After escaping from the hyperacute rejection (HAR), the xenograft has to be faced the challenge of acute vascular, acute cellular and even chronic rejection. Endothelial cells have been confirmed as a kind of antigen processing cell (APC) in allo-rejection. The porcine aortic endothelial cell (PAEC) expressed SLA-II and B7 which are the characteristics of professional APC. PAEC also has plenty of alpha-Gal residues, whether the antigen play any role in the post-HAR is still unknown. Human and porcine peripheral blood lymphocyte (PBLC) were isolated and divided into two parts, one for the effectors and the another were incubated with mitomycin C (MMC) as stimulators. The two kinds of PBLC were mixed-cultured within five days. Cultured PAEC from NJZ Pig was incubated with MMC and divided into two: One digested with alpha-galactosidase. The two kinds of PAEC were taken as stimulators to mixed-culture with human PBLC for five days. All the proliferation was detected with 3H-TdR intermingled in the system. The results showed that allo-MLR was ber than xeno-MLR in the cases. The proliferation was much ber when PAEC was used as the stimulator than that of porcine PBLC. However, the response was remarkably decreased after the digestion of alpha-Gal with alpha-galactosidase. The conclusion was that the low response of porcine-to-human MLR in vitro might be related to the predominant indirect pathway of antigen recognition in this system. While PAEC was used as the stimulator the proliferation in MLR was ber which might be concerned that PAEC itself was an APC as well as xeno-antigen sources, thus the direct pathway was predominant and worked more efficiently. The alpha-Gal might induce T cell proliferation through the linkage with the biological big molecules working as a complete antigen. The other post-HAR antigen might also exist in PAEC such as SLA-II, etc.
ObjectiveTo summarize the advances of precl inical research in xenogeneic (porcine) cell transplantation in recent years. MethodsThe literature about the precl inical research in xenogeneic (porcine) cell transplantation was analyzed and summarized. ResultsWith the application of new immunosuppressive agents and the generation of transgenic pigs, great progress has been achieved in xenogeneic transplantation of pig-derived nerve cells, islet cells, liver cells, and various types of stem cells. The survival time of xenogeneic cell (porcine) significantly prolonged, but there is still a long way to go before cl inical application. ConclusionThe source of xenogeneic (porcine) cells is abundant and the experiments are reproducible. However, how to effectively prevent rejection and prolong the survival time in the host, and avoid the spread of virus between species are still need to be solved in the future research.
Objective To study whether the porcine endothelial cells (PECs) lines transfected by HLA-G1 can alter the lysis mediated by human peripheral blood mononuclear cell (PBMC) and natural killer cell 92(NK-92). Methods By use of liposomes pack, the pcDNA3.0 eukaryotic expression vector carrying HLA-G1 was transfected into PECs. Using indirect immunofluorescence and RT-PCR assays, the HLA-G1 expression in PECs was detected. The alteration of the lysis mediated by PBMC and NK-92 was detected by51Cr-release assays. Results HLA-G1 expression could be detected in PECs after transfection of HLA-G1 at the levels of protein andRNA. It also could be found that the survival rate of transfected PECs was muchhigher than that of non-transfected PECs, when both of them faced the lysismediated by human PBMC and NK-92.After transfecting the expression of HLA-G1 could be found in the transfected PECs and the lysis mediated by PBMC and NK-92 to PECs decreased obviously (Plt;0.05). Conclusion The PECs- transfected by HLAG1 can decrease the NK lysis, so that it may provide us a new thought to inhibit the xeno-cell-rejection.
