Objective To study a simple and practical method of isolation, culture and identification of hepatic oval cells from adult rat. Methods Wistar adult rats were fed by 2-acetaminofluorere （AAF） and were stimulated by partial hepatectomy to activate the proliferation of hepatic oval cells. After operation 12 days, the livers were resected for isolating oval cells. Hepatic tissue was digested by 0.10% collagenase Ⅳ and the obtained heterogeneous liver cells were then isolated and purified by density gradient centrifugation. The expressions of albumin and CK19 mRNA in hepatic oval cells were analyzed by immuno-fluorescence and RT-PCR. Results The survival rate of the newly isolated oval cells was more than 90%. The hepatic stem cells were shown by immuno-fluorescence of stem cell’s antigen c-kit. The expressions of mRNA CK19 and albumin of the oval cell were also detected by PCR. The proliferation activity of the newly isolated oval cells was significantly high and they could be induced to differentiate into both hepatic and bile ductal cells by some growth factors. Conclusion The successful development of the simple and feasible isolation and purification procedure as well as the identification method for hepatic oval cells may provide a fundamental for further studies about bionomics of the hepatic stem cell and the relation between stem cells and hepatic carcinoma.
Cardiopulmonary bypass(CPB) is associated with thrombocytopenia and platelet dysfunction. The primary cause of acquired platelet defect is thought to be activation and release of alpha granules during CPB. Before CPB, platelet-rich plasma (PRP) was prepared by obtaining the required amount of patient’s whole blood by autologous plateletpheresis. PRP could be reinfused after operation in order to protect the function and quantities of the platelets. On the other hand, PRP could be made into autologous platelet gel (APG). APG contains supraphysiologic amounts of growth factors, and has adequate tensile strength and adhesive ability. Therefore, it can be used for hemostasis in operation, sealing wound and enhancing incision or dehiscent sternal wounds healing.
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.
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 The purity and activity of islets will greatly affect the outcome of xenotransplantation therapy of type 1 diabetes mell itus. To set up an improved method of the isolation and purification of rat islets, which can obtain highpurity,high-yield, and high-viabil ity islets. Methods Ten healthy and adult male SD rats, weighing 250-300 g were used asorgan donors. Collagenase V was perfused into pancreas via pancreatic duct. Pancreas was digested with collagenase in water bath at 38℃ about 15 minutes, islet purification was performed using two techniques: with Ficoll 400 density gradient (group A), and Ficoll-Paque™ PLUS (group B). Dithizone (DTZ) was util ized for identifying islets, counting islets equivalent quantity (IEQ) and islets’ purity. Trypan blue staining was used to detect the viabil ity of islets. Islets of group B was encapsulated with alginate/poly-L-lysine/alginate (APA). Islets function of microencapsulated and nonmicroencapsulated was evaluated by the insul in release test. Results DTZ staining showed that islets shape were round, ell ipse and irregular with a clear edge and a diameter range of 50-300 μm. The IEQ values were 338.04 ± 76.61 and 834.80 ± 54.00 in groups A and B, respectively, showing significant difference (P lt; 0.05). The purities were 88.31% ± 2.67% and 95.63% ± 1.96% in groups A and B, respectively, showing no significant difference (P gt; 0.05). The activities of islets were 67.40% ± 5.15% and 86.05% ± 2.52% in groups A and B, showing significant difference (P lt; 0.05). Islet APA microcapsules had round shape, unified size, and its diameter was between 1.5 and 2.0 mm. Each microcapsule was encapsulated of 1 to 3 islets. The result of insul in release assay was that the concentrations of insul in secretion with islets of microencapsulated and nonmicroencapsulated were (5.53 ± 1.64) ng/ mL and (4.76 ± 0.26) ng/mL in low glucose, and its concentrations of insul in secretion in high glucose were (11.95 ± 2.07) ng/ mL and (14.34 ± 3.18) ng/mL. Stimulated insul in secretion in high glucose was 2 times more than that in low glucose (P lt; 0.05), but there was no significant difference (P gt; 0.05) in the stimulation index between group A (2.16 ± 0.30) and group B (3.01 ± 0.59). Conclusion The method of islets isolation and purification using Ficoll-Paque™ PLUS own the virtues of more convenient, high islet yield, and high islet purity. Both microencapsulated and nonmicroencapsulated islets show high-viabil ity while culture in vitro.
Objective To review the general approaches in isolation and purification of pancreatic islets and progress in several aspects. Methods The latest l iterature concerning acquisition of pancreatic islets was reviewed and analyzed interms of the choice of pancreatic islet donors, the digestion and isolation of pancreas, the purification of islet and the assay of outcome. Results The profile of the isolation and purification depends on the selection of reagents and methods of operation in every step and l inkup between every step. Conclusion Pancreatic islet transplantation is the most effective method to treat type 1 diabetes, the problem of inadequate sources of pancreatic islets could be resolved by the optimal process and the establ ishment of standardized operation.
【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.