Objective To establish the rat orthotopic liver transplantation model by characterizing the blood supply of hepatic artery with the Cuff skill and the modified arterial sleeve anastomosis, to explore the possible mechanisms of acute rejection and the express of Fractalkine (Fkn) in the early stage after hepatic allograft operation. Methods SD rats were selected as donors and Wistar rats as receptor for the rejection model of orthotopic liver transplantation. Recipient rats were divided into 2 groups randomly after operationand the drugs were given intraperitoneally once a day in each group. In the experimental group, cyclosporine A (CsA) was delivered with 3 mg/kg. In the control group, only normal saline was given with 3 ml/kg. Condition of survivals were observed. The rejection actvity index (RAI) and the expression of Fkn of liver tissue were observed after 3rd, 5th and 7th days in 5 rats. The rest of rats in each group were fed and given drug or normal saline until they were died and the mean survival time were recorded. Results There were 18 survivals in control group, and 19 in experimental group after liver transplantation. Condition of survivals in experimental group was better than that of control group. The mean survival times of experimental group(19.50±4.51 days) was significantly longer than that of control group(7.60±1.60 days), showing statistically significant difference (P＜0.05). After 3rd, 5th and 7th days of transplantation, RAI of control group were 3.80±0.35,5.90±0.87 and 7.50±1.30,respectively;RAI of experimental group were 3.10±0.21,3.90±0.41 and 4.50±0.52.Therewasstatistically significant difference in RAI between 2 groups on the 7th day after transplantation (Plt;0.01). On the 3rd,5th and 7th days after transplantation, the Fkn of control group was 8.20±0.57,21.30±3.30 and 25.70±4.91, and that of experimental group was 8.30±0.56,10.30±0.67 and 11.70±1.23. There were statistically significant differences in Fkn between 2 groups on the 5th, 7th days after transplantation (Plt;0.01). Conclusion Fkn is a participant inacute rejection after the rat orthotopic liver transplantation and can be chosen as a useful target in the diagnosis of acute rejection. CsA has immunosuppressive property in the condition of acute rejection in the rat orthotopic liver transplantation, which may be result from the decreased the level of Fkn.
Insufficient supply of organ for allotransplantation made the study on finding new organ resources from animal progress. Pig is regarded as one of the optimal donor animals for human. The major obstacle in this field is hyperacute reaction (HAR), which is triggered after the xenogenic natural antibodies preexisting in recipient blood combine to the antigens on the surface of the endothelium and activate the complement system. alpha-Galactose residues (alpha-Gal) on the endothelial cell have been identified as the major xenoantigens. NJZ Pig has been closely breed since 1938, whose family history is clear. Tissue samples from heart, liver, kidney, pancreas, lung, small intestine, skin, spleen, thymus and lymph node were obtained and embedded in paraffin. The sections were performed the immunohistochemical staining with the sera from health volunteers (including all the blood types) as the primary antibodies as well as the biotin labeled bandeirae simplicifolia I isolectin B4 (BS I-B4), which has specific affinity to alpha-galactose. All the staining sections were compared with the tissues digested with alpha-galactosidase. There was no difference between the antigens recognized by sera of different blood types. alpha-Gal was still the major xenoantigen on the endothelial cells. There might exist non-alpha-Gal antigens on the distal convoluted tubules and collecting tubules of the kidney. There was no alpha-Gal distributing on the secreting part of pancreas, either the islet cells or the matrix cells, but surely on pancreatic duct and vessels. All the antigenity was destroyed after the enzyme digestion except that the small intestine gland still positive with the BS I-B4. alpha-Gal is the major xenogenic antigen in NJZ Pigs. There exist some unknown antigens on the distal convoluted tubules and collecting ducts of the kidney. The blood type of recipient is not the first affair to be considered in pig-to-human xenotransplantation. The specificity of BS I-B4 for the alpha-galactose needs more detail research.
Limitation of donor source for allograft makes the research on xenograft progress. Pig is regarded as one of the ideal donor animals. The major obstacle in xenograft is hyperacute rejection, which is caused by complements after they are activated by xenogeneic antigens combined with natural antibodies. It has been confirmed that alpha-Gal is the major target antigen, whose expression is incharged by alpha-1,3 galactosyltransferase (alpha-GT). The approaches to overcome hyperacute rejection against alpha-Gal included: immunoadsorption of xenogeneic natural antibodies, lysis of antigen by enzyme and genetic manupilation to obtain animal lack of alpha-GT. Besides alpha-Gal, there were other antigens binding to human serum antibody, such as gp65 and gp100, which was expressed on PAEC after induced by TNF, the A-like antigen. But their function was still unknown. It was debatable on the role of MHC in xenograft. Both direct and indirect pathway were involved in cellular response in xenograft.
