Objective：To establish the polytransgenic mice expressing the human HT and complement regulatory proteins （CRPs） and discuss their ability to resist the hyperacute rejection （HAR） and delayed xenograft rejection ...Objective：To establish the polytransgenic mice expressing the human HT and complement regulatory proteins （CRPs） and discuss their ability to resist the hyperacute rejection （HAR） and delayed xenograft rejection （DXR） of heterogenic transplantation. Methods ：Transgenic mice were produced by microinjection to construct gene for human HT, delay acceleration factor （DAF） and/or CD59 into the male pronucleus of zygote. PCR and Southern blot were used to screen the positive trarisgenic mice. Flow cytometry （FCM） was used to detect the expression of HT, ct-Gal and DAF or CD59 on the PBMCs of transgenic mice. The survival time and function of the heart of transgenic mice were determined by a modified Langendorff cardiac perfusion apparatus： The change of proteinosis on IgM,IgG, C3c and C9 from different cardiac vascular iendothelial cells of transgenic mice were detected by immunohistochemistry. Results：HT, DAF or CD59 were highly expressed on the positive transgenic mice by FCM. The deposition of IgM,IgG,C3c or C9 in the cardiac vascular endothelial cells of the positive transgenic mice were de- creased. The survival time and function of the heart of the co-transgenic mice with AB serum perfusion were significantly longer and higher than that of the single HT positive transgenic mice（P 〈0.05）. Conclusion ：The mice co-expressing HT/DAF or HT/CD59 could resist the HAR,which was better than those expressing HT alone. It is feasible to use HT and CRPs co-transgenic methods to resist the HAR and DXR.展开更多
Objective To explore the strategies which reduce the amount of xenoantigen Galα1, 3 Gal. Methods Human α-galactosidase gene and α1,2-fucosyltransferase gene were transferred into cul-tured porcine vascular endothel...Objective To explore the strategies which reduce the amount of xenoantigen Galα1, 3 Gal. Methods Human α-galactosidase gene and α1,2-fucosyltransferase gene were transferred into cul-tured porcine vascular endothelial cells PEDSV.15 and human α-galactosidase transgenic mice were produced. The Galα1,3Gal on the cell surface and susceptibility of cells to human antibody-mediated lysis were analyzed. Results Human α-galactosidase gene alone reduced 78% of Galα1,3Gal on PEDSV.15 cell surface while human α-galactosidase combined with α1,2-fucosyltransferase genes removed Galα1,3Gal completely. Decrease of Galα1,3Gal could reduce susceptibility of cells to human antibody-mediated lysis, especially during co-expression of α-galactosidase gene and α1,2-fucosyltransferase gene. RT-PCR indicated positive human α-galactosidase gene expression in all organs of positive human α-galacto-sidase transgenic F1 mice including heart, liver, kidney, lung, and spleen, the amount of Galα1,3Gal antigens on which was reduced largely. 58% of spleen cells from F1 mice were destroyed by comp-lement-mediated lysis compared with 24% of those from normal mice. Conclusions Human α-galactosidase gene and α1,2-fucosyltransferase gene effectively reduce the expression of Galα1,3Gal antigens on endothelial cell surface and confers resistance to human serum-mediated cytolysis. The expression of human α-galactosidase in mice can also eliminate the Galα1,3Gal antigens in most tissues and decrease the susceptibility of spleen cells to human serum-mediated cytolysis.展开更多
The expression of humanα-1,2-fucosyltransferase(HT)or complement regulatory proteins has been proved as an strategy to overcome hypercute rejection in discordant xenogeneic organ transplantation.In this study,we exam...The expression of humanα-1,2-fucosyltransferase(HT)or complement regulatory proteins has been proved as an strategy to overcome hypercute rejection in discordant xenogeneic organ transplantation.In this study,we examined whether peripheral blood mononuclear cells(PBMCs)from polytransgenic mice expressing the human HT,and complement regulatory proteins(DAF and CD59),can provide more effective protection against xenograft rejection.Transgenic mice were produced by co-injection of gene constructs for human HT,DAF and/or CD59.Flow Cytometry(FCM)was used to screen the positive transgenic mice.PBMCs from transgenic mice were incubated with 15%human serum to evaluate natural antibody binding,complement activation and expression of adhesion molecules.Three transgenes were strongly expressed in PBMCs of transgenic mice,and HT expression signifi-cantly reduced expression of the major xenoepitope galactose-α-1,3-galactose(α-Gal).Functional studies with PBMCs showed that co-expression of HT and DAF or CD59 markedly increased their re-sistance to human serum-mediated cytolysis when compared with single transgenic PBMCs.Moreover,the combined expression of triple transgenes in PBMCs led to the greatest protection against human serum-mediated cytolysis,avoided hyperacute rejection and reduced expression of adhesion mole-cules.Strong co-expression of triple transgenes was completely protected from xenograft hyperacute rejection and partially inhibited acute vascular rejection.The studies suggest that engineering mice to express triple molecules represents an critical step toward prolonging xenograft survival and might be more suitable for xenotransplantation.展开更多
In a recently published study in Nature Medicine,Jianxing He et al.,performed the first successful pig-to-human lung xenotransplantation using a six-gene-edited Bama Xiang pig,in which the lung graft maintained viabil...In a recently published study in Nature Medicine,Jianxing He et al.,performed the first successful pig-to-human lung xenotransplantation using a six-gene-edited Bama Xiang pig,in which the lung graft maintained viability and gas exchange for 216 hours in a brain-dead human recipient without evidence of hyperacute rejection.1 This achievement demonstrates the potential of xenogeneic lungs as a future solution to the global shortage of donor organs while highlighting the unique immunological and physiological barriers that must be overcome for clinical translation.展开更多
基金Tianjin Municipal Science and Technology CommissionGrant number:043803411
文摘Objective：To establish the polytransgenic mice expressing the human HT and complement regulatory proteins （CRPs） and discuss their ability to resist the hyperacute rejection （HAR） and delayed xenograft rejection （DXR） of heterogenic transplantation. Methods ：Transgenic mice were produced by microinjection to construct gene for human HT, delay acceleration factor （DAF） and/or CD59 into the male pronucleus of zygote. PCR and Southern blot were used to screen the positive trarisgenic mice. Flow cytometry （FCM） was used to detect the expression of HT, ct-Gal and DAF or CD59 on the PBMCs of transgenic mice. The survival time and function of the heart of transgenic mice were determined by a modified Langendorff cardiac perfusion apparatus： The change of proteinosis on IgM,IgG, C3c and C9 from different cardiac vascular iendothelial cells of transgenic mice were detected by immunohistochemistry. Results：HT, DAF or CD59 were highly expressed on the positive transgenic mice by FCM. The deposition of IgM,IgG,C3c or C9 in the cardiac vascular endothelial cells of the positive transgenic mice were de- creased. The survival time and function of the heart of the co-transgenic mice with AB serum perfusion were significantly longer and higher than that of the single HT positive transgenic mice（P 〈0.05）. Conclusion ：The mice co-expressing HT/DAF or HT/CD59 could resist the HAR,which was better than those expressing HT alone. It is feasible to use HT and CRPs co-transgenic methods to resist the HAR and DXR.
