【作者】 沈晓霞；

【导师】 李光伟； 李宏亮；

【作者基本信息】 中国协和医科大学 ， 内科学， 2009， 博士

【摘要】 第一部分:PI3K/Akt通路在高糖毒性诱导的TC1-6细胞和胰岛α细胞胰岛素抵抗中发挥的作用目的:探讨慢性葡萄糖毒性对α细胞胰高糖素分泌的影响及α细胞胰岛素抵抗对其影响。方法:胰岛细胞和TC1-6细胞（α细胞株）分别培养于含低浓度（5.5mM）、中浓度（11.1mM）和高浓度（25mM）葡萄糖的培养基中1,3,5d,分别检测胰高糖素（Glc）分泌及其mRNA表达;加入不同浓度胰岛素6h后,观察对培养5d的原代胰岛和TC1-6细胞Glc分泌和基因表达的影响并用Western Blot方法检测高糖对α细胞Akt磷酸化的影响。结果:（1）培养1d时,低、中和高浓度葡萄糖培养的原代胰岛细胞和TC1-6细胞Glc分泌没有显著差别,培养3d时,高糖培养的胰岛细胞胰高糖素分泌明显增加42%[（2252.64±41.71）pg/mg protein vs（3205.97±45.53）pg/mg protein,P<0.05)],而TC1-6细胞胰高糖素的分泌无明显变化,试验第5d,中浓度和高糖培养的原代胰岛细胞胰高糖素均较低糖培养明显升高,分别增加52%[（2452.22±67.89）pg/mg proteinvs（3735.44±54.89）pg/mg protein,P<0.05)和117%[（2452.22±67.89）pg/mg protein vs（5320.20±398.51）pg/mg protein,P<0.05)],而TC1-6细胞仅在高糖培养5d时胰高糖素分泌较低糖培养明显增加,为74%(（78.62±4.72）ng/10⁶ protein vs（136.8±10.94）ng/10⁶ protein,P<0.05]。（2）与低糖培养1d相比,中、高浓度葡萄糖培养1d的原代胰岛细胞和TC1-6细胞Glc基因表达没有明显差别,但培养3d后,高糖培养的胰岛细胞胰高糖素基因表达较低糖培养升高125%（P<0.05）,而TC1-6细胞胰高糖素基因表达此时没有明显变化;当培养5d时,25mM糖浓度培养的原代胰岛和TC1-6细胞Glc基因表达明显升高,比低糖培养分别增高272%和78%;（3）10^-7M胰岛素可以抑制低糖培养的原代胰岛和TC1-6细胞Glc的分泌,分别为60%[（2315.02±48.12）pg/mgprotein vs（915.99±32.54）pg/mg protein;P<0.05]和61%[（55.12±3.86）ng/10⁶ proteinvs（21.59±1.30）ng/10⁶ protein,P<0.05],但仅能轻微抑制高糖培养下原代胰岛和TC1-6细胞Glc的分泌;当胰岛素增至10^-5M时,高糖培养下原代胰岛和TC1-6细胞的Glc的分泌也明显受到抑制,分别为66%[（3872.34±29.66）pg/mg protein vs（1307.65±23.34）pg/mg protein;P<0.05]和61%[（118.61±10.68）ng/10⁶ protein vs（46.55±3.72）ng/10⁶ protein;（4）加入10（?）胰岛素2h后,高糖和低糖培养下原代胰岛和TC1-6细胞P-Akt水平分别升高137%和70%以及200%和180%,但高糖组明显低于低糖组,给予PI3K抑制剂-wortmannin,低糖培养的原代胰岛和TC1-6细胞P-Akt水平抑制率明显高于高糖组。±结论:高糖可以引发原代胰岛和TC1-6细胞胰高糖素的分泌升高,其可能的机制与原代胰岛和TC1-6细胞胰岛素介导的PI3K/Akt信号通路受阻而产生的原代胰岛和TC1-6细胞胰岛素抵抗有关。第二部分:Syntaxin1A/SNAP-25蛋白在逆转α细胞高糖毒性中的作用目的:探讨逆转高糖毒性对α细胞胰高糖素分泌和合成的影响以及可能的机制。方法:TC1-6细胞（α细胞株）分别给予5.5mmol/L（低糖组）和25mmol/L（高糖组）葡萄糖的培养基培养10d,25mmol/L葡萄糖培养5d/5.5mmol/L葡萄糖培养5d（高糖.低糖组）以及5.5mmol/L葡萄糖培养5d/25mmol/L葡萄糖培养5d（低糖-高糖组）,检测各组细胞胰高糖素分泌及其mRNA的表达;Western印迹检测持续高糖以及由高糖降至低糖后TC1-6细胞Snare蛋白（syntaxin1A和SNAP-25）的表达水平;结果:（1）与高糖组TC1-6细胞相比,高糖-低糖组TC1-6细胞Glc分泌下降29%[（2.68±0.21）ng/mg protein vs（3.74±2.99）ng/mg protein,P<0.05];（2）与高糖组TC1-6细胞相比,高糖-低糖组TC1-6细胞Glc mRNA表达下降52.6%±2.8%（P<0.05）;（3）高糖组TC1-6细胞表达syntaxin1A和SNAP-25的水平较低糖组TC1-6细胞syntaxin1A和SNAP-25的表达分别升高36%和69%（P<0.05）,而高糖-低糖组syntaxin1A和SNAP-25蛋白表达与高糖组相比明显下调,分别下降49%和56%（P<0.05）。结论:解除高糖毒性α细胞胰高糖素分泌异常明显改善,促进胰高糖素分泌相关的syntaxin1A和SNAP-25蛋白表达相应降低。更多还原

