转录因子PDX-1在猪胰干细胞体外诱导分化过程中的表达及意义

目的检测转录因子PDX-1在体外诱导胰干细胞分化过程中的表达,探讨PDX-1表达的意义.方法自动胰岛分离系统分离胰腺组织,获得纯化的胰管上皮细胞,在有血清培养基(RPMI1640)中进行细胞原代培养,而后在无血清培养基(DMEM/F12)中加入表皮生长因子(EGF)和尼克酰胺(nicotinamide)促进胰管上皮细胞中的干细胞分化.Western Blotting检测分化各个阶段贴壁细胞、衍生胰岛和成熟胰岛PDX-1蛋白表达;逆转录-多聚酶链反应(RT-PCR) 检测PDX-1 mRNA和上皮细胞标志抗原CK-19(cytokine-19)mRNA在分化各个阶段的表达.结果转录因子PDX-1在分化的各个阶段表达逐渐增加,上皮细胞标志抗原CK-19在各个分化阶段表达逐渐减少.结论转录因子PDX-1可能在胰干细胞分化衍生为胰岛的过程中起重要作用.     

Glucagon-like peptide-1: a major regulator of pancreatic beta-cell function

Glucagon-like peptide-1 (GLP-1) is a gut hormone synthesized by post-translational processing in intestinal L-cells, and it is released in response to food ingestion. GLP-1 stimulates insulin secretion during hyperglycemia, suppresses glucagon secretion, stimulates (pro)-insulin biosynthesis and decreases the rate of gastric emptying and acid secretion. GLP-1 has also been shown to have a pro-satiety effect. In addition, it has been demonstrated that a long-term infusion with GLP-1, or exendin-4, a long-acting analog of human GLP-1, increases beta-cell mass in rats. In conclusion, GLP-1 appears to regulate plasma glucose levels via various and independent mechanisms. GLP-1 is an excellent candidate option for the treatment of patients with type 2 diabetes mellitus.     

转录因子PDX-1在猪胰干细胞体外诱导分化过程中的表达及意义

目的 检测转录因子PDX-1在体外诱导胰干细胞分化过程中的表达,探讨PDX-1表达的意义。方法 自动胰岛分离系统分离胰腺组织,获得纯化的胰管上皮细胞,在有血清培养基(RPMI1640)中进行细胞原代培养,而后在无血清培养基(DMEM/F12)中加入表皮生长因子(EGF)和尼克酰胺(nicotinamide)促进胰管上皮细胞中的干细胞分化。Western Blotting检测分化各个阶段贴壁细胞、衍生胰岛和成熟胰岛PDX-1蛋白表达;逆转录-多聚酶链反应(RT-PCR)检测PDX-1mRNA和上皮细胞标志抗原CK-19(cytokine-19)mRNA在分化各个阶段的表达。结果 转录因子PDX-1分化的各个阶段表达逐渐增加,上皮细胞标志抗原CK-19在各个分化阶段表达逐渐减少。结论 转录因子PDX-1可能在胰干细胞分化衍生为胰岛的过程中起重要作用。     

Differentiation of iPSCs into insulin-producing cells via adenoviral transfection of PDX-1, NeuroD1 and MafA

Aims

The aim of this study was to evaluate the effect of PDX-1 (pancreatic and duodenal homeobox-1), NeuroD1 (neurogenic differentiation-1) and MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homolog A) in the differentiation of induced pluripotent stem cells (iPSCs) into insulin-producing cells and to explore this new approach of cell transplantation therapy for type 1 diabetes in mice.

Methods

iPSCs were infected with adenovirus (Ad-Mouse PDX-1-IRES-GFP, Ad-Mouse NeuroD1-IRES-GFP and Ad-Mouse Mafa-IRES-GFP) and then differentiated into insulin-producing cells in vitro. RT-PCR was applied to detect insulin gene expression, immunofluorescence to identify insulin protein, and mouse insulin enzyme-linked immunosorbent assay (ELISA) was used to evaluate the amount of insulin at different concentration of glucose. Insulin-producing cells were transplanted into the liver parenchyma of diabetic mice. Immunohistochemistry, intraperitoneal glucose tolerance test (IPGTT) and fasting blood glucose (FBG) were performed to assess the function of insulin-producing cells.

Results

Insulin biosynthesis and secretion were induced in iPSCs and insulin-producing cells were responsive to glucose in a dose-dependent manner. Gene expression of the three-gene-modified embryoid bodies (EBs) was similar to the mouse pancreatic β cell line MIN6. Transplantation of insulin-producing cells into type I diabetic mice resulted in hyperglycemia reversal.

