The Effection Of PEGFP-N1-H2B1 Plasmid Vector Transfection To The Proliferation And Apoptosis Of Mus Vascular Endothelial Cell

Objective Heart transplantation is the unique method to cure terminal stage heart failure. However, the urgent and chronic reject reaction after heart transplantation restrict the method widely used. It is significant that investigate the method to induce immune tolerance. Fetus immune tolerance is the ideal model of alloimmunity tolerance. The most important mechanism of fetus immune tolerance is that chorionic vesicle express nonclassic HLA classⅠmolecule ( including HLA-G) , however , nonexpress classic HLA classⅠm olecule. We extracted mus fetus specific H2Bl gene and constructed pEGFP-N1-H2B1 plasmid vector, then investigated the effection of mus vascular endothelial cell proliferation and apoptosis that pEGFP-N1-H2B1 plasmid vector transfected to mus vascular endothelial cell and approached the mechanism of reject reaction after heart transplantation which utilize fetus immune tolerance. Method This reasech separated two part: the first part we confirm recombined pEGFP-N1-H2B1 plasmid vector with the help of XhoI and BamHI enzyme digestion, then was confirmed by gene sequencing; the second part we investigate the effection of mus vascular endothelial cell proliferation and apoptosis that pEGFP-N1-H2B1 plasmid vector transfected to mus vascular endothelial cell. In the course of pEGFP-N1-H2B1 plasmid vector, first we extracted RNA from mus fetus and amplificated H2B1 gene by PCR; then we linked the production of PCR to T vector with the help of EcoRI and BamHI enzyme digestion and confirmed by gene sequencing; finally we subcloned H2B1 to pEGFP-N1with the help of EcoRI and BamHI enzyme digestion and confirmed recombined pEGFP-N1-H2B1 plasmid vector with the help of XhoI and BamHI enzyme digestion. The transfection pEGFP-N1-H2B1 plasmid vector to mus vascular endothelial cell, we observed mus vascular endothelial cell. with inverted microscope and confirmed by immunohistochemistry. We selected blank group and experiment group and intervene mus vascular endothelial cell with different density of pEGFP-N1-H2B1 plasmid vector: Cytositimulation experiment is that intervened mus vascular endothelial cell with 2.5μg/mL,5μg/mL,10μg/mL of pEGFP-N1-H2B1 plasmid vector and observed the quantity of mus vascular endothelial cell; MTT experiment is that detected mus vascular endothelial cell proliferation and intervened mus vascular endothelial cell with 2.5μg/mL,5μg/mL,10μg/mL of pEGFP-N1-H2B1 plasmid vector and drawed cell growth curve which is time for abscissa axis and photoabsorption value for ordinate axis; Flow cytometry detected mus vascular endothelial cell apoptosis and intervened mus vascular endothelial cell with 2.5μg/mL,5μg/mL,10μg/mL of pEGFP-N1-H2B1 plasmid vector and disposed mus vascular endothelial cell at 24 h,48 h,72 h after transfection and drawed cell growth curve. Result In the course of pEGFP-N1-H2B1 plasmid vector, we detected the production of PCR by 1% agarose gel electrophoresisc and observed a brght band at 1070bp which corresponding to the molecular weigh of H2bl; we detected TS-H2B1 vector by 1% agarose gel electrophoresisc and observed a brght band at 1070bp from three cloned sequence and confirmed three cloned sequence is correct cloning; the result of gene sequencing is that two basic group mutation; we confirmed pEGFP-N1-H2B1 plasmid vector with the help of XhoI and BamHI enzyme digestion and observed a brght band at 1070bp from three cloned sequence. we observed mus vascular endothelial cell. with inverted microscope: adherent cell conjuncted with reticulation when cell cultivation for 48h; endotheliocyte island appeared when cell cultivation for 3-5day; cell conjugated with flakiness and cell rank as monolayer inlay,cobblestone or slabstone,which called as contact inhibition. The result of confirmation of mus vascular endothelial cell is that vascular endothelial cell nucleolus clearly appeared with the help of rabbit antimouse antibody. The result of Cytositimulation experiment is that the quantity of mus vascular endothelial cell decreased as the density of pEGFP-N1-H2B1 plasmid vector increased and time extended. The result of MTT experiment is that there is difference between blank group and experiment group, it has statistical significanc(eP<0.05). The result of cell apoptosis detected by flow cytometry is that there is difference between blank group and experiment group, it has statistical significance ( P<0.05 ) . Conclusion The construction of pEGFP-N1-H2B1 plasmid vector succeed. The culture in vitro of mus vascular endothelial cell succeed. The transfection pEGFP-N1-H2B1 plasmid vector to mus vascular endothelial cell, inhabit cell proliferation and promote cell apoptosis.