Isolation Culture And Identification Of The Endothelial Cells From The Humanized Genetically Modified Pigs

Xenotransplantation is one important way to solve the shortage of donor organs and clinical pigs are considered as the ideal donor for human xenotransplantation. However,usage of pig organs in transplants of human organs faces with various rejections. The porcine a-1,3 galactosyl transferase gene (?-1,3GT) knockouts that were transfected with the human complement regulatory protein CD46 can significantly delay the hyperacute rejection and the transfered thrombomodulin TBM can obviously overcome coagulation disorders. The vascular endothelial cell injury is the key step in mediating the amplification of immune rejection reactions and coagulation disorders.Liver transplantation from pig to baboon showed that the platelet phagocytosis reduce is associated with liver sinusoidal endothelial cells. Our study isolated and cultured the pAEC and pLSEC cells, generating the methods for the pLSEC isolation and providing in vitro model for the study pLSEC platelets are expected to further prolong xenograft survival in time.The previously constructed xenograft miniature pig model with GGTA1, ASGR1 and CMAH knocked out and with hCD46 and hTBM transgenes was used for the pAEC and pLSEC isolation and culturing. CD31 is an important endothelial cell surface membrane molecules, and thus identified as a specific marker for endothelial cells. The CD31 detection by FITC was applied to evaluate the purity for pAEC and LSEC. The pLSEC special aperture structures were observed under scanning electron microscopy and transmission electron microscopy.The pAEC, pLSEC uptake of the labeled acetylated low density lipoprotein (DiI-Ac-LDL) of DiI was used to detect their endocytic functions through immunofluorescence method.The real-time quantitative PCR (Real-time PCR, qRT-PCR) was used to detect the expression of pLSEC, pAEC, pEFC of CD31, CD 146, VIII factor and vWF factor.The telomerase reverse transcriptase enzyme hTERT were applied to generate the immoralized pAEC.The results were as follows:(1) pAEC showed typical endothelial cells morphology with cobblestone arrangement and well growth; pAEC absorbed DiI-Ac-LDL and flow cytometry results showed that the expression of CD31 was positive.(2) The isolated pLSEC showed the typical morphology of endothelial cells with cobblestone arrangement and aperture structures; pLSEC absorbed the labeled acetylated low density lipoprotein (DiI-Ac-LDL); the expression of CD31 in pLSEC was as 96.3 percent.The expression of CD31, CD146, ? factor, vWF was significantly in pLSEC than pAEC and pig ear fibroblasts (Porcine ear fibroblast cell, pEFC).(3) The pCI-neo-hTERT transfected pAEC cells were passaged by 14th generations and the immortalized pAEC immortalize kept the biological characteristics of the normal vascular endothelium.(4) The flow cytometry analysis showed that GGTA1, ASGR1 and CMAH were knocked out and hCD46 gene were transferred successfully in the pig model.In conclusion, this study successfully established various genetically modified porcine aortic endothelial cells and sinusoidal endothelial cells by using CD31 and purified pAEC, transferres to the pCI-neo-hTERT simultaneously, get immortalized pAEC. The CD31 molecule can be used as a specifically marker of separation and purification pLSEC. pAEC and pLSEC provide a good cell model of platelet phagocytosis problems for the study of non-Gal antigens and sinusoidal endothelial cells. Flow cytometry analysis methods for multi-gene-modified pig GGTA1, ASGR1, CMAH knockout and hCD46 gene transfer for testing, provide a rapid, accurate identification genetically modified pigs methods.