The Studies Of Human α (1,2) Fucosyltransferase Gene Transferring And Its Function In Rejection Of Xenotransplantation

PART Ⅰ Transgenic expression of human α 1 , 2-fucosyltransferase (HT) in porcine cellsObjective Through recombinant plasmid pcDNA3-HTcDNA constructed by molecular biology and gene engineering technique to express HT in porcine cells in order to produce H antigen and reduce the expression of xenoantigen α -Gal simultaneously. Methods pRc/CMV-HTcDNA and pcDNA3 plasmids were digested by Hind Ⅲand Xba Ⅰ , then HTcDNA fragment was recovered and cloned into pcDNA3 plasmid. The recombinant plasmid pcDNA3-HTcDNA was identified using PCR, multidigestion and sequence analysis. Porcine aortic endothelial cells, porcine ilioarterial endothelial cells (P1EC) lines and porcine kidney PK-15 cell lines were cultured in vitro. The recombinant plasmid was transfected into porcine cells by lipofectamine mediated method. Stable transfectants were selected using G418. The integration of HT gene was assayed by PCR. The expression of HT gene mRNA was assayed by RT-PCR. The expression of H antigen and α-Gal were assayed by flow cytometry (FCM) . Normal accordingly porcine arterial endothelial cells and human umbilical vein endothelial cells(HUVEC) were control cells. Results The three kinds of porcine cells transfected successfully were selected by G418 and survived. The HTcDNA gene fragment was produced by PCR. The HT mRNA fragment was produced by RT-PCR. The transfected porcine cells expressed high H antigen and lower α -Gal through FCM assay. Conclusions Recombinant HT gene could be expressed in porcine cells, which produced H antigen and reduced α -Galproduction simultaneously. Human CMV initiator could express recombinant HT gene in all kinds of porcine cells.PART II The study of human a 1, 2-fucosyltransferase (HT) transgenic porcine arterial endothelial cells to overcomerejection in xenotransplantation.Objective Implore the function and mechanism of human HT transgenic PAEC in overcoming xenogenous rejection. Methods 1. Transgenic and normal porcine arterial endothelial cells (PAEC) were exposed to 20 %.. 40 % and 60 % concentration human serum respectively for two hours, then the percentages of two kinds of lyzed cells were compared using non-radioactive cytotoxicity assay. 2. Transgenic and normal PAEC were stimulated by 5 % human serum, 5 % human serum of inactivated complement and 1000 U/ml human TNF- a respectively. E-selectin and ICAM-1 expression on the PAEC were assayed by cell Elisa after 2 hours, 4 hours, 8 hours, 12 hours and 24 hours respectively. At same time E-selectin release in cell culture liquid was assayed by Elisa. NF-kappa B expression differentiation on the two kinds of PAEC was assayed by immunocytochemistry. Results 1. The percentage of lyzed cells of transgenic PAEC was lower than that of normal after exposed to human serum (all P<0. 05) . 2. PAEC could be activated by three stimulators. E-selectin and ICAM-1 expression on the PAEC increased according to exposed time and got to the peak after 8 hours and 12 hours respectively. After PAEC stimulated 2 hours E-selectin and ICAM-1 expression on the cells of the test group were low and differentiation compared to that of the control group had nosignificant sense (P > 0.05) . But after PAEC stimulated 4 hours E-selectin and ICAM-1 expression on the cells of the test group were lower than that of the control group (all P<0. 05) , which implicated the activation of PAEC was inhibited. 3. E-selectin release of two kinds of PAEC under three stimulating conditions increased smoothly and got to the.peak after 8 hours. After PAEC stimulated 2 hours E-selectin release of the test group were low and differentiation compared to that of the control group had no significant sense (P>0. 05) . But after PAEC stimulated 4 hours E-selectin release of the test group were lower than that of the control group (all /7< 0. 05) , which implicated the activation of PAEC was inhibited. 4. The differentiation of the percentage of NF-kappa B positive cells between the two kinds of PAEC under different stimulating conditions had significant sense (all P < 0. 05) . The number of HT transgenic PAEC was lower than that of mormal PAEC. Conclusions 1. Transgenic PAEC expressing human HT could resist human serum lysis more efficiently and human HT transgene could reduce hyperacute rejection (HAR) in a degree. 2. II type activation of transgenic PAEC expressing human HT was inhibited. Human HT transgene might overcome acute vascular rejection(AVR) in a degree. NF-kappa B might play a important role during this process. The factors of endothelial cells II type activation were multiple.PART IH The production of microinjected DNA fragment for human a 1, 2-fucosyltransferase(HT) transgenic mouseObjective To produce microinjected DNA fragment for human HT transgenic mouse. Methods The end of primer was designed to haveunique EcoR I or Bamll I site. A DNA fragment encoding the full length6...