IGF2在小鼠胚胎干细胞定向分化为胰岛样细胞过程中的表达

【摘要】目的观察印迹基因胰岛素样生长因子（IGF）2在小鼠胚胎干细胞定向诱导分化为胰岛样细胞过程中的表达情况。方法体外诱导胚胎干细胞向胰岛样细胞分化,逆转录-PCR（RT-PCR）和细胞免疫荧光检测胰岛细胞标志基因表达;聚合酶链式反应-限制性内切酶片段长度多态性(PCR-RFLP)检测印迹基因IGF2在诱导分化前后细胞中的亲本表达情况。结果诱导分化终末细胞能表达胰岛素、胰高糖素及C肽等胰岛细胞特异性标志物,PCRRFLP检测分析显示,体外诱导分化的细胞的印迹基因IGF2呈双等位基因表达,印迹丢失。结论体外诱导培养可导致胚胎干细胞印迹基因IGF2表达的改变。     

地塞米松诱导大鼠胰岛素抵抗机制的形态学研究

为了探讨胰岛素抵抗的发病机制及胰岛素抵抗与非胰岛素依赖性糖尿病的关系，我们选用了地塞米松处理大鼠1周和3周并设置了对照组，观察了大鼠胰岛B细胞的变化，结果显示：地塞米松处理1周后，大鼠体重下降（P＜0.01），大鼠胰岛素细胞增殖明显。地塞米松处理3周后大鼠体重进一步下降（P＜0.01），胰岛B细胞数目减少，功能下降。     

罗格列酮对2型糖尿病大鼠肝脏线粒体功能障碍的影响

目的探讨罗格列酮对2型糖尿病大鼠肝脏线粒体功能障碍的影响,为阐明糖尿病的发病机制以及罗格列酮治疗糖尿病的药理作用提供新的实验依据。方法采用高脂饲养加腹腔注射小剂量链脲佐菌素(35 mg.kg-1)诱导2型糖尿病大鼠;检测肝脏线粒体琥珀酸脱氢酶及细胞色素C氧化酶活性、线粒体膜电位、肝ATP含量等指标以评价线粒体功能;测定过氧化物酶体增殖物激活受体γ辅激活因子1α(PGC-1α)的mRNA水平及线粒体基因细胞色素C氧化酶亚基Ⅰ(COXⅠ)与核基因-βactin的拷贝数之比来反映线粒体的生物合成;并检测解偶联蛋白2(UCP2)的基因转录、NO含量及NOS活性、脂质过氧化产物MDA含量和抗氧化酶SOD活性等指标以探讨糖尿病大鼠肝脏线粒体功能障碍的可能机制。结果糖尿病大鼠血糖、血胰岛素水平显著升高,胰岛素敏感性指数明显降低,表明2型糖尿病大鼠模型建立成功。与正常大鼠相比,2型糖尿病大鼠肝脏线粒体琥珀酸脱氢酶及细胞色素C氧化酶活性下降、线粒体膜电位降低、ATP生成减少并伴有肝脏线粒体生物合成抑制,提示线粒体功能损害;此外,肝脏UCP2转录上调,同时伴肝MDA含量增加,SOD及NOS活性降低,NO含量减少。罗格列酮治疗8周后,不仅明显改善糖尿病大鼠肝脏线粒体功能的损害,而且增加肝脏线粒体生物合成。进一步研究揭示罗格列酮对糖尿病大鼠肝脏线粒体功能的保护作用可能与下调UCP2 mRNA水平,上调PGC-1α转录表达,降低体内氧化应激水平有关。结论罗格列酮对糖尿病大鼠肝脏线粒体功能障碍具有保护作用。     

