Establishment And Identification Of Porcine Aorta Endothelial Cell Lines

Xenotransplantation is the best way to solve the shortage of clinical organs. Nowdays,pig is suitable donor for xenograft, and the major obstacle is acute vascular rejection. Thevascular endothelial cells in xenografts are attacked by the immune system of receptorfirstly. The injury of endothelial cells is mediated and amplified of immune rejection andblood coagulation disorders. In order to protect and prolong survival of xenografts, themechanism of porcine artery endothelial cell injury and activation, and the role of porcineartery endothelial cell in xenotransplantation should be studied. Althought the porcineartery endothelial cell lines are vital to study xenotransplantation, there are no goodporcine artery endothelial cell lines in China. In the experiment the porcine arteryendothelial cells were obtained by collagenase. The cell line was established following thetransfection of primary endothelial cells isolated from the aortas of the pig with Lentiviruscarrying genes for Simian Virus40Large T (SV40LT) antigen. After that, the cells were identified by the morphology, surface markers, the ability of tube forming andimmunologic function. The basic characteristics of cell line were similar with primarycells. So the long-term and stable phenotype cultured porcine aortic endothelial cells linewas established. The cell line has the most of function as vascular endothelial cell in vivo.It provides a good platform for studying xenotransplantation.ObjectiveThe pig aortic endothelial cell lines were established for providing necessary cellmodel for xenotransplantation research.Methods1. To extract primary cells and establish cell line: The cell line was established followingthe transfection of primary endothelial cells isolated from the aortas of the pig bycollagenase with Lentivirus carrying genes for Simian Virus40Large T (SV40LT)antigen.2. To identify basic characteristics of cells: In morphology, the gross morphologicalstructure and ultrastructure of the cells were observed by inverted microscope andelectron microscope. The growth curve of the cells was tested by MTT. The expressionof SV40LT gene was detected by RT-PCR. The expression of vWF was observed byimmune cell fluorescent chemical method. The phagocytic capacity of cells forDiI-Ac-LDL was observed fluorescence microscope. The ability of tube forming wasdetected in matrigel matrix.3. To detct the immunologic characteristics of cells: The expression of α-1,3-galactosyltransferase (α-1,3-GT) gene was determined by RT-PCR. The α-1,3-galactose antigen was detected by flow cytometry. At last, the response was testedbetween the cells and human serum after co-culture. The effect of human serum on theproliferation of cells was measured by MTT. The effect of human serum on theapoptosis of cells was detected by flow cytometry. Then the ability of cells to combinewith human IgG antibody and C3, C5b-9were tested by immunocytochemistry. Results1. The porcine aortic endothelial cells was isolated and cultured successfully. The cellsgrew as a monolayer, spindle, oval or irregular in shape in inverted phase contrastmicroscope. In electron microscope, the surface of cells grew villous projections andW-P bodies. The cells had ability to express vWF and take in DiI-Ac-LDL. The cellshad ability to form tube.2. The cell line was established following the transfection of primary endothelial cellswith Lentivirus carrying genes for SV40LT antigen. After transfection, the cells hadability to express SV40LT gene highly. Doubling times of the primary cells and thecell lines was about21h and18h, respectively. The cell line also displayedendothelial cell characteristics since they endocytosed acetylated DiI-Ac-LDL andsteadily continuously expressed the vWF.3. In immunologic properties, the high expression of α-1,3-GT gene and α-1,3-galactoseantigen (the fluorescence intensity was (498.2士3.5) and (500.6士3.2)，respectively)had no difference in the two types of cells (P>0.05). But the fluorescence intensity oftypes of cells was higher than that of human umbilical endothelial cells (P <0.05).When co-incubation with human serum, the two types of endothelial cells proliferationand apoptosis rate increased along with the concentration of huanm serum rising. Theablity of the two types of endothelial cells to combine human IgG and C3and C5b-9hadno difference (P>0.05).ConclusionThe primary porcine artery endothelial cells that we extracted have the same basicsame characteristics in morphological, phenotypic and function with common vascularendothelial cells. The porcine artery endothelial cell line established with Lentiviruscarrying genes for Simian Virus40Large T (SV40LT) antigen has well kept the variousfeatures of primary vascular endothelial cells, and has the ability to response with humanserum immune. The cell line has the most of functions as vascular endothelial cells in vivo.It can provide a platform for the study of xenotransplantation.