor Ⅸ-deficient skin fibroblasts, we constructed four retroviral vectors containing factor Ⅸ cDNA driven by retroviral LTR promoter, SV40 early promoter and mouse MT-I promoter, respectively. These retroviral vectors were transfected into an amphotropic packaging cell line, PA317 cells, by electroporation, and a human iibrosarcoma cell line, HT1080 cells, was used to assay the factor Ⅸ-virus titers of these four virus-producing PA317 cells, which ranged from 2×10~4 to 5×10~5 cfu/ml. The factor Ⅸ proteins produced by bulk population of four virus-producing PA317 cells were determined by ELISA. Results showed that LTR promoter directed the highest production of factor Ⅸ at the rate of 584 rig/10~6 cells/24h, while SV40 early promoter and MT promoter directed about 10 and 20 times less production of factor Ⅸ than LTR promoter. The highest expressed retroviral vector XL-Ⅸ was used to infect a line of factor Ⅸ-deficie human primary skin fibroblasts, FDⅨ cells. The factor Ⅸ secretion rate of the infected FDⅨ cells was about 549 ng/10~6 cells/24h and over 75% of secreted factor Ⅸ was biologically active. We are convinced that this factor Ⅸ-deficient human primary skin fibroblast had been cured, or genetically corrected, by retroviral-mediated gene therapy in vitro.
文摘Double-copy retroviral vector containing human factor Ⅸ cDNA driven by human cytomegalovirus enhancer-promoter was constructed. The vector was introduced into the amphotropic packaging cell line PA317. The recombinant virus produced in PA317 was used to transduce skin fibroblasts from a hemophilia B patient. The infected cells produced high levels of biologically active human factor Ⅸ at a rate of 3420 ng/10~6 cells/24 h. These cells were embedded in a collagen matrix and implanted into the peritoneal cavity or subcutaneous space of mice. It was demonstrated that human factor Ⅸ was produced by the implants for at least 12 days in vivo, reaching a peak of 105 ng/ml plasma. Over 90% of the protein was functionally active. This technique has the potential to be developed into a new approach for gene therapy for hemophilia B.
基金supported by The Commission on Higher Education,Ministry of Education of Thailand,The National Research University Project of Thailand(NRU)Office of Higher Education Commission,Thammasat University(Center of Excellence in Pharmacology and Molecular Biology of Malaria and Cholangiocarcinoma)+1 种基金Liverpool School of Tropical Medicine,University of Liverpool,UKThe Royal Golden Jubilee PhD Programme,Thailand Research Fund-Thammasat University Joint Fund and Graduated Student Grant to P.Thongdee(Grant No.PHD/0365/2552)
文摘Objective: To investigate the role of human host heme-oxygenase-1(HO-1) in pathogenesis of cerebral malaria in the in vitro model,Methods: The effect of human host HO-1 [human brain microvascular endothelial cell(HBMEC)] on hemoglobin degradation in the co-culture model of HBMEC and ITG Plasmodium falciparum-infected red cells(i RBC) through measurement of the enzymatic products iron and bilirubin,Results: Following exposure to the HO-1 inducer Co PPIX at all concentrations,the HBMEC cells apoptosis occurred,which could be prominently observed at 15 μM of 3 h exposure,In contrast,there was no significant change in the morphology in the non-exposed i RBC at all concentrations and exposure time,This observation was in agreement with the levels of the enzymatic degradation products iron and bilirubin,of which the highest levels(106.03 and 1 753.54% of baseline level,respectively) were observed at 15 μM vs,20 μM at 3 h vs,24 h exposure,For the effect of the HO-1 inhibitor Zn PPIX,HBMEC cell morphology was mostly unchanged,but significant inhibitory effect on cell apoptosis was seen at 10 μM for the exposure period of 3 h(37.17% of baseline level),The degree of the inhibitory effect as reflected by the level of iron produced was not clearly observed(highest effect at 10 μM and 3 h exposure),Conclusions: Results provide at least in part,insight into the contribution of HO-1 on CM pathogenesis and need to be confirmed in animal model.