【Abstract】 PartⅠInvolvement of the PI3K/Akt pathway in high glucose-induced insulin resistance in rat islets and TC1-6 cellsobjective In order to clarify whether long-term exposure to high glucose affects a cell function and induces hyperglucagonemia via insulin resistance.Method We intraperitoneal injected Sprague Dawley rats with dose of 100mg/kg streptozotocin and administrated the diabetic rats with NPH insulin to keep their glucose level from 5mmol/L-10mmol/L to establishβcell deleted rat model,then we isolated rat islet and cultured for 1d,3d and 5d at 37℃with RPMI 1640 medium containing 5.5mM,11.1mM and 25mM glucose,similarly,TC1-6 cells were exposed to DMEM media containing 5.5mM,11.1mM and 25mM glucose for 1d,3d and 5d.On the following experiment day,the secretion and gene expressions of glucagon were measured and we also detected the glucagon secretion when rat islets and TC1-6 cells cultured for 5 d stimulating with three different concentrations of insulin.Then we used Western Blot method to confirm the effect of high glucose on phosphorylated of Akt in rat islets and TC1-6 cells.Result（1） There was no significant difference in glucagon secretion in response to the three glucose levels in the rat islets or in TC1-6 cells at the end of one day’s high glucose exposure,At day 3,compared with the control group（5.5 mM）,the glucagon secretion in rat islets exposed to 25 mM glucose was markedly increased by 42%（2252.64±41.71 vs 3205.97±45.53 pg/mg protein;P<0.05）,but there was no difference in TC1-6 cells.After 5d exposure,compared with the control group（5.5mmol/l）,the glucagon secretion in rat islets was increased by 52%in response to 11 mM glucose（2452.22±67.89 vs 3735.44±54.89 pg/mg protein;P<0.05） and by 117%in response to 25 mM glucose （2452.22±67.89 vs 5320.20±398.51 pg/mg protein;P<0.05）.However,with respect to TC1-6 cells,compared with 5.5 mM glucose,glucagon secretion was increased by 74% at day 5 in response to 25 mM glucose（78.62±4.72 vs 136.8±10.94 ng/10⁶ protein;P< 0.05）.（2） Acute（1 day） exposure of TC1-6 cells and rat islets to high glucose had no effect on glucagon mRNA levels measured by RT-PCR relative to cells and islets incubated with 5.5 mM glucose（control group）.However,at day 3,the glucagon mRNA levels of rat islets incubated with 25 mM glucose increased by 125%compared with the control group（P<0.05）,whereas there was little change in glucagon mRNA levels in TC1-6 cells.Meanwhile,at day 5,the glucagon mRNA levels of rat islets and TC1-6 cells exposed to 25 mM glucose increased by 272%and 78%,respectively,relative to the control groups（both:P<0.05）.