OBJECTIVE: To construct tissue engineering bone with bio-derived materials and bone marrow stromal cells (MSCs), and to investigate the effect of allogeneic engineering bone implants on healing of segmental bone defects. METHODS: MSCs being aspirated aseptically from tibial tuberosities of young rhesus monkeys were induced into osteoblasts in vitro and then were cultured and marked with 5-bromo-2-deoxyuridine (BrdU). Tissue engineering bones were constructed with these labeled osteoblasts being seeded onto bio-derived materials made from fresh human bones which were treated physically and chemically, Then the constructs were implanted in 15 allogeneic monkeys to bridge 2.5 cm segmental bone defects of left radius as experimental groups, bio-derived materials only were implanted to bridge same size defects of right radius as control group. and, 2.5 cm segmental bone defects of both sides of radius were left empty in two rhesus monkeys as blank group. Every 3 monkeys were sacrificed in the 1st, 2nd, 3rd, 6th and 12th weeks postoperatively and both sides of the implants samples were examined macroscopically, histologicaly, and immunohistochemicaly. The two monkeys in blank group were sacrificed in the 12th week postoperatively. RESULTS: Apparent inflammatory reactions were seen around both sides of the implants samples in the 1st, 2nd, 3rd weeks, but it weakened in the 6th week and disappeared at the 12th week. The labeled osteoblasts existed at the 6th week but disappeared at the 12th week. The bone defects in experimental group were repaired and the new bone formed in multipoint way, and osteoid tissue, cartilage, woven bone and lamellar bone occurred earlier when compared with control group in which the bone defects were repaired in ’creep substitution’ way. The bone defects in blank group remained same size at the 12th week. CONCLUSIONS: Engineering bones constructed with bio-derived materials and MSCs were capable of repairing segmental bone defects in allogeneic monkeys beyond ’creep substitution’ way and making it healed earlier. Bio-derived materials being constituted with allogeneic MSCs may be a good option in construction of bone tissue engineering.
Objective To study the protective effects of bone marrow mesenchymal stem cells (BMSCs) of rhesus monkeys on porcine islets from hypoxia/reoxygenation (H/R)-induced injury. Methods BMSCs were isolated and cultured from the marrow of 5 adult rhesus monkeys (weighing, 6-10 kg) by adherent monocytes. Islets were isolated and purified from the pancreas of 5 neonatal porcine (3-5 days old) by collagenase V digestion method, and were cultured with or without BMSCs, and exposed to hypoxia (1%O2) for 12 hours and reoxygenation for 24 or 48 hours, respectively. The experiment was divided into 4 groups: normal islet group (group A), normal islet + BMSCs group (Group B), H/R islet group (group C), and H/R islet + BMSCs group (group D). The survival rate of islets was calculated by fluorescein diacetate/propidium iodide (PI) staining. The viability of the islet cells was detected by cell counting kit 8. Apoptotic rate of islet cells was tested using Annexin V-FITC/PI labeling and flow cytometry. The stimulation index (SI) of islet function was analyzed by glucose-stimulated insulin secretion assay. Results The islet cell cluster of group C was more dispersed than that of groups A and B, and group C had more death cells; and the islet cell cluster of group D was more complete and the survival rate was higher than those of group C. The survival rate of islet was 90.2% ± 9.1%, 88.3% ± 5.9%, 52.3% ± 12.1%, and 71.4% ± 11.5% in groups A, B, C, and D respectively, it was significantly lower in groups C and D than in groups A and B (P lt; 0.05), but it was significantly higher in group D than in group C (P lt; 0.05). After coculture of BMSCs and islet at the ratio of 1 ∶ 10 and 1 ∶ 20 in group D, the viability of islet cells was significantly higher than that in group C (P lt; 0.05). The apoptotic rate was 27.1% ± 3.2%, 24.0% ± 1.0%, 64.3% ± 1.8%, and 46.2% ± 1.4% in groups A, B, C, and D respectively, it was significantly higher in groups C and D than that in groups A and B (P lt; 0.05), but it was significantly lower in group D than in group C (P lt; 0.05). There was no significant difference in SI between groups A and B at each time point (P gt; 0.05), but it was significantly lower in group C than in groups A and B (P lt; 0.05); and it was significantly higher in group D than in group C at 24 and 72 hours (P lt; 0.05). Conclusion BMSCs of rhesus monkeys can protect islet vitality and function from H/R-induced injury.
