Objective To establish effective method for large-scale purification of islet cells from pig pan-cress. Methods Pig pancreas tissue was digested with collagenase P followed by purification in a HCA-Fi-coil dis continuous gradient using Cobe2991 cell separator. After isolation, the islet cell yield and purity were evaluated with light microscope with DTZ staining, and the islet function assessed by insulin release as-say in vitro. Results The number of the islets coll ected from each pancreas averaged (275 000±20 895)islet equivalents (IEQ) before purification, and (230 350±26 679) IEQ after the purification with discon-tinuous gradient centrifugation. From each gram of the pancreatic tissue, (2710±229) IEQ were obtained with an average purity of (50.2±1.95) %. The purified islets responded well to high-concentration (16.7 mmol/L) glucose stimulation with a 4. 74-fold increase of insulin secretion over the basal level (3.3 mmol/L, P <0.001). Conclusion The established method can be applicable for large-scale purifi-cation of fully functional islet cells from pig pancreas.

Objective To study the role of glucagon-like peptide-1 (GLP-1) analogue liraglutide played in the proliferation of CD4+CD25 T cells in normal people and newly-onset type 1 diabetic patients,and to evaluate the possible immune regulatory role of liraglutide in the therapy of type 1 diabetes.Methods CD4+ CD25-T cells of 10 normal people and 10 newly-onset type 1 diabetic patients were separated from peripheral blood by MACS immunomagnetic beads and stimulated by Human T-Activator CD3/CD28 Dynabeads to proliferate.CFSE labeling technique was used to evaluate the proliferation of CD4+ CD25-T cells by flow cytometry.Liraglutide of different concentrations(0,25,50,and 100 nmol/ml) was added to the proliferation system,then the proliferation of CD4+CD25-T cell was measured.Results (1) Liraglutide suppressed the proliferation of CD4+ CD25-T cells from either normal people or type 1 diahetic patients with dose-dependent manner (P ＜ 0.05).(2) Under the different concentrationsofliraglutide,the proliferation ofCD4+CD25 T cells from diabetic patients was mueh more robust than that of normal people (P＜0.01).(3) The inhibitory effects of liraglutide on CD4+ CD25-T cells proliferation in normal people and diabetic patients were similar (P＞0.05).Conclusion The proliferation of CD4+ CD25 T cells in type 1 diabetic patients was more robust than normal people,which indicated cellular immune dysfunction in type 1diabetes.Liraglutide inhibits the proliferation of CD4+ CD25-T cells of type 1 diabetic patients in vitro.The immunosuppression effect of liraglutide may have potential value in the treatment of type 1 diabetes.

ObjectiveTo ascertain the safety and function of the transplantation of neonatal pig islets (NPIs) for diabetic patients.MethodsNPIs were injected into the hepatic artery of 22 patients.After the transplantation,the patients were treated with a multiple drug immunosuppressive regimens.The first 14 patients were treated with cyclosporine (CsA),mycophenolate mofetil (MMF) and prednisolon,and porcine C-peptide was not monitored,the following 2 patients were given cyklosporin and MMF only,while the next 6 patients were given a quadruple drug regimen consisting of OKT3,takrolimus,sirolimus and prednisolon.The blood glucose levels,exogenous insulin requirement,HbA1c,porcine endogenous retrovirus (PERV) and liver function were assessed before and after NPI transplantation.The serum porcine C peptide were monitored in last 8 patients.ResultsThe first 14 patients required less insulin and the HbAlc dropped after the transplantation.In the 2 subsequent patients,the metabolic parameters remained unchanged and monitor of porcine C-peptide was negative.Insulin requirements were reduced in all 6 patients,and HbAlc was normalized 3 months after the transplantation.Significant levels of porcine C-peptide were detected in the patient serum.Two of the patients were given a second injection of NPIs,and one of them became insulin independent for 7 d.No serious adverse events were noted after the transplantation.There was no evidence of PERV transmission.Six out of the 22 patients were followed up for 4-6 years after the NPIs injection,immunosuppressive treatment was stopped 1 year after the transplantation.The patients started to take insulin at the time of follow up.Four patients restricted the intake of sugar,while the other 2 did not.One patient had ketoacidosis twice and slight diabetic retinopathy,and another patient had ketoacidosis induced by acute gastroenteritis.The remaining 4 patients did not have any complications.Assays for PERV were again negative.ConclusionXenogenic islets can survive and function in the human body.No serious adverse events are noted.

OBJECTIVE: To determine the genetic modification on neonatal porcine islet cell clusters (NICC) by small interfering RNA (siRNA)-mediated tissue factor (TF) knockdown in vitro. METHODS: Porcine NICC were transfected with 5 pairs of designed siRNA respectively or in different combinations with lipofectamine 2000. Transfected NICC were analyzed for TF gene by real-time PCR to select the siRNA which worked best. Meanwhile, the viability of NICC after the TF siRNA transfection was examined by FACS. The efficiency of TF gene and protein suppression was measured by real-time PCR and and FACS respectively. RESULTS: Real-time PCR and FACS showed that a 60% reduction in the TF gene expression and a 50% reduction in the protien level of TF on NICC were achieved by transfecting 3 pairs of selected siRNA. The siRNA transfection had no significant effect on the viability of NICC which was analyzed by FACS. CONCLUSION The expression of TF on porcine NICC is efficiently suppressed by 3 pairs of designed siRNA in vitro.
Animals ; Animals, Newborn ; Cells, Cultured ; Female ; Gene Knockdown Techniques ; Gene Silencing ; Islets of Langerhans ; cytology ; metabolism ; Male ; RNA, Small Interfering ; genetics ; Real-Time Polymerase Chain Reaction ; Swine ; Thromboplastin ; genetics ; metabolism ; Transfection

OBJECTIVE: To establish a method for in vitro expansion of human natural CD4⁺CD25⁺ T regulatory cell (Treg) cells for clinical study and immunotherapy. METHODS: Human natural CD4⁺CD25⁺ Treg were isolated from peripheral blood monocyte cells (PBMCs) by magnetic activated cell sorting (MACS) and expanded by CD3/CD28 expansion beads, IL-2 and rapamycin. The number and the viability of the freshly isolated and expanded Treg were detemined by trypan blue staining. The phenotype and the purity of the freshly isolated and expanded Treg were analyzed by FACS. Treg suppression activity was assessed by mixed lymphocyte reaction (MLR) assay. RESULTS: Human natural Treg were expanded up to 2 000 folds after 3 weeks in culture, and the activity was more than 97%. The expanded Treg retained Treg phenotype as shown by their freshly isolated counterparts, and the purity of CD4⁺CD25⁺FoxP3⁺ Treg was (94.22 ± 2.12)%. The expanded Treg demonstrated a similar potent suppression of both proliferating auto- and allo- CD4⁺CD25⁻ effector T cells in vitro in a cell number-dependent manner. CONCLUSION An in vitro expansion of human natural Treg was established to obtain large numbers of human Treg with highly suppressive phenotype and function, thereby providing a solution to the availability of sufficient human natural Treg in clinical study and immunotherapy.
Cell Culture Techniques ; Cell Separation ; Cells, Cultured ; Humans ; Interleukin-2 ; Leukocytes, Mononuclear ; Lymphocyte Culture Test, Mixed ; T-Lymphocytes, Regulatory ; cytology