（3） 10^-7mmol/L insulin can significantly inhibit glucagon secretion from rat islets and TC1-6 cells cultured with containing 5.5mM glucose by 60% （2315.02±48.12 vs 915.99±32.54 pg/mg protein;P<0.05） and 61%（55.12±3.86 vs 21.59±1.30ng/10⁶ protein;P < 0.05）,accordingly,but just can slightly inhibit glucagon secretion both in rat islets and TC1-6 cells containing 25mM glucose（P>0.05）.when insulin concentration added to 10^-5 mmol/L,glucagon secretion of rat islets and TC1-6 cells cultured with 25mM were both suppressed by 66%（3872.34±29.66 vs 1307.65±23.34 pg/mg protein;P<0.05）and 61%（118.61±10.68 vs 46.55 + 3.72 ng/10⁶ protein;P<0.05）;（4） when 10^-5mmol/L insulin were administrated for 2h,the phosphorylated ser473-Akt protein levels of rat islets and TC1-6 cells exposure to 5.5mM glucose increased by 200%and 180%accordingly（P<0.05）,those of rat islets and TC1-6 cells exposure to 25mM glucose also increased by 137%and 70%in response to 5.5mM after 5d（P<0.05 ）.While pretreatment with 10^-5mmol/L wortmannin, phosphorylated ser473-Akt protein expressions of rat islets and TC1-6 cells exposure to 5.5mM glucose and 25mM glucose were both down regulated（P<0.05）,but the inhibition in low high group is more significant than high glucose group.Conclusion Chronic glucose toxicity can cause hypersecretion of glucagon,which may be due toαcell insulin resistance induced by impaired activity of the a cell insulin signaling pathway mediated by glucose toxicity. PartⅡThe role of Syntaxin1A/SNAP-25 proteins in reversing hyperglycemia-induced a cell toxicityObjective To examine the effects of reversing hyperglycemia-induced a cell toxicity and explore the possible mechanisms.Method TC1-6 cell were cultured 10 days in media containing 5.5mmol/L glucose（Low glucose groups,LG）,10 days in media containing 25mmol/L glucose（High glucose groups,HG）,5 days in 25mmol/L glucose plus 5 days in 5.5mmol/L glucose（HL groups）, 5 days in 5.5mmol/L glucose plus 5 days in 25mmol/L glucose（LH groups）,The secretion and gene expression of glucagon were measured,we also used Western Blot analysis to confirm the effects of high glucose and reversible glucose induced effects on the expression of Snare proteins（syntaxinl A and SNAP-25）.Result（1）compared with TC1-6 cells of HG group,HL group cells Glucagon secretion dropped by 29%（P<0.05）;（2）compared with TC1-6 cells of HG group,HL group cells Glucagon mRNA was decreased by 52.6%±2.8%（P<0.05）;（3）compared with TC1-6 cells of LG group,the expressions of syntaxin1A and SNAP-25 of HG group cells were increased by 36%and 69%,and HL group cells were decreased by 49%and 56% respectively（P<0.05）;Conclusionα-cells abnormal glucagon were ameliorated after removing glucose toxicity, also the expressions of syntaxinlA and SNAP-25 proteins which regulating secretion of glucagon were reduced accordingly.更多还原