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Objective This study was conducted to establish a stable and highly efficient method for isolation and purification of pancreatic islets from NOD mice and to evaluate their characteristics in vitro and in vivo. Methods The is-lets were isolated from mouse pancreas using modified collagenase digestion and Ficoll density gradient centrifugation. The endocrine secretory function was assessed by insulin secretion in either low or high dose glucose stimulation. To evaluate the function of the graft,body weight and blood glucose were monitored,and IVGTT was performed. In addition,to assess sur-vival of the implanted islets,Pathology using HE staining and insulin immunostaining of the graft were performed. Results The average islet yield was 116 ± 12 islets/pancreas and purity was higher than 90%. Compared with islets from Kunming mice,the islets isolated from NOD mice were poorly responsive to glucose challenge. Blood glucose levels and body weight changes of the islet-transplanted diabetic mice were significantly improved compared with the sham-operated mice. In addi-tion,blood glucose levels in vivo after an IVGTT also significantly improved. However,these improvements were only main-tained for 2 weeks. Furthermore,HE staining and immunostaining assays demonstrated that there were insulin-positive cell clusters and lymphocyte infiltration in the graft-bearing kidney. Conclusions A large number of quality islets can be isola-ted and purified from NOD mice by using the modified mouse islet isolation method, which can be used to develop thera-peutic strategies to protect transplanted islets from rejection and autoimmune attack.
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[Summary] The epithelial‐mesenchymal transition (EMT) is a biological process in which epithelial cells are converted into cells with mesenchymal phenotype in specified physiological and pathological conditions. EM T plays a critical role in proper embryonic development ,tissue regeneration ,cancer metastasis and organ fibrosis. EM T can be divided into three subtypes (Ⅰ ,II and Ⅲ ) based on different biological context ,of which type II EMT contribute importantly to the development of organ fibrosis.Renal interstitial fibrosis (RIF) is an important pathological feature of diabetic nephropathy (DN). The understanding of molecular mechanisms of this process tubular EM T may provide a clue to intervene the development of DN through suppressing EM T and reversing RIF.
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Mesenchymal stem cells (MSCs) have received considerable attention for various therapeutic approaches in recent years. MSCs are also easy to genetically modify to express therapeutic genes by using lentiviral vectors. Because of the similarities in genetics, physiology and metabolism between non-human primates (NHPs) and humans, NHPs models are invaluable for researching human disorders and for developing therapeutic strategies. Therefore, MSCs derived from NHPs could be a powerful tool for cell therapy and genetic engineering. Studies from captive and free-ranging adult NHPs show that up to 100% were infected with simian foamy virus (SFV). In this study, we found that all cultured MSCs derived from adult cynomolgus monkey were infected with SFV by RT-PCR. Therefore, antiviral drugs must be added in MSCs culture. However, because of SFV infection and additive antiviral drugs, the infection efficiency of the lentiviral vectors reduced significantly. In this study, we improved the infection efficiency by disabled antiviral drugs before lentiviral infection. It might be provide technical assistance for the culture of adult cynomolgus monkey MSCs as genetically engineered cells applied to clinical and experimental research.
Subject(s)
Animals , Bone Marrow Cells , Cell Biology , Cell Differentiation , Genetic Vectors , Genetics , Green Fluorescent Proteins , Genetics , Lentivirus , Genetics , Macaca fascicularis , Mesenchymal Stem Cells , Cell Biology , Virology , Simian foamy virus , Physiology , Transduction, GeneticABSTRACT
Objective To investigate the value of MRI and~1H-MRS in diagnosis of early stage of diabetic encephalopathy by detecting regional metabolite in cynomolgus diabetes models. Methods Five pathogen-free male adolescent cynomolgus were made type 1 diabetes mellitus models (T1DM) by intravenous injection of streptozotocin (STZ) (100 mg/kg), and the reliability and stability of the modes were assessed with long term follow-up of blood glucose and intravenous glucose tolerance tests. MRI and ~1H-MRS were performed to evaluate the volume, signal intensity and metabolic ratios of NAA/Cr, mI/Cr and Cho/Cr at hippocampus, lateral temporal lobe and occipital lobe 3 years after model establishment. Cortisol in serum was detected with immunoradiometric assay. In addition, 5 normal adult cynomolgus monkeys were selected in the control group and accepted the same examination above. Results ①Intravenous administration of STZ could made stable T1DM monkey model. ②Only mI/Cr ratio increased at hippocampus of diabetic monkeys compared to the control group (P<0.05). ③There was no statistical difference of cortisol in serum between the diabetic group and the control group (P＞0.05). Conclusion ~1H-MRS may detect the metabolic changes of the hippocampus in STZ-induced diabetic adolescent cynomolgus monkeys and may contributes to the early diagnosis of diabetic encephalopathy.
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Objective To investigate the biological characteristics of neural stem cells from the striatum of human embryos. Methods Neural stem cells were isolated from the striatum of 16-20 weeks human embryos.These cells were cultured to proliferate and then differentiate without mitogens or inductive factors.At various time points the progeny of neural stem cells differentiation were analyzed.Using immunocytochemistry stainings,the biological characteristics of neural stem cells were examined. Results Neural stem cells in the striatum of 16-20 weeks human embryos proliferated rapidly in vitro within the first one month of culture at an average doubling time of 3-4 days.Upon mitogen withdrawal,the differentiation of neural stem cells to neurons exceeded 50%.After 8 weeks in culture,however,the proliferation speed of neural stem cells lowered significantly.The proportion of neurons induced by mitogen removal was under 20%.There were about 20% of cells within neurosphere continue to divide.Conclusion The proliferation and self-renewal ability of neural stem cells in the striatum of 16-20 weeks human embryo is robust.In vitro,the rate of proliferation went down with time,and the ability of differentiation to multiple neural cells varied.The different mitogen factors in media have different effects on neural stem cell.Within neurospheres,neural stem cells are not homogenous,as only a portion of cells can divide.