Objective To establish the swine to monkey auxiliary heterotopic liver transplantation model and to summarize surgical points. Methods Five healthy landrace piglets and five rhesus monkeys were adopted to establish the xenogenic auxiliary heterotopic liver transplantation model. In our trail landrace piglets were treated as donor andrhesus monkeys were treated as receptor. Putting the transplanted liver which included right posterior segment and part of right anterior segment into rhesus’ nest and left paracolic sulcus， and then blocking abdominal aorta and inferior vena cava shortly for end-to-side anastomosis in portal vein of transplanted liver and abdominal aorta of receptor， in infra-hepatic vena cava of transplanted liver and inferior vena cava of receptor. Ligation was performed at hepatic artery with no recon-struction. The postoperative situation and survival time of the receptors were observed. Results Four pairs of transplan-tation models were successfully established. The duration of donor liver harvesting， donor liver preparation， occlusion of recipient inferior vena cava， occlusion of abdominal aorta， and liver transplantation were （30±5） min （24-35 min），（40±10） min （31-51 min）， （30±6） min （23-36 min）， （30±8） min （22-38 min）， and （220±80） min （130-310 min）， respectively. The blood loss during transplantation was （42±6） mL （35-48 mL）. There were no thrombosis of the anas-tomosis and biliary fistula in receptors after operation， but 4 receptors died at 48， 54， 88， and 96 hours after liver transp-lantation respectively. Conclusions Swine to monkey auxiliary heterotopic liver transplantation model has b repea-tability， operation procedure is easy， and transplanted organ of it have good perfusion， which can be available for various kinds of basic and preclinical researches.
Objective To construct the rhesus monkey Schwann cells (SCs) modified with human glial cell derived neurotrophic factor (hGDNF) gene. Methods The coding sequence of hGDNF amplified by PCR from pUC19-hGDNF was inserted into eukaryotic expression vector pBABE-puro. The recombinant eukaryotic expression vector pBABE-puro-hGDNF was identified with restriction enzyme digestion and DNA sequencing. The SCs were isolated from rhesus monkeys, cultured and purified. The SCs were transfected with the recombinant retrovirus vector containing hGDNF gene. The mRNA and protein expressions of hGDNF were analyzed by real-time fluorescent quantitative PCR and Western blot. Results The PCR product of hGDNF coding sequence was a 596 bp specific segment. The recombinant eukaryotic expression vector was digested into a 596 bp specific segment by specific restriction enzyme and another segment. The 596 bp segment confirmed by DNA sequencing was consistent with hGDNF sequence on GenBank. Restriction enzyme digestion and sequencing results showed that the coding sequence of hGDNF was successfully inserted into the recombinant retrovirus vector and the mRNA and protein expressions of hGDNF were significantly higher in transfected SCs than non-transfected SCs (P lt; 0.05). Conclusion The rhesus monkey SCs modified with hGDNF gene are successfully constructed and hGDNF can be released continuously and stably, which will provide a foundation for the further research about cell therapy of hGDNF-SCs in the repair of injured nerve.
ObjectiveTo investigate the early diagnostic value of transforming growth factor-β1(TGF-β1) on acute rejection after liver transplantation in rhesus by detecting the expression of TGF-β1 in the liver tissue. MethodsLiver transplantation models in rhesus were constructed by the improved vascular dual cuff, supporting tube of biliary tract, and artery anastomosis method.The successful models were randomly divided into experimental group (no immunosuppressant treatment in perioperative period) and control group (treated by immunosuppressant in perioperative period).Then the blood samples and liver tissues were collected at 6, 12, 24, and 72 hours after surgery.Allograft rejections of liver tissue after liver transplantation were monitored by liver function test, hematoxylin-eosin staining and Banff score.Finally, the expression level of TGF-β1 was detected by Western blot analysis or immunohistochemistry technique. Results①The acute rejection happened in all the rhesus at 12 h, 24 h and 72 h after liver transplantation, especially at 72 h after liver transplantation in the experimental group, the Banff grade levels of acute rejection in the liver tissue was more severe than that in the control group (P < 0.05).②The levels of ALT, AST, and TBIL after liver transplantation was gradually increased, which were similar at 6 h and 12 h after transplantation between the two groups, but which at 24 h and 72 h after transplantation in the experimental group were significantly higher than those in the control group (P < 0.05).③The results of TGF-β1 protein expression using immunohistochemical detection:The percentage of positive area of TGF-β1 of liver tissue at 12 h in the experimental group was significantly higher than that in the control group (P < 0.05).With the extension of time, it was gradually increased and significantly higher than that in the control group at 24 h or 72 h (P < 0.05).④The semi-quantitative results of TGF-β1 protein expression using Western blot detection:The TGF-β1 protein expressions began to increase at 6 h after liver transplantation in the experimental group and the control group, and the magnitude of increase was more obvious in the experimental group.The TGF-β1 protein expressions at different time (6 h, 12 h, 24 h, and 72 h) in the experimental group were significantly higher than those in the control group (P value was 0.003, 0.001, 0.001, and 0.001, respectively). ConclusionsThe elevated level of TGF-β1 of liver tissue after liver transplantation might suggest the enhanced cellular immune function, it might have certain significance for early diagnosis of acute rejection after liver transplantation.
