【作者】 吴志香；

【导师】 刘国良；

【作者基本信息】 中国医科大学 ， 内科学， 2007， 博士

【摘要】 目的本研究通过对不同浓度葡萄糖和高浓度游离脂肪酸协同作用一定时间的β细胞的胰岛素基因表达及分泌功能的检测，β细胞凋亡的形态学检测及凋亡相关基因表达来观察β细胞功能障碍的表现模式。并进一步对功能受损的β细胞进行分子生物学检测，通过观察游离脂肪酸在不同浓度葡萄糖协同作用下对PI-3K、P-PKB、PDX-1活性及表达的影响来揭示这种协同作用导致β细胞功能损伤的分子机制。方法Wistar大鼠胰岛β细胞原代培养及β细胞功能损伤模型的建立采用明尼苏达大学改良方法，原位灌注消化、梯度密度离心分离纯化胰岛。经DTZ特异染色鉴定并过夜孵育后，将细胞分为九组：分别给予A组：5.5mM Glucose；B组：5.5mM Glucose，0.4mM PA；C组：5.5mM Glucose，0.4mM OA；D组：11mM Glucose；E组：11mM Glucose，0.4mM PA；F组：11mM Glucose，0.4mM OA；G组：25mM Glucose；H组：25mM Glucose，0.4mM PA；I组：25mM Glucose，0.4mM OA：的系列浓度孵育，在孵育24h、48h后，各取一组细胞(n=6)用放免法测定在16.7mM葡萄糖刺激后1h时培养液上清中的胰岛素含量。用半定量RT-PCR检测上述七组细胞在16.7mM葡萄糖刺激后5min的细胞内胰岛素mRNA水平(n=6)。利用Western技术检测上述九组细胞24h、48h时PI-3K的p85亚单位的蛋白含量以及磷酸化的PKB的蛋白含量(n=4)。半定量RT-PCR检测九组细胞24h、48h时bax、myc以及PDX-1的mRNA表达量(n=6)。Western免疫印迹分析检测PDX-1的蛋白表达量(n=4)。间接免疫荧光染色显示PDX-1在不同状态下细胞内的原位表达。结果胰岛细胞收获量、纯度及成活率分别为438±27个胰岛／胰腺：87.68±3.2％：92.3±2.3％。胰岛细胞的胰岛素分泌功能：培养的胰岛细胞在基础条件下和16.7mM的葡萄糖刺激1h后胰岛素分泌量为39.74±2.73mM，128.94±8.72mM；SI为3.25±0.26，说明胰岛功能良好。经不同糖和游离脂肪酸糖浓度孵育后实验组胰岛细胞在16.7mM的葡萄糖1h后的胰岛素分泌量：在培养24h后，对照组两个时相分泌量在高浓度组(G组)有增高趋势，组间无明显差别(p＞0.05)；在油酸存在时，随着葡萄糖浓度的增高，基础时相分泌量组间比较无明显差别，刺激相分泌量有下降趋势，但无统计学意义；在软脂酸存在时，随着葡萄糖浓度的增高，基础分泌量及刺激后分泌量均明显下降(p＜0.01)。在培养48h后不同葡萄糖浓度孵育的两个时相分泌量组间无明显差别(p＞0.05)；在油酸存在时，随着葡萄糖浓度的增高，两个时相分泌量组间比较均有下降(p＜0.05)；在软脂酸存在时，随着葡萄糖浓度的增高，两个时相的分泌量组间比较下降更明显(p＜0.01)。同一浓度葡萄糖水平时，在培养24h后油酸组两时向下降无统计学意义(p＞0.05)；软脂酸组两时向均明显下降(p＜0.01)。在培养48h后油酸组刺激后时相分泌量减少(p＜0.05)；软脂酸组两时向差别显著(p＜0.01)各组细胞在16.7mM葡萄糖刺激5min时的胰岛素mRNA表达：在培养24h及48h后，葡萄糖对照组胰岛素mRNA表达组间无明显差别(p＞0.05)；油酸组培养24h胰岛素mRNA表达组间无明显差别(p＞0.05)，培养48h后mRNA表达(C、I)组间比较存在统计学差异(p＜0.05)；软脂酸组培养24h时胰岛素mRNA表达量下降(p＜0.05)，培养48h后胰岛素mRNA表达量下降明显(p＜0.01)。