Objective To investigate the feasibility of using the porcine small intestinal submucosa (SIS) as a kind of the new tissue engineered materials to repair the rat full skin defect. Methods Twenty-eight 6-week-old SD rats weighing 300-350 g were selected in this experimental study. Two 2-cm-diameter round full skin defects were made on the rat back. The upper round defect was used as the blank group, which had no coverings, and the lower round defect was used as the SIS group. SIS that had been produced earlier was transplanted in the defected area. At 3 days, 1, 2, 3, 4, 6 and 8 weeks after the transplantation, the observation was made on the repaired skin conditions, the HE stain, and the repaired skin proportion. Results There was no infection in the two groups. The repairing speed in the SIS group was faster than that in the blank group at 2, 3, 4 and 6 weeks after the transplantation. The skin repaired by SIS was soft and elastic in texture, which had the same high level as the normal skin. The scar tissues in the SIS group were thinner than those in the blank group. The repaired skin proportions at 1, 2, 3, 4, 6 and 8 weeks after the transplantation were 15.72%±3.64%, 43.81%±4.87%, 65.35%±5.63%, 87.95%±4.78%，96.90%±6.89% and 100%, respectively in the SIS group, and 13.42%±5.63%，58.74%±4.48%，76.50%±5.23%，92.30%±5.75% and 100%, respectively in the blank group. Therewas a statistically significant difference between the two groups at 1, 2, 3 and 4 weeks after the transplantation(P＜0.05). Under the microscope, the SIS-repaired skin was observed to have more keratinocytes and collagen tissues, whichwas familiar to the normal skin.Conclusion Porcine SIS can be used as a new kind of the tissue engineered materials to repair the full skin defect.
【Abstract】 Objective To investigate the feasibil ity of applying enzymatic method to prepare decellularizedporcine aorta and to evaluate its biomechanical properties, immunogenicity and cell compatibil ity. Methods 0.1% trypsin- 0.01% EDTA was appl ied to extract cells from porcine aorta under 37 continuously vibrating condition and its histology and microstructure were observed. The thickness, stress-strain curve, ultimate tension stress (UTS) and strain of failure (SOF) were compared before and after decellularization for 48, 96 and 120 hours under uniaxial tensile tests, respectively. The histological change was observed at 1, 3 and 6 weeks after the decellularized tissue was implanted subcutaneously in 3 dogs. According to the HE stains and a semi-quantitative Wakitani grading method, gross changes, category and amounts of infiltrated cells and neo-capillaries were compared between pre- and post-decellularization of porcine aortae. Endothel ial cells from canine external jugular vein were seeded onto the decellularized patches to observe the cell compatibil ity. Results Microscopy and electron microscopies examination identified that cell components was completely removed from the fresh porcine aorta and Masson’ strichrome showed that the structure of matrix (fibrins) was maintained intact at 96 hours using the decellularization method. There were no significant differences in the thickness, UTS and SOF between before and after decellularization (P gt; 0.05). However, The UTS values showed a decrease tendency and SOF showed an increase tendency. The stress-strain curve also verified a decrease tendency in mechanical intensity and an increase one in ductil ity after decellularization. After implanting the acellularized matrix subcutaneously in canine, moderately lymphocyte infiltration was seen at the 1st week and the infiltration was replaced by fibroblasts accompanied by neocapillary formation at the 6th week. A semi-quantity histological evaluation showed that there were differences in gross observation, category and the numbers of the infiltrated cells between decellularized and non-decellularized tissues（P lt; 0.05). A cell monolayer was identified by HE staining and scanning electron microscopywhen the endothel ial cells were seeded onto the inner luminal surface of the scaffold, al igned at the same direction on the whole. Conclusion The decellularized porcine aortic scaffold, prepared by trypsin-EDTA extraction under continuously vibrating condition, could meet the requirements of tissue-engineering graft in biomechanical properties, immunogenicity and cell compatibil ity.
