Objective To investigate the changes of differentially expressed genes before and after in the liver xenotransplantation, and they were screened and verified. Methods Four cases of xenograft splenic fossa heterotopic auxiliary liver transplantation were performed with a -1，3- galactoside transferase gene knockout（GTKO） mini-pig as donor and Tibetan macaque as recipient. Appropriate preoperative liver specimens were harvested from the donor liver at the back table procedure，and postoperative liver specimens were harvested by biopsy at 48 h afteropening the blood flow of the graft. High throughput chip analysis of miRNAs and mRNAs was performed on the transplanted liver specimens. The key differential expression of miRNAs and mRNAs were screened，and real-time quantitative PCR and Western Blot tests were performed. Results There were 165 miRNAs differentially expressed after surgery，and 112 of them were up-regulated and 53 were down-regulated. Twelve differentially expressed miRNAs，such as mi-23b，were screened and verified. In addition to miR-150 and miR-126-3p，the expression changes of other miRNAs were basically consistent with the results of miRNAs chip. Compared with preoperative data，2 035 mRNAs showed significant differential expression after operation，922 of them were up-regulated and 1 113 were down-regulated，5 differentially expressed genes which were closely related to liver transplantation, such as IGFBP6，CDK2AP1，TNFAIP6，FⅦ and NKG2D，were screened at mRNA and protein levels. Except for NKG2D，the changes in the protein level of the other 4 genes were basically consistent with the changes in the mRNA level（P ＜ 0.05），but the changes in the CDK2AP1 protein were small，and there was no statistical difference （P ＝ 0.073）. Conclusion The expression of miRNAs and mRNAs in transplanted liver after xenotransplantation has greatly changed. These differentially expressed genes may be related to immune injury and coagulation dysfunction.

Objective To perform comprehensive preoperative testing in order to reduce the risk of hyperacute rejection（HAR）and acute rejection（AR）in the Wuzhishan miniature pig-Tibetan monkey xenotransplantation. To Minimize the influence of immunological factors on recipients survival in order to prolong the recipients survival. Methods The donor GGTA1 gene knockout type was identified by PCR and lectin cell staining，respectively. A total number of 30 Tibetan monkey and Wuzhishan miniature pigs were tested for lymphocyte toxicity and ABO blood group identification. Results The donor was GGTA1 knockout pigs，and the fluorescence was not detected in GTKO pig peripheral blood mononuclear cell（PBMC）with lectin cell staining，it indicated that the GGTA1 gene was successfully knocked out，which was consistent with the genotype identification results. The Tibetan monarch monkey 28# with the least toxicity to the donor PBMC through lymphocyte toxicity test was selected as a candidate recipient. Blood type test showed identical blood type between donor and recipients. Conclusion The preoperative test of GTKO Wuzhishan miniature pigs-Tibetan monkey xenotransplantation has greatly reduced the risk of HAR and AR after xenotransplantation.

Objective To detect the functional significance of surface inhibitory receptor leukocyteassociated immunoglobulin like receptor-1（LAIR-1）of NK cells in xenogeneic immune rejection. Methods The infiltration of immune cells was detected by the immunohistochemical staining in the donor liver tissue after xenotransplantation. The soluble LAIR-1（sLAIR-1）in the recipient plasma and membrane LAIR-1（mLAIR-1）in recipient immune cells were detected by the enzyme linked immunosorbent assay（ELISA）and RNA sequencing， respectively. Results In tissue level, the donor liver was infiltrated of lymphocytes. In molecular level, the sLAIR-1 and mLAIR-1 were in equilibrium. Conclusion The mLAIR-1of NK cell and sLAIR-1 in plasma were in balance and related to xenogeneic immune rejection. Taken all together these data suggest that the LAIR-1 represents an important target in inducing immunological tolerance.

Objective To establish an effective method for the extraction and culture of vigorous primary porcine hepatocytes and aortic endothelial cells, and provide the basis for the research of porcine vascular endothelial cells and hepatocytes which are the target cells in xenograft rejection. Methods Liver and aortic blood vessel were isolated from wild Bama pigs, followed by primary porcine hepatocytes extraction using peristaltic pump perfusion and type Ⅱ collagenase for digestion. Then porcine hepatocytes were purified by low speed centrifugation and differential adherence methods. Primary hepatocytes were identified by PAS staining, immunofluorescence staining for albumin and hepatocyte nuclear factor 4α. Endothelial cells of porcine aorta were extracted by type I collagenase digestion, and authenticated by testing factor Ⅷ associated antigen vWF and endocytosis of acetylated DiI-Ac-LDL. Results A large number of highly viable primary porcine hepatocytes and aortic endothelial cells were extracted, and they can express hepatocyte and endothelial cell marker proteins respectively. Conclusion The primary pig endothelial cells and hepatocyte extraction methods provided in this study are reliable methods for preparing a large number of highly viable primary endothelial cells and hepatocytes.

