ObjectiveTo explore the optimal conditions of rat model of hyperuricemia (HUA) induced by different doses of potassium oxanate (PO) combined with adenine, and to provide reference for the treatment of HUA.MethodsMale Sprague-Dawley rats (220-240 g body weight) were divided into normal control group, potassium oxanate (1000, 1500 mg/kg) and adenine (0, 50, 100 mg/kg) combined model groups, with 8 rats in each group. After 5 weeks of intragastric administration, blood were collected from tail vein of rats every week, and serum uric acid, creatinine and blood urea nitrogen level were measured. At the 6th week, the changes of the pathological characteristics, expression of inflammatory and fibrosis-related factors in the kidneys were observed.ResultsIn the 1500 mg/kg potassium oxanate combined with 100 mg/kg adenine group, rats died after 2 weeks of molding, and the survival rate at the 6th week was 62.5%; but there was no significant difference between the other groups and the normal control group in survival rate (P>0.05). Compared with the normal group, the level of serum uric acid in each model group increased significantly after 1 week of molding (P<0.05), but recovered to the pre-model level after stopping intragastric administration in week 6. After 5 weeks, in model groups the levels of serum creatinine and blood urea nitrogen were higher than those in the normal control group; and the inflammation and fibrosis-related factors mRNA and protein expression of kidney tissue in model groups increased with the increase of ademine dose, and there was a significant difference in the PO 1 000 mg/kg with adenine 100 mg/kg group, PO 1 500 mg/kg with Adenine 50 mg/kg group compared to the normal control group (P<0.05). The results of renal anatomy and histology testing in rats showed that with the increased of the dosage of PO and adenine in the model groups, the increase of white deposition of renal medulla, tubulointerstitial fibrosis, and tubular epithelial cell necrosis was found, and the glomerular atrophy aggravated. Compared with the indexes in the normal control group, the expression levels of inflammation and fibrosis related genes and proteins in the 50 mg/kg adenine combined with 1 500 mg/kg PO group were higher, and inflammatory cell infiltration and fibrosis were observed, which was consistent with the clinical manifestation of hyperuricemia induced renal injury.ConclusionPO (1500 mg/kg) combined with adenine (50 mg/kg) can establish a stable hyperuricemic nephropathy model in rats.
【Abstract】 Objective To explore good methods for isolation and purification of rat islets. Methods The isletswere isolated from male SD rat pancreata by a collagenase perfusion method and purified by a modified method: added 4 kinds of Euro-Ficoll of different densities (F1: D=1.132, F2: D=1.108, F4: D=1.069, F5: D=1.023), discontinuous density gradient centrifuge the tube at 2 000 r/min for 20 minutes at 4℃ , then the islets between F1 and F2 were collected. The purity of islets was assessed by dithizone staining with islets counted and scored for size. Islets viabil ity was assessed by fluorescin diacetate / propidium iodide. The function of purified islets was judged by the test of insul in release and islets transplantation. Results After an improved method for optimized isolation and purification, (920±122) IEQ purified islets were obtained from one rat. Both the purity and viabil ity of islets were over 90%. The amount of insul in secretion was (18.25±0.32) mU/L and (36.70±3.57) mU/Lat 2.2 mmol/ L and 22.2 mmol/L concentration of glucose respectively, there was significant difference between the two phases(P lt; 0.05). The insul in release index was 2.01±0.15. Under 1 000 IEQ islets transplantation, the normal glucose level could beremained in diabetic rats. Conclusion High purity and high viabil ity islet cells can be got through improved collagenase perfusion and centrifugation on gradients method.