To overcome the disadvantages of the artificial materials, to design pedicled demucosal small intestinal sheet to repair full-thickness abdominal wall defect. Methods The porcine model of full-thickness abdominal wall defect by resecting 10 cm × 7 cm abdominal wall tissue (from skin to peritoneum) in 20 female animals, which were randomizedto jejunum and ileum sheet groups(n=10). Defect of abdominal wall were repaired with pedicled demucosal jejunum/ileum sheet respectively and immediate spl it-thickness free skin grafting. The general condition was observed and the tension strength of the repaired abdominal wall was measured 30 days postoperatively. In another 5 models, defect was repaired with pedicled demucosal small intestinal sheets and immediate spl it-thickness free skin grafting. The histological change and tissue thickness of the pedicled demucosal small intestinal sheet, spl it-thickness free skin graft and the repaired abdominal wall were observed and measured respectively after 30 days of operation. Results The operations were successful and no operative death occurred in all animals. All pedicled demucosal small intestinal sheets primarily healed to the edge of defected abdominal walls. Neither infection nor wound dehiscence occurred. All the spl it-thickness free skin grafting were successful. Regeneration of the intestinal mucosa occurred 4 days to 5 days postoperatively in 3 animals (2 of jejunum sheet group and 1 of ileum sheet group) at the initial stage andwere successfully treated. No postoperative herniation occurred in all animals. The cel iac pressure of herniation of the repaired abdominal wall jejunum/ileum sheet was (24.8 ± 3.4) kPa in jejunum sheet group and (21.3 ± 2.8) kPa in ileum sheet group, and the difference was significant (P lt; 0.01). No rupture of the repaired abdominal wall occurred in jejunum and ileum sheet groups when the cel iac pressure was 40 kPa. Before repairing the abdominal wall defects, there was a l ittle residual mucosal tissue on the surface of all pedicled demucosal small intestinal sheets. At the 30th day after operation, conspicuous hyperplasia and thickening occurred in all parts of tissue of the repaired abdominal walls and the residual mucosal tissue disappeared completely. Conclusion Because of simple operation, satisfactory achievement ratio, good effect, no important compl ication, and no use of expensive prosthetic materials, it is a feasible method to repair the full-thickness abdominal wall defect with pedicled demucosal small intestinal sheet.
Objective To review the common methods of isolation and purification of porcine islets and research progress. Methods Domestic and abroad literature concerning the isolation and purification of porcine islets was reviewed and analyzed thoroughly. Results The efficacy of the isolation and purification depends on the selection of donor, the procurement and cryopreservation of high-quality donor pancreas, and the selection and improvement of the operation. Conclusion The shortage of transplanted islets could be resolved by the establishment of standardized and optimal process, which may also promote the development of porcine islet xenograft.
Objective To evaluate the feasibility of poly-L-lactide(PLLA)/porcinederived xenogeneic bone(PDXB) composite as a scaffold for the bone tissue engineering. Methods The film and the scaffold of the PLLA-PDXB composite were respectively prepared by a solution casting method and a solution casting-particle leaching method. The composite film and scaffold were further treated by the surface alkaline hydrolysis. The surface morphology of the composite was observed by the scanning electron microscopy, and hydrophilicity degree of the composite was measured. The OCT-1 osteoblastlike cells were cultured and amplified in vitro as the seeding cells, which werethen implanted on the film and scaffold. The adherence rate, adherence shape,proliferating activity, and growing morphology of the OCT-1 osteoblastlikecells were observed on the film. Results The PDXB particle 50 μm in diameter on average had a similar phase structure to that of hydroxyapatite. But its Ca/P ratio was lower than that of hydroxyapatite. After the surface alkaline hydrolysis, the PDXB particle could be exposed on the surface of the PLLA-PDXB composite. The surface roughness and hydrophilicity of the PLLAPDXB composite were obviously enhanced. The cell adherence rate and the cell proliferation activity of the PLLAPDXB composite were higher than those of the pure PLLA material. The cells tended to grow on the exposed surface of the PDXB particles. The cells seeded on the composite scaffold could migrate to the inside of the composite scaffold and grew well. Conclusion The PLLA-PDXB composite has a good cell affinity, and this kind of composite can hopefullybecome a new scaffold material to be used in the bone tissue engineering.