Human lymphocyte function-associated antigen 3 (hLFA3) has been identified as an important T cell accessory molecule. Rhesus monkeys (Macaca mulatta) have been widely used as animal models for human immune disorders. Due to the species-specificity of immune system, it is necessary to study M. mulatta LFA3 (mmLFA3). In this study, the gene encoding mmLFA3 CD2-binding domain (mmLFA3Sh) was amplified by polymerase chain reaction (PCR) and genetically fused to human IgG1 Fc fragment in pPIC9K to construct the expression plasmid pPIC9K-mmLFA3Sh-Ig. Approximately 3-4 mg mmLFA3Sh-Ig protein was recovered from 1 L of inductive media, and mmLFA3Sh-Ig produced by the P. pastoris can bind to the CD2 positive cells, and suppress the monkey and human lymphocytes proliferation induced by Con A and alloantigen in a dose-dependent manner. These results suggested that mmLFA3Sh-Ig might be used as a novel tool for pathogenesis and experimental immunotherapy of Rhesus monkey immune disorders.
ObjectiveTo analyze the relationship between the bone mineral density (BMD) and lumbar intervertebral disc degeneration in rhesus macaques by using T1ρ-MRI. MethodsTwenty female rhesus macaques at the age of 10.9 years on average (rang, 4-20 years) were selected. The lumbar intervertebral discs were classified by Pfirrmann grading system and the T1ρ relaxation time (T1ρ value) was examined by using MRI (Philips 1.5 Tesla), and then BMD values of the L4,5 vertebrae and femoral ward's triangle were detected by using Osteocore dual energy X-ray absorptiometry. Finally, the relationship of T1ρ value of the lumbar intervertebral discs and Pfirrmann grading with age, weight, BMD of lumbar vertebrae and femoral ward's triangle was analyzed. ResultsThe BMD values of lumbar vertebrae and femoral ward's triangle were (0.64±0.17) g/cm2 and (0.67±0.19) g/cm2 respectively, showing no significant difference (t=2.893, P=0.128). According to Pfirrmann grading system, there were 7 cases of grade I, 8 cases of grade Ⅱ, and 5 cases of grade Ⅲ at L4,5 intervertebral discs. The T1ρ value of the lumbar intervertebral disc was (104.08±18.65) ms; the T1ρ values of grades I, Ⅱ, and Ⅲ were (121.31±13.44), (104.73±15.01), and (77.41±11.87) ms, respectively. There was a negative correlation between T1ρ value and the age and the BMD of lumbar vertebrae and femoral ward's triangle. There was a positive correlation between Pfirrmann grading and the variables as listed above. Significant negative linear correlation was also observed between T1ρ value and Pfirrmann grading. ConclusionThe T1ρ value is a reliable index when quantifying lumbar intervertebral disc degeneration, and there is a significant positive correlation between BMD and lumbar intervertebral disc degeneration in rhesus macaques.
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.