【摘要】 目的 探討同種異基因骨髓間充質干細胞（bone mesenchamal stem cells，BMSC）靜脈輸注對大鼠到小鼠胰島移植物的功能保護和小鼠糖尿病狀態改善。 方法 全骨髓培養法獲得C57BL/6小鼠BMSC。不連續梯度離心法分離純化Sprague-Dawley（SD）大鼠胰島，將300胰島當量的胰島單獨或與BMSC聯合移植入鏈脲菌素誘導的糖尿病BALB/c小鼠腎包膜下，并通過尾靜脈在移植后0、3和5 d注射CM-DiI標記的BMSC 5×105/只，對照組給于磷酸鹽緩沖溶液。移植后監測血糖，第9天處死小鼠，取肝、脾、胸腺、淋巴結和移植胰島的腎臟，冰凍切片，熒光顯微鏡觀察CM-DiI標記細胞的組織分布；免疫熒光法觀察移植物中胰島素和胰高血糖素表達，評價胰島的功能。 結果 BMSC靜脈輸注后主要分布于胸腺，其次是脾臟和淋巴結，腎和肝組織中未觀察到BMSC；BMSC聯合胰島移植組血糖控制水平優于其他組，且在第7天的口服糖耐量實驗優于單純胰島移植組。 結論 與胰島聯合移植的BMSC對受者免疫器官和組織有明顯的趨向性，且對胰島細胞的體內存活有一定保護作用。【Abstract】 Objective To research on the protection function by the allogeneic rat bone mesenchymal stem cells (BMSC) on rat to mouse islet transplantation and the improvement of diabetic state in mouse. Methods BMSC were prepared from C57BL/6 mouse bone marrow cells and identified by flow cytometry (FCM). Islets were isolated from Sprague-Dawley (SD) rats with Ficoll discontinuous centrifugation. CM-DiI labeled BMSC at 5×105 for one mouse were intravenously infused into STZ induced diabetic BALB/c mice after rat to mouse islet transplantation at day 0, 3 and 5. Mice with PBS intravenously infused after islet transplantation were set as the negative controls. Blood glucose was monitored every day at the first 3 days after transplantation, and then monitored every two days. At day 9 after transplantation, spleen, thymus, lymph nods, liver and islets recipient kidney were harvested. Ice slices were prepared and CM-DiI labeled cells were investigated with fluorescence microscope. Results CM-DiI-labeled BMSC were mainly distributed in thymus followed by spleen and lymph nodes. In liver and kidney, there was no red fluorescence observed. The blood sugar control for combined BMSC infusion group was superior to other groups, and the control level of islet combined BMSC infusion group were better than single islet transplantation group in OGTT at day 7. Conclusion Allogeneic BMSC can sustain the insulin secretion of islets in vivo and tend to distribute in immune organs or adenoid tissues after infusion.
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 a method to isolate the CD4+CD25+ regulatory T cells (Tregs) and to identify the purity and function of these cells.
Methods
The peripheral blood (8 mL) were collected from the great saphenous vein of 10 rhesus monkeys (4 females and 6 males, aged 4-5 years, and weighing 5-8 kg). The mononuclear cells were isolated with density gradient centrifugation. CD4+ T cells were separated by the Magnetic cell sorting (MACS) negative selection and MACS positive selection. The cell yield rate, the cell viability, and the cell purity were compared between MACS negative selection and MACS positive selection. In CD4+ MACS negative selection, the anti-biotin MicroBeads and biotin-antibody cocktai in CD4+CD25+ Tregs isolation kit non-human primate were used, and in MACS positive selection, the anti-APC MicroBeads in CD4+CD25+ Tregs isolation kit non-human primate and CD4-APC were used. The CD4+ T cells separated by positive selection were selected to obtain CD4+CD25 Tregs with CD25 MicroBeads. The purity, activity, the FoxP3 level, and the suppressive function to concanavalin A (ConA) activated autologous CD4+CD24- effective T cells (Teffs) of CD4+CD25+ Tregs were detected by flow cytometry.
Results
After CD4+ T cells were separated by MACS negative selection and MACS positive selection, the cell viabilities were all up to 95%, showing no significant difference (P gt; 0.05). The cell yield rate and purity of CD4+ T cells by positive selection were significantly higher than those of CD4+ T cells by negative selection (P lt; 0.05). CD4+CD25+ Tregs can be successfully isolated by MACS double positive selection. The classifying purity was 76.2% ± 8.6%; the cell activity was 93.3% ± 4.7%; and the level of FoxP3 was 74.2% ± 6.9%. The CD4+CD25+ Tregs had suppressive effect on ConA activated autologous CD4+CD25- Teffs.
Conclusion
MACS double positive selection can be used to isolate high-purity CD4+CD25+ Tregs from the peripheral blood of rhesus monkeys and the process does not influence the activity of CD4+CD25+ Tregs.