OBJECTIVE:To investigate the index of the rejection of lJle retinal pigment epithelium(RPE)cells transplantation. METHOD:Allogenic RPE transplantation on rahbits by transcleral technique, the changes of interleukin-2 (IL-2) activity in peripheral blood and the effect of immunoinhibitor (methylprednisonlone)were detected. RESLILTS:In the group of simple transplantation,the IL-2 activity in peripheral blood begin to rise in the first day after operation. The peak value occured in the third day,and is still much higher than that of the control group in the 14th day,whereas in the group treated with immunoinhibitor ,there was no obvious difference in the first day after operatlon,in the third day,the IL-2 activity rises slightly,and returned to normal level in the 7th day. CONCLUSION: After RPE transplantation, the level of IL-2 activity in peripheral blood might serve as an important index to determining and detecting the rejective response. （Chin J Ocul Fundus Dis,1996,12: 239-241）
Objective To establ ish the modified model of cervical heterotopic cardiac transplantation in rats for investigation of cardiac chronic rejection. Methods Forty healthy male Wistar rats, aged 10 weeks, weighing 250-300 g, were appl ied as the donor group, and forty healthy male SD rats, aged 10 weeks, weighing 300-350 g, served as the recipient group. The donors’ pulmonary artery was anastomosed to the reci pients’ right external jugular vein by non-suture cuff technique while the donors’ innominate artery was anastomosed to the recipients’ right common carotid artery by suture microvascular anastomosis. All recipients received cyclosporin to prevent acute allograft rejection. Results Forty consecutive successful transplantations were performed. Neither anastomosis leakage nor vessel obstruction occurred. The total operation time was 40-50 minutes. The time of cuff vascular anastomosis was 2-3 minutes and that of microvascular anastomosis was 9-12 minutes. All recipients survived for more than 30 days and all allografts were examined at 30 days after the transplantation. Pathological manifestations of allograft vessels were chronic rejection. Conclusion This modified model of cervical heterotopic cardiac transplantation is simple, practical and highly reproducible and is appl icable for investigation of chronic rejection in various organ transplantation studies.
Objective To study the effect of recombinant lentiviral vector mediated human hepatocyte growth factor (hHGF) gene-modified bone marrow mesenchymal stem cells (BMSCs) on the immunological rejection after allograft l iver transplantation in rats, and to reveal the mechanism of immune tolerance. Methods Eight male Sprague Dawley (SD)rats of clean grade (aged 3 to 4 weeks, weighing 75-85 g) were selected for the isolation and culture of BMSCs; 64 adult male SD rats of clean grade (weighing 200-250 g) were used as donors; and 64 adult male Wistar rats of clean grade (weighing 230-280 g) were used as receptors. After establ ishing a stable model of rat allogeneic l iver transplantation, 1 mL sal ine, 2 ×106/mL of BMSCs 1 mL, 2 × 106/mL of BMSCs/green fluorescent protein 1 mL, and 2 × 106/mL of BMSCs/hHGF 1 mL were injected via the portal vein in groups A, B, C, and D respectively. Then the survival time of the rats was observed. The hepatic function was determined and the histological observation of the l iver was performed. The hHGF mRNA expression was detected by RT-PCR, the level of cytokine including hHGF, interleukin 2 (IL-2), IL-4, IL-10, and interferon γ (IFN-γ) by ELISA assay, the level of apoptosis by TUNEL method, and the expression level of prol iferating cell nuclear antigen (PCNA) by immunohistochemical method. Results The survival time of group D was significantly higher than that of groups A, B, and C (P lt; 0.01); the survival time of groups B and C was significantly higher than that of group A (P lt; 0.01), but there was no significant difference between group B and group C (P gt; 0.05). RT-PCR demonstrated the transcription of hHGF mRNA in the grafts of group D; the serum cytokine hHGF reached to (6.2 ± 1.0) ng/mL. Compared with groups B and C, group D exhibited significant inhibitory effect, significantly improved l iver function, and showed mild acute rejection. In addition, the levels of cytokine IL-2 and IFN-γ decreased; the levels of cytokine IL-4 and IL-10 increased; the level of apoptosis reduced; and the expression level of PCNA increased. Except for the expression of IL-4 (P gt; 0.05), there were significant differences in the other indexes between group D and groups B, C (P lt; 0.05). Conclusion BMSCs/hHGF implanting to rat l iver allograft via portal vein can induce immune tolerance. Compared with injection of BMSCs alone, BMSCs/hHGF treatment can alleviate acute rejection and prolong the survival time significantly. The immunosuppressive effect of BMSCs/hHGF is correlated with Th2 shifts up of Th1/Th2 shift, reduced apoptosis, promoted l iver regeneration.