Objective To investigated whether exosomes from human umbilical cord-derived MSCconditioned medium (hu-MSC-CM) could increase the survival and function of neonatal porcine islet cell clusters （NICCs）exposed to hypoxia. Methods NICCs were cultured with Hu-MSC-CM and native medium RPMI1640（control）under hypoxic conditions（1% O2 ），with or without exosomes. The effects of exosomes on NICCs viability and function in vitro were examined by FACS and the extracellular Flux assay，respectively. The expression of HIF-1α, PDH2 and VEGF related with hypoxia tolerance genes in NICCs were detected by real-time quantitative PCR. Results Compared with NICCs cultured group with RPMI-1640 medium and Hu-MSC-CM group without exosomes，the survival rate，viability and function were increased in Hu-MSC-CM group with exosomes〔IEQ ： 7 800±210 vs. 5 894±188 4 740±273，P ＜ 0.001 ；β cell viability（%）：77.2±7.0 vs. 68.8±1.8、61.5±5.1， P ＜ 0.05〕. The expression of HIF-1α, PDH2 and VEGF in Hu-MSC-CM group with exosomes was significantlyhigher than the other two groups. Conclusion Hu-MSC-CM could protect NICCs from hypoxia-induced dysfunction, and exosomes played an important role in hypoxic resistance.

Objective To detect the reliability of α-1，3-galactosyl（α-Gal）antigen knockout and the expression of human protein CD46（hCD46）on the surface of genetically modified pigs at the protein level, to provide reliable donors for xenotransplantation. Methods A total of 500 μl blood from wild type（WT），α-1，3-galactosyl transferase knockout（GTKO），hCD46 and GTKO/hCD46 Bama miniature swine were collected for peripheral blood mononuclear cell（PBMC）isolation. Expression of α-Gal antigen was detected by flow cytometry after coincubation with fluorescein isothiocyanate-Griffonia simplicifolia isolectin B4（FITC-GSIB4）and PBMC. Expression of hCD46 protein was detected by flow cytometry after co-incubation of PBMC with fluorescein isothiocyanate-CD46 membrane protein antibody（anti-CD46-FITC）. Results The α-Gal expression on the surface of PBMC were negative in GTKO and GTKO/hCD46 Bama pigs and positive in WT and hCD46 Bama pigs，consistent with the sequencing results. The hCD46 protein was detected positively on the surface of PBMC of hCD46 and GTKO/hCD46 Bama pigs and was negative in WT and GTKO Bama pigs，which was consistent with the results of genotype identification after birth. Conclusion A method for visually detecting the expression of α-Gal antigen and hCD46 protein in donor porcine using microscale blood was established.

Objective To investigate the impact of recombinant human erythropoietin（rhEPO）pretreatment on liver ischemia-reperfusion injury（IRI）in rats and explore the molecular mechanism. Methods Thirty male Sprague-Dawley（SD）rats were randomly divided into three groups equally, including Sham，control and rhEPO groups. Rats in rhEPO group were treated with rhEPO（5 000 U/kg）1 hour before IR induction while sham and control groups were pretreated with saline. 70% liver IR rat model was established in control and rhEPO groups except for sham group. Rat liver tissues and serum were harvested for hematoxylin and eosin（HE）staining，Western Blot，qPCRand enzyme linked immunosorbent assay（ELISA）assay, respectively. Liver histology，transaminases（AST，ALT）， pro-inflammation cytokines（TNF-α，IL-6，IL-1β）and PI3K/AKT signaling were investigated. Results Distinct liver injury was observed with a higher histological damage score and the release of AST and ALT in serum after 70% tissue IR process. rhEPO significantly decreased the pathologic changes of liver and the release of AST and ALT in serum〔AST（U/L）：582.0±52.5 vs. 245.0±31.7 ；ALT（U/L）：388.0±39.6 vs. 166±24.3，both P ＜ 0.05〕. The expression of p-p85 and p-AKT were increased in rhEPO group compared to control group. TNF-α，IL-6 and IL-1β mRNA levels were demonstrated to ascend after IR but lower expression was observed in rhEPO group. Remarkable IRI-induced secretion of TNF-α，IL-6 and IL-1β proteins in serum was verified but rhEPO inhibited the secretion〔TNF-α（pg/ml）：425.0±31.2 vs. 227.0±19.7；IL-6（pg/ml）：353.0±26.4 vs. 189.0±16.3；IL-1β （pg/ml）：511.0±39.6 vs. 328.0±23.2 ，all P ＜ 0.05）〕. Conclusion rhEPO pretreatment can reduce the release of pro-inflammation cytokines and protect liver from IRI in rats through enhanced activation of PI3K/AKT signaling.