Objective To study the method of obtaining a large number of dendritic cells （DC）. To study the specific cytotoxicity T lymphocyte (CTL) effect against tumor cells initiated by DC pulsed with peptide of cancer cell. Methods Development of cells with cytologic features of DC in bone marrow cultures supplemented with granulocyte macrophage-colony stimulus factor (GM-CSF) and IL-4. Determining the DC phenotype and the specific structure by electronic microscopy. The CTL effect against pancreatic carcinoma leading by the DC pulsed with tumor cells lysate in vitro was observed. Results A large number of typical DC was proliferated by supplementing with GM-CSF and IL-4 cytokines. DC had specific cell appearance and structure, and highly expressed various cell surface molecules. TNF-α had the ability of stimulating DC mature, the mature DC had the enhancing abilities of antigen presenting and IL-12 self-secreting, as well as, expressed higher levels of CD54， MHC-Ⅱ and CD86 molecules than control group (P＜0.05). T lymphoid cell stimulated by DC without tumor antigen could not recognize and kill the target cells, only if DC pulsed with peptide of cancer cell can lead a b immune response to special tumor cells. The inhibiting ratio of CTL was significantly higher than that in other groups (P＜0.01). Conclusion Bone marrow DC has b ability of inducing special CTL against determined cancer cells after they are pulsed with tumor cell lysate. DC vaccine is probably a new immunotherapeutic method against tumor in the near future.
Objective To observe the systemic and local immune response after repair of nerve defect with acellular nerve xenograft laden with allogenic adipose-derived stem cells (ADSCs) in rhesus monkey so as to evaluate the safety of the proposed material for nerve reconstruction. Methods Bilateral tibial nerves were taken from a healthy adult male landrace (weighing 48 kg) to prepare acellular nerve xenograft by chemical extraction. ADSCs were isolated from a healthy adult male rhesus monkey (weighing 4.5 kg), and were seeded into the acellular nerve grafts. The radial nerve defect models with 25 mm in length were established in 10 healthy adult female rhesus monkeys (weighing 3-5 kg), and they were divided into cell-laden group (n=5) and non-cell-laden group (n=5) randomly. Defect was repaired with acellular nerve xenograft laden with allogenic ADSCs in cell-laden group, with acellular nerve xenograft only in non-cell-laden group. The blood samples were taken from peripheral vein preoperatively and at 14, 60, and 90 days after operation for lymphocyte analysis; at 5 months after operation, the grafts were harvested to perform histological examination for local immune response and nerve regeneration. The nerve autograft in rhesus monkey was used as control. Results In cell-laden group and non-cell-laden group, no significant difference was found in the count of lymphocytes and T lymphocytes, the percentage of T lymphocytes, CD8+ T lymphocytes, as well as the ratio of CD4+ T lymphocytes to CD8+ T lymphocytes between pre- and post-operation (P gt; 0.05); in cell-laden group, the percentage of CD4+ T lymphocytes at 14 days was significantly lower than that at 60 and 90 days postoperatively (P lt; 0.05). The percentage of CD4+ T lymphocytes in cell-laden group was significantly lower than that in non-cell-laden group at 14 days (P lt; 0.05), but no significant difference was found in the other indexes at the other time between 2 groups (P gt; 0.05). At 5 months after operation, mild adhesion was found on the surface of nerve xenografts; the epineurium of nerve xenografts was thicker than that of nerve autografts; and neither necrosis nor fibrosis was found. CD3+, CD4+, CD8+, CD68+, and CD163+ T lymphocytes were scattered within the grafts, in which regenerative axons were revealed. CD3+, CD4+, CD8+, CD68+, and CD163+ T lymphocytes were comparable in cell-laden group, non-cell-laden group, and autograft group. Conclusion Repair of nerve defect with acellular nerve xenograft elicits neither systemic nor local immune response in rhesus monkeys. Implantation of allogenic ADSCs might result in transient depression of CD4+ T lymphocytes proliferation early after surgery, no immune response can be found.
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 observe the changes of immune status in recipient after implanting with xenogeneic acellular bone matrix (ACBM). Methods Twenty rabbits were randomly divided into 4 groups. Autograft，ACMB and bone soaked in alcohol were implant into the 3 experimental groups separately, and No-treatment was done as control group. The CD4+,CD8+,CD25+T lymphocytes in blood were detected by flow cytometer at 1, 2, 4 and 6 weeks after operation. After 2 and 6 weeks of implantation, the changes of bone and tissue were observed by histology. Results After 2-6 weeks, CD4+ and CD8+ T cells were significantly higher in the implantedgroup of bone soaked in alcohol than that in the other 3 groups(Plt;0.05) and there wasno statistically significant difference in the other 3 groups(Pgt;0.05). After 2 weeks, CD25+ T cells were significantly higher in the implanted group of bone soakedin alcohol than that in the other groups. In the 2nd week, there were inflammatory infiltration with a predominance of granulocytes. In the 6th week, there were many fibroblasts instead of granulocytes with a few lymphocytes and cartilage island formed in the implanted groups of autograft and ACMB. Conclusion ACBM implanting has low influence on cellular immunity in recipient.