人脂肪间充质干细胞体外定向诱导分化为胰岛样细胞的实验研究

目的体外定向诱导人脂肪间充质干细胞向胰岛样细胞的分化。方法人脂肪间充质干细胞分3个阶段进行诱导,第一阶段培养在含适当浓度的2-巯基乙醇的高糖-达氏修正依氏培养基(HG-DMEM)培养2d,第二阶段在含适当浓度的B27、碱性成纤维细胞生长因子(bFGF)及表皮生长因子(EGF)的HG-DMEM培养基中诱导6d,第三阶段在含适当浓度的2-巯基乙醇和B27和尼克酰胺高糖无血清DMEM培养基诱导细胞向胰岛样细胞分化。对照组用HG-DMEM培养。在相差显微镜下观察细胞的形态;用反转录-聚合酶链反应(RT-PCR)法检测诱导前后nestin、胰岛素基因的表达;用免疫荧光染色法检测诱导前后nestin、胰岛素的表达;诱导第三阶段进行双硫腙染色鉴定胰岛B样细胞团。结果未经诱导的脂肪间充质干细胞呈长梭形贴壁生长,诱导后细胞逐渐变圆,并聚集成团。诱导8d细胞nestin基因呈阳性表达,诱导14d细胞nestin基因表达量下降,胰岛素基因表达呈阳性。诱导后的细胞团双硫腙染色呈棕红色。结论2-巯基乙醇、EGF、bFGF及尼克酰胺等可在体外诱导人脂肪间充质干细胞分化为具有分泌胰岛素功能的胰岛样细胞。     

胰淀素对胰岛细胞凋亡及相关基因表达的影响

目的：观察胰淀素对胰岛细胞凋亡和凋亡相关基因表达的影响。方法：应用放免法检测胰淀素培养后大鼠胰岛细胞和胰岛β细胞瘤细胞系(βTc3)高糖刺激后上清液的胰岛素水平；应用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)技术检测胰淀素培养后大鼠胰岛细胞和小鼠βTc3细胞凋亡百分率；应用定量RT-PCR(QRT-PCR)检测培养后bcl-2、bax mRNA的表达。结果：胰淀素使原代培养的大鼠胰岛细胞和βTc3胰岛素分泌功能明显下降，使原代培养的大鼠胰岛细胞和βTc3的凋亡细胞比例明显增加；胰岛细胞凋亡过程中，诱导凋亡的基因bax mRNA表达水平明显升高，抵抗凋亡基因bcl-2 mRNA表达水平明显下降。结论：胰淀素可能通过诱导胰岛细胞凋亡导致或加重糖尿病，其中bcl-2／bax mRNA表达比率变化可能起重要作用。     

体外培养人脐带血间充质干细胞向胰岛细胞分化及对糖尿病治疗的实验研究

目的研究人脐带血间充质干细胞向胰岛素分泌细胞分化的潜能及其移植后对糖尿病大鼠的治疗效果。方法体外分离培养HUCB-MSCs,在胰岛细胞培养条件下经药物定向诱导其分化;免疫组化对诱导细胞进行胰岛β细胞标记鉴定;双硫腙染色鉴定锌离子表达及检测胰岛样细胞的移植效果。结果 HUCB-MSCs经诱导后,免疫细胞化学染色显示表达人胰岛素;双硫腙染色呈棕红色;移植后2周,胰岛样细胞组血糖浓度明显降低。结论 HUCB-MSCs在体外诱导培养条件下,具有向胰岛素分泌细胞分化的潜能,这种细胞可能为Ⅰ型糖尿病提供一条新的治疗途径。     

高浓度葡萄糖对胰岛细胞凋亡及凋亡相关基因的影响

目的：观察高浓度葡萄糖体外对胰岛细胞凋亡及凋亡相关基因表达的影响。探讨葡萄糖毒性及其分子机制。方法：应用TUNEL法检测高浓度葡萄糖培养后大鼠胰岛细胞和小鼠βTc3细胞凋亡百分率；应用定量RT-PCR（QRT-PCR1）检测培养后大鼠胰岛细胞和小鼠βTc3细胞bcl-2和baxmRNA的表达；应用定量RT-PCR检测低剂量链脲佐菌素糖尿病大鼠胰岛bcl-2和baxmRNA的表达。结果：高浓度葡萄糖使原代培养的大鼠胰岛细胞和βTc-3的凋亡细胞比例明显增加；胰岛细胞凋亡过程中。诱导凋亡基因bax的mRNA表达水平明显升高，抵抗凋亡基因bcl-2 mRNA表达水平明显下降．致使bcl-2／bax比率明显降低；对低剂量链脲佐菌素糖尿病大鼠胰岛凋亡相关基因QRT-PCR检测也显示同样的结果。结论：高浓度葡萄糖可能通过诱导胰岛细胞凋亡增加而加重糖尿病。其中bcl-2／bax mRNA表达比率变化可能起重要作用。     