文摘Objective To explore the strategies which reduce the amount of xenoantigen Galα1, 3 Gal. Methods Human α-galactosidase gene and α1,2-fucosyltransferase gene were transferred into cul-tured porcine vascular endothelial cells PEDSV.15 and human α-galactosidase transgenic mice were produced. The Galα1,3Gal on the cell surface and susceptibility of cells to human antibody-mediated lysis were analyzed. Results Human α-galactosidase gene alone reduced 78% of Galα1,3Gal on PEDSV.15 cell surface while human α-galactosidase combined with α1,2-fucosyltransferase genes removed Galα1,3Gal completely. Decrease of Galα1,3Gal could reduce susceptibility of cells to human antibody-mediated lysis, especially during co-expression of α-galactosidase gene and α1,2-fucosyltransferase gene. RT-PCR indicated positive human α-galactosidase gene expression in all organs of positive human α-galacto-sidase transgenic F1 mice including heart, liver, kidney, lung, and spleen, the amount of Galα1,3Gal antigens on which was reduced largely. 58% of spleen cells from F1 mice were destroyed by comp-lement-mediated lysis compared with 24% of those from normal mice. Conclusions Human α-galactosidase gene and α1,2-fucosyltransferase gene effectively reduce the expression of Galα1,3Gal antigens on endothelial cell surface and confers resistance to human serum-mediated cytolysis. The expression of human α-galactosidase in mice can also eliminate the Galα1,3Gal antigens in most tissues and decrease the susceptibility of spleen cells to human serum-mediated cytolysis.
基金Supported by the Doctor Initiation Foundation of Zhengzhou University
文摘The expression of humanα-1,2-fucosyltransferase(HT)or complement regulatory proteins has been proved as an strategy to overcome hypercute rejection in discordant xenogeneic organ transplantation.In this study,we examined whether peripheral blood mononuclear cells(PBMCs)from polytransgenic mice expressing the human HT,and complement regulatory proteins(DAF and CD59),can provide more effective protection against xenograft rejection.Transgenic mice were produced by co-injection of gene constructs for human HT,DAF and/or CD59.Flow Cytometry(FCM)was used to screen the positive transgenic mice.PBMCs from transgenic mice were incubated with 15%human serum to evaluate natural antibody binding,complement activation and expression of adhesion molecules.Three transgenes were strongly expressed in PBMCs of transgenic mice,and HT expression signifi-cantly reduced expression of the major xenoepitope galactose-α-1,3-galactose(α-Gal).Functional studies with PBMCs showed that co-expression of HT and DAF or CD59 markedly increased their re-sistance to human serum-mediated cytolysis when compared with single transgenic PBMCs.Moreover,the combined expression of triple transgenes in PBMCs led to the greatest protection against human serum-mediated cytolysis,avoided hyperacute rejection and reduced expression of adhesion mole-cules.Strong co-expression of triple transgenes was completely protected from xenograft hyperacute rejection and partially inhibited acute vascular rejection.The studies suggest that engineering mice to express triple molecules represents an critical step toward prolonging xenograft survival and might be more suitable for xenotransplantation.
基金supported by the KRIBB Research Initiative Program(KGM4252533,KGM5362521,JHM0022511)the National Research Foundation(NRF)funded by the Korean government(MSIT)(RS-2021-NR057659,RS-2022-NR067578,RS-2025-25460035)the National Research Council of Science&Technology(NST)grant by the Korea government(MSIT)(No.GTL24022-000).
文摘In a recently published study in Nature Medicine,Jianxing He et al.,performed the first successful pig-to-human lung xenotransplantation using a six-gene-edited Bama Xiang pig,in which the lung graft maintained viability and gas exchange for 216 hours in a brain-dead human recipient without evidence of hyperacute rejection.1 This achievement demonstrates the potential of xenogeneic lungs as a future solution to the global shortage of donor organs while highlighting the unique immunological and physiological barriers that must be overcome for clinical translation.