Objective To evaluate the host immune reaction against adenovirus mediated human bone morphogenetic protein 2 (Adv-hBMP-2) gene therapy in repairof tibial defects. Methods Twelve goats were made 2.1 cm segmental defects in he tibial diaphysis and divided into 2 groups. AdvhBMP2 transfected marrow mesenchymal stem cells(MSCs) and untransfected MSCs were implanted into the defect sites of transfected group(n=7) and untransfected group (n=5), respectively. The defect repair was observed by X-ray films after 4, 8, 16 and 24 weeks of transplantation and cellular and humoral immune reactions to adenovirus were assayed before implantation and after implantation. Results More bony callus was found in the bone defects of transfected group. The healing rates were 6/7 in transfected group and 2/5 in untransfected group, respectively at 24 weeks after implantation. The mixed culture of lymphocytes and MSCs showed that the lymphocytes stimulation indexes (SI) increased 14 days after implantation, and there was significant difference between the transfected group (4.213±1.278) and the untransfected group(－0.310±0.147，Plt;0.05); SI decreased after 28 days, but there was no significant difference between the transfected group (2.544±0.957) and the untransfected group (3.104±0.644，Pgt;0.05). After 14, 28, 49, and 120 days of treatment, the titer values of neutralizing antibody against Adv-hBMP-2 (log0.1) were 2.359±0226, 2.297±0.200, 2.214±0.215 and 2.297±0.210 in transfected group, and -0.175±0.335, -0.419±0.171, 0±0.171 and 0.874±0.524 in untransfected group, being significant differences betweentwo groups(Plt;0.05). Conclusion Adenovirus mediated BMP-2gene therapy can cause cellular and humoral immune reactions against adenovirus, which can eliminate the influence of adenoviral genes and proteins within a certain period.
Objective To study the effect of transforming growth factor β1 (TGF-β1) plasmid on poly frosted-defrosted allogenic nerve transplantation. Methods Forty Wistar rats were randomly divided into two groups equally. A 2.0 cm sciatic nerve segment, 5 mm away from infrapiriformis muscle space, was removed and the defect was repaired with poly frosteddefrosted allogenic nerve. The TGF-β1 plasmids were injected into the nerve anastomosis and adjacent muscles in the experimental group, normal saline in the control group. The nerve specimens were sectioned for staining in the 6th and 12th weeks . Axonal count and statistical analyses were done. Results The grafted and distal nerve segments showed regenerated fibers in both groups. In the experimental group,less edema and more nerve fibers were observed in the 6th week. The grafted nerve segment was filled with regeneration axons, the myelinated nerve fibers arranged regularly, and the axons and the myelin sheaths developed well in the 12th week. There was significant difference in the number of regenerating axons between the experimental group 98.6±4.8／μm2 and control group 75.8±5.1／μm2 (Plt;0.01). Conclusion Multiple frost-defrost of allogenic nerve can reduce its antigenicity and increase itsusefulness in repairing nerve defects. Local use of TGF-β1 plasmid can enhance immunosuppression to reduce immuno rejection.
OBJECTIVE To detect the immunoreaction after osteoblast xenotransplantation and to investigate the possibility of heterogenic osteocyte transplantation and tissue engineered bone reconstruction. METHODS: Rat osteoblasts were isolated by two-part bony digestion/elements in culturing, and incubated in vitro at 37 degrees C, 5% carbon dioxide for 5 days until they multiplied and formed a monolayer on the bottom of dish. Twenty-eight rabbits were divided into 3 groups. Autograft of osteoblasts(group A), xenograft of osteoblasts(group B) and normal saline(group C) were implanted into the rectus abdominus muscle. The immunological and histological observations were performed after 1, 2, 4 and 8 weeks of transplantation. RESULTS: Cultured cells reached confluence within 5 days and was identified as osteoblasts by ALP staining and Bon kossa staining. The result of host versus graft reaction was negative. In group B, specific antibody reaction was detected 2 weeks and 4 weeks after transplantation. Cell mediated cytotoxicity was detected after 2 weeks, reached the peak value 4 weeks later, and then began to decline 8 weeks later. HE staining showed mass inflammatory cells and no ectopic ossification after 8 weeks. CONCLUSION: Heterogenic osteoblast transplantation will lead to an obvious change in host humoral and cellular immunity and lost the ability of bone formation. So, it can not be used for the reliable cell sources for osteocyte transplantation or tissue-engineering bone reconstruction.
OBJECTIVE: To investigate the influence of tissue engineered tendon on subgroup of T lymphocytes and its receptor in Roman chickens. METHODS: The flexor digitorum profundus of the third toes of right feet in 75 Roman chickens were resected and made 2.5 cm defects as experimental model. They were randomly divided into five groups according to five repair methods: no operation (group A), autograft (group B), fresh allograft (group C), polymer combined with allogenous tendon cells (group D), derived tendon materials combined with allogenous tendon cells (group E). The proliferation and transformation of lymphocytes and contribution of CD4+, CD8+, CD28 and T cell receptor (TCR) were detected to study the immune response. RESULTS: The CD4+, CD8+ and TCR of group D and E were increased slightly than that of group B after 7 days, while after 14 days, those data decreased gradually and no significant difference between tissue engineered tendon and autografts (P gt; 0.05), and there was significant difference between fresh allograft and tissue engineered tendon (P lt; 0.05). Lymphocytes transformation induced by conA also showed no significant difference between tissue engineered tendon and autografts (P gt; 0.05). CONCLUSION: Tendon cells are hypoantigen cells, there are less secretion of soluble antigen or antigen chips dropped out from cells. Tissue engineered tendon has excellent biocompatibility.