GLP-1对小肠上皮细胞株IEC-6向胰岛素分泌细胞分化的作用

目的：探讨胰高血糖素样肽-1（GLP-1）对小肠上皮细胞株IEC-6向胰岛素分泌细胞分化的作用。方法：采用不同浓度的GLP-1作用于IEC-6细胞24h,MTT法检测细胞活性;GLP-1诱导IEC-6细胞5d后,RT-PCR法检测胰腺十二指肠同源框1（PDX-1）,胰岛素基因增强子结合蛋白1（Isl-1）,神经源性分化蛋白（NeuroD）和胰岛素的mRNA表达,ELISA法检测培养基中是否存在胰岛素。结果：GLP-1可提高IEC-6细胞活性;使Isl-1和NeuroD的mRNA表达上调,并可以诱导PDX-1和胰岛素的表达;另外,GLP-1诱导5d后培养基中可检测到胰岛素的表达。结论：GLP-1促进IEC-6细胞增殖,并可以诱导IEC-6细胞向胰岛素分泌细胞分化,而且PDX-1可能和GLP-1的促分化作用有关。     

胰高血糖素样肽1和Extentin-4体外诱生人骨髓间充质干细胞分化为分泌胰岛素细胞

目的 探讨并优化人骨髓间充质干细胞（hBMSCs）向分泌胰岛素细胞（IPCs）定向分化的条件,为糖尿病替代疗法寻找新出路.方法 hBMSCs复苏培养后,联合应用胰高血糖素样肽1（GLP-1）、Ex-tentin-4等细胞因子通过三步诱导方案进行体外向IPCs诱导分化.应用反转录-聚合酶链反应检测与β细胞发育和功能相关的基因表达;免疫细胞化学、双硫腙染色证实IPCs的生成;电化学发光法检测细胞胰岛素的分泌量.结果 诱导分化18d后,镜下观察到胰岛样细胞团的生成,双硫腙及免疫细胞化学染色胰岛素均呈现阳性反应;诱导分化的细胞在第18天则可以观察到胰十二指肠同源盒基因1、胰岛素的高表达;诱导分化的细胞接受葡萄糖刺激后能够分泌胰岛素,且胰岛素的分泌量随葡萄糖浓度的提高而增加,高糖胰岛素分泌量[（188.7±1.5）mU/L]与低糖胰岛素分泌量[（60.1±0.9）mU/L]相比,差异有统计学意义（P＜0.05）.结论 体外联合应用GLP-l、Extentin-4、尼克酰胺等细胞因子采用三阶段诱导方案可以大大提高胰岛素细胞诱导分化率,并能促进IPCs的成熟和胰岛素的释放.     

IL—1β诱导的NO产生没有参与其细胞毒性作用

目的：研究白细胞介素－1β（IL－1β）对原代培养大鼠肝细胞的作用并进一步观察IL－1β是否通过诱导NO产生发挥作用的。方法：所有实验使用雄性Wistar大鼠（5－7周龄，体重170－230g），无菌条件下原位胶原酶灌注肝脏分离肝细胞。观察IL－1β的细胞毒性作用（对肝细胞LDH释放的影响），以及IL－1β诱导产生的NO是否参与了这一作用。结果：IL－1β显促进了原代培养大鼠肝细胞LDH的释放，这种作用是剂量和时间依赖的；IL－1β剂量和时间依赖地诱导iNOS的表达和NO的产生，L-Arginine（NO合成底物）显促进了IL－1β刺激的NO产生，L－NMMA和L－NAME（iNOS抑制剂）完全抑制了IL－1β刺激的NO产生；IL－1β诱导的NO产生不影响IL－1β对肝细胞LDH释放的促进作用。结论：IL－1β对于原代培养大鼠肝细胞具有细胞毒性作用，而其诱导产生的NO没有参与这一作用。     

胰腺-十二指肠同源框-1与糖尿病的研究新进展

糖尿病是以胰岛素绝对或相对不足为主要特征的临床综合征,而胰岛素是由胰岛β细胞分泌的体内唯一降血糖激素。胰腺-十二指肠同源框-1(PDX-1)在胰腺的早期发育和晚期分化、胰岛β细胞正常功能的维持及胰岛素的分泌等方面均有重要作用。此外,PDX-1可诱导非胰岛细胞如肝细胞、成肌细胞等向胰岛素分泌细胞分化,提示PDX-1在糖尿病的治疗领域有重要作用。     

Assessment of diabetogenic drug activity in the rat: 5,5-diphenyl-2-thiohydantoin.