基金Project supported by grant from the State High Technology Development Program 863-102-17-40 and grant from State Education Commission Doctoral Foundation 32780266.
文摘Clotting factor Ⅸ is an essential member in the intrinsic dotting pathway and its defect may cause an inherited X-chromosome linked bleeding disorder——hemophilia B, or Christmas disease. Factor Ⅸ is synthesized in liver hepatocytes where it undergoes complex vitamin K-dependent post-translational modifications which are essential for the activation of factor Ⅸ. Recently, several groups have cloned factor Ⅸ cDNA and reported that, in the presence of vitamin K, active factor Ⅸ could be secreted in human and mouse fibroblasts,
基金Project supported by the National High Technology Program
文摘Hemophilia B is an inherited bleeding disorder due to a deficiency of factor Ⅸ.Currently, conventional therapy depends on the infusion of human plasma or factor Ⅸ concentrate. The therapeutic effect is temporary and periodic infusions are required. This approach of treatment is expensive, and is associated with a significant risk of AIDS and hepatitis. Recent advances in gene-transfer techniques have provided a new approach of therapy for genetic diseases, including factor Ⅸ deficiency. Previously, we constructed sev-
文摘In this study, rabbits were used as a model for gene therapy for hemophilia B, Human factor Ⅸ cDNA was transferred to cultured normal rabbit skin fibroblasts (RSF) by a recombinant plasmid (pCMVIX) or retrovirus(XL-IX or N2CMVIX) constructed in our laboratoy. Infected fibroblasts capable of synthesizing and secreting high levels of biologically active human factor Ⅸ protein were selected and embedded in a collagen matrix. The latter was surgically implanted into rabbits as autografts or allografts. Human factor Ⅸ protein was detected in the plasma of all the grafted rabbits, and its expression has been maintained for more than 10 months at the time of writing. In addition, we have improved and simplified the method of implantation from surgically grafting the tissue-like matrix to the injection of the infected cell-collagen mixture subcutaneously. Using the latter method, human factor Ⅸ in rabbits injected with RSF-N2CMVIX reached a peak of 480 ng/ml plasma, and its expression has continued for more than 3 months at the time of writing. We suggest that the simplified method of transplantation by subcutaneous injection would offer an effective and acceptable approach to somatic cell gene therapy and may be practical for human trials.
基金This work was supported by a grant from National High Technology Program 863-102-17-40.
文摘Ⅰ. INTRODUCTION Clotting factor Ⅸ is an essential member in the intrinsic clotting pathway, and the deficiency of factor Ⅸ may cause hemophilia B. An important subject on gene therapy for hemophilia B is to increase the protein products of factor Ⅸ in human cells. In our earlier studies, we have constructed several recombinant retroviral vectors with factor
基金Project supported by High Technique Research of the State.
文摘Polymerase chain reaction (PCR) is a recently devoloped molecular biological technique, using specific oligonucleotide primers and DNA polymerase to amplify the specific fragment of DNA in vitro. This technique has been successfully introduced in gene diagnosis, mutation detection, polymorphism analysis and nucleotide sequencing. In the study of gene transfer and gene therapy, it is necessary to analyse DNA in the
文摘Factor IX is an important component in human clotting system.Its deficiency ordysfunction leads to hemophilia B.Recently,with the advance of efficient gene transfertechniques,great progress has been made in the gene therapy of hemophilia B.It hasbeen moved from labs to clinics and positive results have been reported.In this study,a double-copy retroviral vector(N2CMVIX)and a recombinant plasmid(pCMVIX),bothcontaining human factor IX cDNA,were constructed.The vectors were transferred into cul-tured rabbit skin fibroblast(RSF).The cells capable of secreting high level of human fac-tor IX protein were embedded in collagen matrix and implanted in rabbits allograft
