Study on SCARB1 mediated coagulation dysregulation in recipients after liver xenotransplantation / 器官移植

To investigate the changes in the expression levels of scavenger receptor class B member 1 (SCARB 1) in the liver tissues before and after liver xenotransplantation and analyze the relationship between the variations in the SCARB1 expression and coagulation regulating dysfunction in the recipients.Methods The Wuzhishan miniature pig with α-1,3-galactosyltransferase gene-knockout(GTKO) was utilized as the donor and Macaca thibetana was chosen as the recipient.Heterotopic auxiliary liver xenotransplantation models were established.The liver tissue specimen was collected before and after liver xenotransplantation.Primary hepatocytes were extracted from the pig using collagenase digestion method.Human peripheral blood mononuclear cells were obtained by immunomagnetic bead sorting.These two types of cells were co-cultured and supplemented with human plasma to establish cell models with coagulation regulating dysfunction following liver xenotransplantation.Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were performed to quantitatively measure and statistically compared the expression levels of messenger ribonucleic acid (mRNA) and protein of SCARB1 in the tissue and cell samples.At the cellular level,the expression of SCARB 1 was interfered by lentiviral vector.The coagulation time was detected to validate the effect upon coagulation function.Results The expression levels of SCARB1 mRNA and protein were significantly down-regulated after liver xenotransplantation (both P＜0.05).In the cell models,the expression levels of SCARB1 mRNA and protein in the porcine hepatocytes co-cultured with human monocytes were significantly down-regulated compared with those in porcine hepatocytes without intervention (both P＜0.05).Compared with the non-intervention group,the coagulation time was significantly prolonged after the expression of SCARB1 was interfered by lentiviral vector (P＜0.05).Conclusions The down-regulated expression of SCARB1 in the liver graft is one of the main causes of mediating coagulation regulating dysfunction.Intervention of SCARB1 expression contributes to resolve the coagulation regulating dysfunction in the recipients after liver xenotransplantation.

Breeding and identification of Wuzhishan miniature pigs with α-1,3-galactosyltransferase gene-knockout / 器官移植

To summarize the breeding and identification of Wuzhishan miniature pig models with α-l,3-galactosyltransferase (GGTA1) gene-knockout (GTKO).Methods The breeding and reproduction perform of GTKO Wuzhishan miniature pigs were assessed and the quantity of piglets was counted.The GTKO Wuzhishan miniature pig models with GGTA1gene knockout were validated by polymerase chain reaction(PCR).The αGal phenotype of peripheral blood mononuclear cells (PBMC) in human,wild-type Wuzhishan miniature pigs and GTKO Wuzhishan miniature pigs was detected by fluorescent microscope and flow cytometry.Routine blood test parameters were statistically compared between the GTKO and wild-type Wuzhishan miniature pigs.Results The inheritance of GGTA1 genotype complied with Mendel's law.Flow cytometry detected no fluorescent expression of PBMC in GGTA1-/-pig models,which were consistent with the genotype identification results.The mean piglets of the primiparous GTKO Wuzhishan miniature pigs were (6.8±1.8) and (8.3±2.2) for the multiparous Wuzhishan miniature pigs.No statistical significance was noted in routine blood test parameters between the GTKO and wild-type Wuzhishan miniature pigs (all P＞0.05).Conclusions Stable inheritance and normal reproductive capacity are observed in two generations of Wuzhishan miniature pigs continuously.GTKO Wuzhishan miniature pig is a reliable donor for heterogeneous organ transplantation.

Detection methods of non-Gal xenoantibody in human serum / 器官移植

Objective To investigate the optimal condition for the detection of anti-non-galactose (Gal) xenoantigen and antibody in human serum.Mehtods Peripheral blood mononuclear cell (PBMC) obtained from Wuzhishan miniature pig models with α-1,3-galactosyltransferase gene knockout (GTKO) were used as target cells,mixed and incubated with healthy human serum of different concentrations (4.8％,16.7％ and 100％) for 0.5,1.0,2.0,3.0 and 6.0 h,respectively.The abilities of PBMC to bind with IgM and IgG were detected by flow cytometry.Results At the serum concentration of 16.7％,the ability ofnon-Gal IgM to bind with PBMC was significantly enhanced from 0.5 h to 3.0 h incubation (P＜0.01),whereas no statistical significance was noted in terms of IgG (P＞0.05).Increasing serum concentration could also enhance the ability of non-Gal IgM to bind with PBMC.At the serum concentration of 100％ and incubation for 3 h,the ability of IgM to bind with PBMC was the highest among all groups (P＜0.01).At the serum concentration of 100％ and incubation for 6 h,the ability of IgG to bind with PBMC was significantly enhanced (P＜0.05).Prolonging incubation time and increasing serum concentration did not affect the activity of PBMC.Conclusions The optimal condition for detection of anti-non-Gal xenoantigen and antibody is determined.A quantity of 1×105 PBMC from pig should be incubated with 100％ human serum for 3 h for detection of IgM level,or incubated with 100％ human serum for 6 h for measurement of IgG level.This optimized condition contributes to screening the donor pigs which lowly express non-Gal antigen.