The diabetogenic activity of 5,5-diphenyl-2-thiohydantoin (DPTH) via oral administration was assessed in both normal and streptozotocin diabetic rats. Rats were fed powdered chow diet with and without 0.1% (w/w) DPTH. Food consumption and body weight were recorded every other day; whole blood glucose concentrations were determined at the start of the study and at the midpoint. At sacrifice, liver and pancreas were excised and blood samples were collected. Protein and lipid levels were determined in liver; insulin in pancreas; and glucose, insulin, and lipid in blood. DPTH treatment caused decreased food consumption and body weight gain. The drug dose, calculated from the food consumption data, was 76.5 mg/kg/day for the normal rats and 107 mg/kg/day for the diabetic rats. DPTH increased liver weight and liver lipid content in both normal and diabetic rats, and markedly lowered serum triglyceride concentration in normal rats but not in diabetic rats. Serum fatty acid concentration was not altered by DPTH. DPTH produced a significant elevation of blood glucose concentration of the diabetic rats that was not, however, correlated with altered pancreatic insulin concentration. In vitro, DPTH infusion inhibited insulin secretion by the perfused pancreas.     

Anti-diabetic activity of SMK001, a poly herbal formula in streptozotocin induced diabetic rats: therapeutic study

The therapeutic anti-diabetic effect of SMK001, a poly herbal formula was evaluated in the streptozotocin (STZ; 60 mg/kg, single intraperitoneal injection) induced diabetic rats. For therapeutic study, test articles were orally dosed once a day from 21 d after STZ-dosing at 100, 200 and 500 mg/kg/5 ml dosage levels for 4 weeks. The body weight changes, blood and urine glucose level changes were monitored with changes on the pancreas weight, and after sacrifice, the histopathological changes of pancreas and the changes of insulin- and glucagon-producing cells were also observed by immunohistochemistry. The results were compared to that of glibenclamide 5 mg/kg-dosing group. Significantly (p<0.01 or p<0.05) decrease of body weight, blood and urine glucose levels were detected in STZ-induced diabetic animals with disruption and disappearance of pancreatic islets. In addition, significantly (p<0.01) increase of glucagon- and decrease of insulin-producing cells were detected in STZ induced diabetic rats. However, these diabetic changes were significantly (p<0.01 or p<0.05) and dose dependently decreased in SMK001-dosing groups, and SMK001 100 mg/kg showed more favorable effects compared to that of glibenclamide 5 mg/kg. Based on these results, it is considered that SMK001 has favorable effect to inhibit the changes on the blood and urine glucose levels, body weight and the histopathological changes of pancreas in STZ induce diabetes.     

胰岛素对糖尿病大鼠肝线粒体氧自由基和氧化磷酸化的影响(英文)

目的:研究胰岛素对糖尿病大鼠肝线粒体功能,质子ATP酶活力及超氧阴离子的影响。方法:大鼠尾静脉注射四氧嘧啶,造成糖尿病动物模型。离心制备线粒体,测氧仪测定态3呼吸率、态4呼吸率、呼吸控制率和磷氧比。用化学发光法测定超氧阴离子生成量,质子ATP酶合成与水解活力分别采用荧光素-荧光素酶法和无机磷法测定。结果:(1)糖尿病大鼠超氧阴离子生成增多,皮下注射胰岛素治疗9周后,Q_2~(·-)生成量减少。(2)在糖尿病状态下,胰岛素治疗能提高质子ATP合成酶活力,降低质子ATP水解酶活力。(3)胰岛素通过改善态3呼吸率调节呼吸控制率和磷氧比。结论:胰岛素能抑制O_2~(·-)生成,提高ATP的合成,改善肝线粒体氧化磷酸化功能。     