Conclusions

The insulin-producing cells we obtained from three-gene-modified EBs may be used as seed cells for tissue engineering and may represent a cell replacement strategy for the production of β cells for the treatment of type 1 diabetes.     

Rac GTPase isoforms Rac1 and Rac2 play a redundant and crucial role in T-cell development

Rac GTPases have been implicated in the regulation of diverse functions in various blood cell lineages, but their role in T-cell development is not well understood. We have carried out conditional gene targeting to achieve hematopoietic stem cell (HSC)- or T-cell lineage-specific deletion of Rac1 or Rac1/Rac2 by crossbreeding the Mx-Cre or Lck-Cre transgenic mice with Rac1(loxp/loxp) or Rac1(loxp/loxp);Rac2(-/-) mice. We found that (1) HSC deletion of both Rac1 and Rac2 inhibited production of common lymphoid progenitors (CLPs) in bone marrow and suppressed T-cell development in thymus and peripheral organs, whereas deletion of Rac1 moderately affected CLP production and T-cell development. (2) T cell-specific deletion of Rac1 did not affect T-cell development, whereas deletion of both Rac1 and Rac2 reduced immature CD4(+)CD8(+) and mature CD4(+) populations in thymus as well as CD4(+) and CD8(+) populations in spleen. (3) The developmental defects of Rac1/Rac2 knockout T cells were associated with proliferation, survival, adhesion, and migration defects. (4) Rac1/Rac2 deletion suppressed T-cell receptor-mediated proliferation, IL-2 production, and Akt activation in thymocytes. Thus, Rac1 and Rac2 have unique roles in CLP production and share a redundant but essential role in later stages of T-cell development by regulating survival and proliferation signals.     

大麻素受体1激动剂和抑制剂对肥胖大鼠胰岛β细胞功能的影响

目的 研究大麻素受体1对肥胖大鼠胰岛β细胞功能的影响.方法 30只8周龄清洁级健康雄性SD大鼠,体质最150～200 g,采用数字表法随机分至正常对照组(n=6)和肥胖组(n=24).正常对照组给予普通鼠饲料喂养,肥胖组给予高脂鼠饲料喂养.8周后,与正常对照组比较,肥胖纽大鼠体质量增加36%,总胆固醇增加52%,提示24只肥胖大鼠制作成功.肥胖大鼠以数字表法随机分至生理盐水组(n=8)、WIN55212-2组(n=8)、AM251组(n=8).测定大鼠体质量、血脂、胰岛素、胰岛素原等指标,采用高葡萄糖钳央试验评价胰岛β细胞功能和胰岛素敏感性.采用LSD检验进行统计学分析.结果 腹腔注射药物2周后,生理盐水组、WIN55212-2组体质量、血脂、卒腹血糖、C肽、胰岛素、胰岛素原、胰岛素原/C肽、胰岛素原/胰岛素、胰岛素10～90 min分泌量及胰岛素最大分泌量均高于正常对照组(P<0.05),WIN55212-2组上述指标均高于生理盐水组(P<0.05),AM251组上述指标均低于生理盐水组和WIN55212-2组(P<0.05).生理盐水组、WIN55212-2组胰岛素0～10 min分泌量、匍萄糖输注率低于正常对照组(P<0.05),WIN55212-2组胰岛素0～10 min分泌量、葡萄糖输注率低于生理盐水组(P<0.05).AM251组胰岛素0～10 min分泌量、葡萄糖输注率高于生理盐水组和WIN55212-2组(P<0.05).结论 大麻素受体1受体激动可增加肥胖大鼠体质量,升高血脂水平,加重胰岛素抵抗和胰岛β细胞功能减退,抑制大麻素受体1可逆转此效应.     