Objective To evaluate the immunological reaction and the outcome of allogeneic chondrocyte transplantation in repairing articular cartilage defects in porcins. Methods Full articular cartilage from the knee of two Shanghai white porcins about one-month-old was removed and cut mechanically, digested by 0.25% trypsin and 0.2% type Ⅱ collagenase and cultured in 10％ DMEM medium. Defects of 0.5 cm×0.5 cm involving the subchodral bone were created in both the left and right femur condyloid in 8 two-month-old Yunnai bama porcins. Allogeneic chondrocyte transplantation were implanted in defects at a density of (1.0-2.0)×106,0.2 ml. The lymphocytes from the receivers’ blood were collected before transplantation and after 3, 5, 7 and 12 weeks of transplantation, then mixed with allogeneic chondrocytes to determin the lymphocyte stimulation index(SI) in vitro. The histological observation in vivo was made after 5, 7 and 24 weeks of transplantation. Results Lymphocyte SI at 3, 5, 7 and 12 weeks(1.457±0.062,1.739±0.142,1.548±0.047，1.216±0.028) after transplantation was higher than that before transplantation(1.102±0.034，Plt;0.05). SI began to increase in the 3rd week and reached the peak value in the 5th week, then gradually declined at the 7th and 12th weeks, showing significant differences when compared with in the 5th week (Plt;0.05). Inflammation and lymphocytes infiltration could be seen in subchondral bone and the intergration area between repair tissue and normal cartilage in the 5th week, and then decreased and limited in subchondral bone in the 7th week. Defects were filled with cartilage tissue, which had good intergration with subchondral bone at 24 weeks after transplantation. Conclusion Immunological reactions can be found at early stage of allogeneic chondrocyte transplantation and then decreased with the time, the fullthickness articular cartilage defects could be repaired mainlywith hyaline cartilage by the allogeneic chondrocyte transplantation. This may provide a new method to repair articular cartilage defects clinically.
Objective To construct recombinant lentiviral vectors of porcine bone morphogenetic protein 2 (BMP-2) gene and to detect BMP-2 gene activity and bone marrow mesenchymal stem cells (BMSCs) osteogenetic differentiation so as to lay a foundation of the further study of osteochondral tissue engineering. Methods BMSCs were isolated from bone marrow of 2-month-old Bama miniature porcines (weighing, 15 kg), and the 2nd generation of BMSCs were harvested for experiments. The porcine BMP-2 gene lentiviral vector was constructed by recombinant DNA technology and was used to transfect BMSCs at multiplicity of infection (MOI) of 10, 25, 50, 100, and 200, then the optimal value of MOI was determined by fluorescent microscope and inverted phase contrast microscope. BMSCs transfected by BMP-2 recombinant lentiviral vectors served as experimental group (BMP-2 vector group); BMSCs transfected by empty vector (empty vector group), and non-transfected BMSCs (non-transfection group) were used as control groups. RT-PCR, immunohistochemistry staining, and Western blot were performed to detect the expressions of BMP-2 mRNA and protein. Then the BMSCs osteogenesis was detected by alkaline phosphatase (ALP) staining, ALP activities, and Alizarin red staining. Results The recombinant lentiviral vectors of porcine BMP-2 gene was successfully constructed and identified by RT-PCR and gene sequencing, and BMSCs were successfully transfected by BMP-2 recombinant lentiviral vectors. Green fluorescent protein could be seen in the transfected BMSCs, especially at MOI of 100 with best expression. The immunohistochemistry staining and Western blot showed that BMSCs transfected by BMP-2 recombinant lentiviral vectors could express BMP-2 protein continuously and stably at a high level. After cultivation of 2 weeks, the expression of ALP and the form of calcium nodules were observed. Conclusion The porcine BMP- 2 gene lentiviral vector is successfully constructed and transfected into the BMSCs, which can express BMP-2 gene and protein continuously and stably at a high level and induce BMSCs differentiation into osteoblasts.