The formation process of LDHs (layered double hydroxides) coating on magnesium alloy by the CO2 pressurization method was studied. The micro-structure was observed by OM, SEM and GAXRD. The weighted gain curve, apparent activation energy, and CO2 solubility curve were all calculated by equations. The potentiodynamic polarization curve, hydrogen evolution data, and immersion were analyzed by an electrochemical method. The results show that the LDHs coating was formed layer-by-layer. The formation positions were initially on the α-Mg phase, and then on the β-Mg17Al12 phase. It was found to be the most compact after 30 min. The LDHs coating began to appear to have severe cracks and holes over time. The formation process of the LDHs coating can be divided into three stages: a rapid growth stage (0–10 min), slow growth stage (10–20 min), and periodic growth stage (30 min, 1 h). The apparent activation energies in each of the three stages are 21.78, 31.86 and 34.92 kJ mol−1, respectively. The LDHs coating has a compact micro-structure and better anti-corrosion at a pressure of 3 MPa, a temperature of 50 °C and a time of 30 min. The CO2 pressurization promotes a formation reaction rate and achieves a high formation efficiency and good formation stability under the condition of zero pollution.
Activation of human immune T-cells by swine leukocyte antigens class I (SLA-I) and class II (SLA-II) leads to xenograft destruction. Here, we generated the GGTA1, B2M, and CIITA (GBC) triple-gene-modified Diannan miniature pigs, analyzed the transcriptome of GBC-modified peripheral blood mononuclear cells (PBMCs) in the pig's spleen, and investigated their effectiveness in anti-immunological rejection. A total of six cloned piglets were successfully generated using somatic cell nuclear transfer, one of them carrying the heterozygous mutations in triple genes and the other five piglets carrying the homozygous mutations in GGTA1 and CIITA genes, but have the heterozygous mutation in the B2M gene. The autopsy of GBC-modified pigs revealed that a lot of spot bleeding in the kidney, severe suppuration and necrosis in the lungs, enlarged peripulmonary lymph nodes, and adhesion between the lungs and chest wall were found. Phenotyping data showed that the mRNA expressions of triple genes and protein expressions of B2M and CIITA genes were still detectable and comparable with wild-type (WT) pigs in multiple tissues, but α1,3-galactosyltransferase was eliminated, SLA-I was significantly decreased, and four subtypes of SLA-II were absent in GBC-modified pigs. In addition, even in swine umbilical vein endothelial cells (SUVEC) induced by recombinant porcine interferon gamma (IFN-γ), the expression of SLA-I in GBC-modified pig was lower than that in WT pigs. Similarly, the expression of SLA-II DR and DQ also cannot be induced by recombinant porcine IFN-γ. Through RNA sequencing (RNA-seq), 150 differentially expressed genes were identified in the PBMCs of the pig's spleen, and most of them were involved in immune- and infection-relevant pathways that include antigen processing and presentation and viral myocarditis, resulting in the pigs with GBC modification being susceptible to pathogenic microorganism. Furthermore, the numbers of human IgM binding to the fibroblast cells of GBC-modified pigs were obviously reduced. The GBC-modified porcine PBMCs triggered the weaker proliferation of human PBMCs than WT PBMCs. These findings indicated that the absence of the expression of α1,3-galactosyltransferase and SLA-II and the downregulation of SLA-I enhanced the ability of immunological tolerance in pig-to-human xenotransplantation.
Human hepatocyte transplantation for liver disease treatment have been hampered by the lack of quality human hepatocytes. Pigs with their large body size, longevity and physiological similarities with human are appropriate animal models for the in vivo expansion of human hepatocytes. Here we report on the generation of RAG2-/-IL2Rγ-/YFAH-/- (RGFKO) pigs via CRISPR/Cas9 system and somatic cell nuclear transfer. We showed that thymic and splenic development in RGFKO pigs was impaired. V(D)J recombination processes were also inactivated. Consequently, RGFKO pigs had significantly reduced numbers of porcine T, B and NK cells. Moreover, due to the loss of FAH, porcine hepatocytes continuously undergo apoptosis and consequently suffer hepatic damage. Thus, RGFKO pigs are both immune deficient and constantly suffer liver injury in the absence of NTBC supplementation. These results suggest that RGFKO pigs have the potential to be engrafted with human hepatocytes without immune rejection, thereby allowing for large scale expansion of human hepatocytes.
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