Hypoglycemic effect of Astragalus polysaccharide and its effect on PTP1B

Aim: To examine the effects ofAstragalus polysaccharide (APS), a component of an aqueous extract ofAstragalus membranaceus roots, on protein tyrosine phosphatase 1B (PTP1B), a negative regulator of insulin-receptor (IR) signal transduction, and its potential role in the amelioration of insulin resistance. Methods: Ten-week-old fat-fed streptozotocin (STZ)-treated rats, an animal model of type Ⅱ diabetes mellitus (TIIDM), were treated with APS (400 mg/kg po) for 5 weeks. Insulin sensitivity was identified by the insulin-tolerance test. Further analyses on the possible changes in insulin signaling occurring in skeletal muscle and liver were performed by immunoprecipitation or Western blotting. PTP1B activity was measured by an assay kit. Results: The diabetic rats responded to APS with a significant decrease in body weight, plasma glucose, and improved insulin sensitivity. The activity and expression of PTP1B were elevated in the skeletal muscle and liver of TIIDM rats. Thus the insulin signaling in target tissues was diminished. APS reduced both PTP1B protein level and activity in the muscle, but not in the liver of TIIDM rats. Insulin-induced tyrosine phosphorylation of the IR β-subunit and insulin receptor substrate-1 (IRS-1) were increased in the muscle, but not in the liver of APS-treated TIIDM rats. There was no change in the activity or expression of PTP1B in APS-treated normal rats, and blood insulin levels did not change in TIIDM rats after treatment with APS. Conclusion: APS enables insulin-sensitizing and hypoglycemic activity at least in part by decreasing the elevated expression and activity of PTP1B in the skeletal muscles of TIIDM rats.     

Effects of Hachimi-jio-gan (Ba-Wei-Di-Huang-Wan) on hyperglycemia in streptozotocin-induced diabetic rats

The present study investigated the effects of Hachimi-jio-gan (HJ) on diabetic hyperglycemia in streptozotocin (STZ)-induced diabetic rats. After STZ administration, rats had free access to pellets containing 1% HJ extract powder for four weeks. HJ markedly suppressed hyperglycemia in STZ-induced diabetic rats at three and four weeks after the start of administration. There were also significant increases in serum and pancreatic immunoreactive insulin levels in STZ and HJ co-administering rats. However, in the present study, the number of beta cells in the pancreatic Langerhans' islets did not increase. Next, in order to investigate the action mechanism besides the glycemic control action of insulin, the expression of glucose transporter 2 (GLUT2) protein, which is involved in glucose uptake and release in the liver, was investigated. GLUT2 protein expression was increased by STZ administration but was normalized after four weeks of HJ administration. Therefore, irrespective of the structural changes in pancreatic beta-cells due to STZ, HJ increased insulin production and secretion by the pancreas and significantly suppressed GLUT2 synthesis in the liver. Amylase secretion from the pancreas was measured to assess pancreatic secretion. Amylase activity was decreased by STZ but was increased by HJ. Therefore, the effects of HJ on STZ-induced hyperglycemia in rats could be summarized as follows: besides increasing insulin synthesis and release, HJ normalizes GLUT2 protein expression in the liver to suppress hyperglycemia. Hence, the results of the present study suggest for the first time that HJ affects not only the production and secretion of insulin, but also the release of glucose from the liver.     

The use of animal models in diabetes research

Diabetes is a disease characterized by a relative or absolute lack of insulin, leading to hyperglycaemia. There are two main types of diabetes: type 1 diabetes and type 2 diabetes. Type 1 diabetes is due to an autoimmune destruction of the insulin-producing pancreatic beta cells, and type 2 diabetes is caused by insulin resistance coupled by a failure of the beta cell to compensate. Animal models for type 1 diabetes range from animals with spontaneously developing autoimmune diabetes to chemical ablation of the pancreatic beta cells. Type 2 diabetes is modelled in both obese and non-obese animal models with varying degrees of insulin resistance and beta cell failure. This review outlines some of the models currently used in diabetes research. In addition, the use of transgenic and knock-out mouse models is discussed. Ideally, more than one animal model should be used to represent the diversity seen in human diabetic patients.