胰岛细胞抗体与胰岛β细胞功能关系的探讨

对251例1型糖尿病（T1DM）患者进行胰岛细胞抗体（ICA）检测,发现ICA阳性组中谷氨酸脱羧酶抗体（GADA）和蛋白酪氨酸磷酸酶抗体（IA-2A）阳性率均显著高于ICA阴性组（P〈0.01）,空腹和餐后胰岛素水平明显低于ICA阴性组（P〈0.05,P〈0.01）,胰岛素抵抗指数亦明显下降（P〈0.05）,提示T1DM患者中ICA阳性更易于合并有GADA、1A-2A阳性,且对胰岛β细胞功能的破坏更大。     

Keratinocyte growth factor and beta-cell differentiation in human fetal pancreatic endocrine precursor cells

AIMS AND HYPOTHESIS: Keratinocyte growth factor (KGF) is a member of the heparin-binding fibroblast growth factor family with a high degree of specificity for epithelial cells in vitro and in vivo. Our aim was to study the effect of KGF on beta-cell growth and differentiation on islet-like cell clusters derived from human fetal pancreas. METHODS: We investigated the effects of KGF, in vitro, on beta-cell differentiation from undifferentiated pancreatic precursor cells and in vivo after transplantating human fetal pancreatic cells into athymic rats treated with KGF. RESULTS: Treatment of islet-like cell clusters with KGF in vitro did not change the number of insulin producing cells, as measured by the measurement of insulin content or DNA. The in vivo treatment of recipient rats with KGF increased the number of beta cells within the grafts 8 weeks after transplantation. At this time, glucose-stimulated insulin secretion was evaluated by glucose stimulation tests in rats bearing the transplants. Measurements of human C-peptide concentrations after glucose challenge showed that the newly differentiated beta cells in the KGF-treated group were functionally competent as opposed to the control group, where the graft failed to release insulin appropriately. CONCLUSION/INTERPRETATION: These findings suggest that in vivo, KGF is capable of inducing human fetal beta-cell expansion. The growth promoting effect of KGF on beta cells occurred mainly through the activation of ductal cell proliferation and their subsequent differentiation into beta cells.     

The role of voltage-gated calcium channels in pancreatic beta-cell physiology and pathophysiology

Voltage-gated calcium (CaV) channels are ubiquitously expressed in various cell types throughout the body. In principle, the molecular identity, biophysical profile, and pharmacological property of CaV channels are independent of the cell type where they reside, whereas these channels execute unique functions in different cell types, such as muscle contraction, neurotransmitter release, and hormone secretion. At least six CaValpha1 subunits, including CaV1.2, CaV1.3, CaV2.1, CaV2.2, CaV2.3, and CaV3.1, have been identified in pancreatic beta-cells. These pore-forming subunits complex with certain auxiliary subunits to conduct L-, P/Q-, N-, R-, and T-type CaV currents, respectively. beta-Cell CaV channels take center stage in insulin secretion and play an important role in beta-cell physiology and pathophysiology. CaV3 channels become expressed in diabetes-prone mouse beta-cells. Point mutation in the human CaV1.2 gene results in excessive insulin secretion. Trinucleotide expansion in the human CaV1.3 and CaV2.1 gene is revealed in a subgroup of patients with type 2 diabetes. beta-Cell CaV channels are regulated by a wide range of mechanisms, either shared by other cell types or specific to beta-cells, to always guarantee a satisfactory concentration of Ca2+. Inappropriate regulation of beta-cell CaV channels causes beta-cell dysfunction and even death manifested in both type 1 and type 2 diabetes. This review summarizes current knowledge of CaV channels in beta-cell physiology and pathophysiology.     

Beta1 integrins play an essential role in adhesion and invasion of pancreatic carcinoma cells

To investigate the role of beta1 integrins in pancreatic carcinoma invasion, we analyzed the relationship between the activity of beta1 integrins and the invasive ability of human pancreatic carcinoma cell lines. AsPC1, BxPC3, PANC1, SU8686, KP1NL, KP2, and H48N cells had high expression of beta1 and alpha6 subunits, and various levels of alpha2, alpha3, and alpha5 expression as determined by flow cytometry. Cell adhesion assay revealed that alpha2beta1, alpha5beta1, and alpha6beta1 integrins were the predominant adhesion receptors for collagen, fibronectin, and laminin, respectively. Beta1 integrins on different cell types showed a wide range of constitutive activity. Anti-beta1 monoclonal antibody (MAB) TS2/16 rapidly activated beta1 integrins, and thus TS2/16 requirement in cell adhesion represented the levels of constitutive activity of beta1 integrins. Notably, as the result of in vitro chemoinvasion assay, the levels of constitutive activity of beta1 integrins correlated with the invasive ability of pancreatic carcinoma cells. The inhibitory anti-beta1 MAB 13 completely blocked the invasion of these cell lines. Alternatively, the stimulatory anti-beta1 MAB TS2/16 strongly inhibited the invasion. These results show an essential role of beta1 integrins in invasion of pancreatic carcinoma cells and also suggest subtle regulatory mechanisms of cell invasion.