Objective To investigate the mechanism of hyperacute rejection (HAR) in pig to rhesus monkey vein xenograft. Methods Porcine femoral vein was transplanted into rhesus monkey. Deposits of IgM, IgG, C3 and C4 on the grafts were observed by immunoflurescence. Results Great deal of IgM, C3 and C4 were seen along the endothelium of donor vein, but IgG was not seen. ConclusionIn pig to monkey xenograft model, HAR is intiated by the binding of xenoreactive IgM to donor xenoantigens and followed by the activation of complement via the classical pathway.
Objective To summarize the current advance of xenotransplantation. Methods Relevant literatures about current advance of xenotransplantation published recently domestic and abroad were collected and reviewed. Results Major progress of xenotransplantation had been made in the understanding of xenoimmunobiology in the last two decades and in the threshold of clinical application. However, many problems of immunological rejection were still needed to be explored and resolved. Conclusion Xenotransplantation as a transplantation source has an extensive potential to resolve the shortage of transplanted organs for end-stage organ failure, how to suppress rejection and prolong survival of grafts more effectively is a focal point of search in the future.
Objective To evaluate the long-term function of the traumaticallydamaged joint after its repair with transplantation of a fresh or a frozen allogenic joint. Methods From March 1977 to September 1993, 13 patients (9 males, 4females; age, 17-55 years) with traumatically-damaged joints underwent transplantation of the fresh or the frozen allogenic joints. Five patients had 5 damagedmetacarpophalangeal joints, 6 patients had 9 damaged interphalangeal joints, and 2 patients had 2 damaged elbow joints. So, the traumatic damage involved 13 patients and 16 joints. All the metacarpophalangeal joints and the interphalangeal joints were injured by machines and the 2 elbow joints were injured by road accidents. The patients were randomly divided into 2 groups: Group A (n=7) andGroup B (n=6). The 7patients with 8 joints in Group A underwent transplantation of fresh allogenic joints; the 6 patients with 8 joints in Group B underwent transplantation of frozen allogenic joints. The allogenic joint transplants were performed in the period from immediately after the injuries to 6 months after the injuries. The motion ranges of the transplanted joints and the X-ray films were examined after operation, and the immunological examination was performed at 8 weeksafter operation. Results The time for synostosis was 5-8 months in Group A, but4-6 months in Group B. In Group A, at 2 years after operation the metacarpophalangeal flexion was 30-40° and the interphalangeal flexion was 20-30°; however,at 6 or 7 years after operation the interphalangeal flexion was only 10-20°. The patients undergoing the transplantation with fresh elbow joints had the elbowflexion of 60° and the elbow extension of 0°, and had the forearm pronation of 30°and the forearm supination of 30°. But in Group B, at 2 years after operation the metacarpophalangeal flexion was 6070° and the interphalangeal flexionwas 40-50°; at 6 or 7 years after operation the interphalangeal flexion was still 40-50°. However, the patients undergoing the transplantation with frozen elbow joints had the elbow flexion of 90° and the elbow extension of 0°, and hadthe forearm pronation of 45° and a forearm supination of 45°. The joint motion ranges, the Xray findings, and the immunological results in the patients undergoing the transplantation of the frozen allogenic joints were significantly better than those in the patients undergoing the transplantation of fresh allogenicjoints. There was a significant difference in the immunological examination between Group A and Group B (IL2, 21.64±3.99；CD4/CD8,3.88±0.82 vs.IL-2,16.63±3.11；CD4/CD8, 2.53±0.23, P＜0.01). Conclusion Repairing the traumatically-damaged joints with frozen allogenic joints is a better method of regaining the contour, movement, and complex motion of the hands.
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.