ObjectiveTo establish the degenerative disc animal model in rhesus macaques and to verify its reliability with T1ρ, spin-lock imaging and T2-mapping MRI and histological observation. MethodsTwelve female rhesus macaques (aged 4-6 years, weighing 4.4-6.1 kg) were enrolled in the study. The L5, 6 intervertebral disk was used for the experimental group by injecting 1 mL bleomycin A5 (2 mg/mL) to its adjacent endplates to induce degeneration, and the L4,5 intervertebral disk for the control group by injecting 1 mL normal sodium to its adjacent endplates. T1ρ and T2-mapping relaxation time was examined by using MRI, and the histological observation was performed to evaluate the process of degeneration at 1, 4, and 12 weeks after operation. ResultsThere was no significant change of T1ρ, T2 map relaxation time in the control group at different time points before and after operation (P>0.05). In the experimental group, there was a significant decrease of T1ρ relaxation time from 4 weeks after operation, showing significant difference when compared with the values at pre-operation and 1 week after operation (P<0.05). The T2 map relaxation time decreased significantly at 12 weeks after operation, showing significant difference when compared with the values at the other time points (P<0.05). There was no significant difference between 2 groups at the other time points (P>0.05) except T1ρ relaxation time at 4 and 12 weeks and T2 map relaxation time at 12 weeks (P<0.05). No significant change of the percentage of the high intensity area of the operated discs was observed on T2WI of MRI after operation in 2 groups (P>0.05). The histological results showed that the number of nucleus pulposus decreased and arranged irregularly at 4 weeks, and there were fibrosis changes of nucleus pulposus and cleft of the inner annulus fibrosus at 12 weeks after operation in the experimental group. ConclusionDegeneration of lumbar intervertebral disc in rhesus macaques can be induced by injecting bleomycin A5. T1ρ-MRI may be an effective method to evaluate early degeneration of intervertebral disc.
Objective To discuss the correlation between glutamate receptor 5 (GLUR5) and the pathogenesis of intractable temporal lobe epilepsy (ITLE), through detecting the GLUR5 expression in human with ITLE and Coriaria lactone-induced rhesus monkey temporal lobe epilepsy model. Methods Fifty-four patients with ITLE treated in West China Hospital between January 2007 and December 2015 were regarded as clinical case group in this study. The other 43 patients who underwent temporal lobe removal decompression surgery in the same time period due to trauma, tumor or large area cerebral hemorrhage complicated with cerebral hernia were designated as the clinical control group. Quantitive polymerase chain reaction (PCR) and Western blot methods were used to detect mRNA and protein levels of GLUR5. Western blot was also used to detect the GLUR5 protein level in the hippocampus and temporal lobe tissues of Coriaria lactone-induced rhesus monkey epilepsy model, and the result was compared with that of animal controls. Results Quantitive PCR results showed that the expression ratio (R value) of GLUR5 in the temporal lobe of the clinical case group to the clinical control group was 0.262, without significant difference (P>0.05), while theR value in the hippocampus was 4.896, with a significant difference (P<0.05). The amplification curve showed that the GLUR5 level in the hippocampus of the clinical case group was higher than that of the clinical control group, but the GLUR5 mRNA level in the temporal lobe tissue was not significantly changed. GLUR5 PCR amplified product electrophoresis showed that the amplified fragment was 161 bp. Western blot analysis showed that the GLUR5/actin value of the temporal lobe tissue in the clinical case group was 2.172±0.063, while the value in the clinical control group was 2.142±0.060, and the difference was not statistically significant (P>0.05). The GLUR5/actin value of the hippocampus in the clinial case group was 2.548±0.509, while it was 1.584±0.415 in the clinial control group, and the difference was statistically significant (P<0.05). The GLUR5/actin value of the hippocampus of the rhesus monkey model of epilepsy was 1.007±0.034, and it was 1.001±0.032 in the animal control group, and the difference was not statistically significant (P>0.05). The GLUR5/actin value of the temporal lobe tissue in the animal experimental group of rhesus model of epilepsy was 0.763±0.026, and it was 0.742±0.034 in the animal control group, and the difference was not statistically significant (P>0.05). The target protein bands showed that GLUR5 protein expression in the temporal lobe tissue and hippocampus of the rhesus model of epilepsy and animal controls was not significantly different (P>0.05). Conclusions GLUR5 participates in the pathogenesis of human ITLE by acting on the hippocampus. The expression of GLUR5 in human ITLE is abnormal, but the expression of GLUR5 is not changed in the rhesus model of epilepsy. The abnormal expression of GLUR5 may play a role in the pathogenesis of ITLE.