各组细胞在不同时间凋亡的原位缺口末端标记技术(TUUNEL)检测：在培养24h及48h后，葡萄糖对照组胰岛细胞凋亡数量无明显差异(p＞0.05)，油酸组培养24h凋亡细胞数略有增加，组间比较无统计学差异，48h凋亡细胞数组间比较C组、H组与I组存在差异(p＜0.05)，软脂酸组24h细胞即出现明显的凋亡，组间比较均存在差异(p＜0.05)。48h后凋亡更加明显，组间比较均存在差异(p＜0.01)。各组细胞在不同时间凋亡相关基因bax、myc的mRNA表达：在培养24h及48h后，葡萄糖对照组bax的mRNA几乎不表达，油酸组少量表达，组间无明显差别(p＞0.05)，软脂酸组培养24h时bax的mRNA表达量上升(p＜0.05)，培养48h后bax mRNA表达量增加明显(p＜0.01)。葡萄糖对照组培养24h及48h后及油酸组24h时myc的mRNA表达组间无明显差别(p＞0.05)，油酸组48h时myc mRNA表达开始下降(p＜0.05)，软脂酸组培养24h时myc的mRNA表达量下降(p＜0.05)，培养48h后myc的mRNA表达量下降明显(p＜0.01)。各组细胞在不同时间PI3K、磷酸化PKB／Akt、PDX-1蛋白的表达：在培养24h及48h后，葡萄糖对照组组间无明显差别(p＞0.05)，油酸组随时间的延长及葡萄糖浓度的增高PI3K、磷酸化PKB／Akt及PDX-1蛋白的表达有下降趋势，组间比较48h时C组与I组存在差异(p＜0.05)，软脂酸组随时间的延长及葡萄糖浓度的增高PI3K、磷酸化PKB／Akt及PDX-1蛋白的表达明显下降，组间比较均存在明显差异(p＜0.01)。各组细胞在不同时间PDX-1基因mRNA水平的表达：A、D、G组培养24h及48h的PDX-1基因mRNA水平均无统计学差异(p＞0.05)，B、E、H组培养24h的PDX-1基因mRNA表达减少，存在统计学差异(p＜0.05)，48h的N／C值组间比较统计学差异显著(p＜0.01)。C、F、I组培养24h的PDX-1基因mRNA水平有下降趋势，但无统计学差异(p＞0.05)，48h的PDX-1基因mRNA水平组间比较C组与I组存在统计学差异(p＜0.01)。各组细胞在不同时间PDX-1蛋白的核转位表达：细胞免疫荧光结果显示，在培养24h及48h后，对照组在基础状态下均在胞浆内有少量表达，葡萄糖刺激后几乎全部在核内表达。为量化这种核转位程度，每组计数100个细胞并分别记数有荧光信号在核内表达的细胞数N及荧光信号在核周围表达的细胞核数C，比较各组的N／C值。结果为：A、D、G组培养24h及48h的N／C值均无统计学差异(p＞0.05)，B、E、H组培养24h的N／C值减少，存在统计学差异(p＜0.05)，48h的N／C值组间比较统计学差异显著(p＜0.01)。C、F、I组培养24h的N／C值下降趋势，但无统计学差异(p＞0.05)，48h的N／C值组间比较存在统计学差异(p＜0.01)。结论高浓度游离脂肪酸培养可使β细胞的功能受损，表现为基础胰岛素水平分泌降低，葡萄糖刺激后胰岛素的合成及分泌能力下降。导致β细胞的葡萄糖刺激后的胰岛素基因表达减少，这种损伤随葡萄糖浓度升高和时间的延长而加重。高浓度游离脂肪酸使β细胞出现胰岛素的合成功能损害，同时亦发生胰岛素的分泌功能障碍。在同一浓度水平上，饱和脂肪酸对胰岛细胞功能的损伤大于不饱和脂肪酸，并且这种损伤随着葡萄糖浓度增高和时间延长而加重。高浓度软脂酸可通过抑制PI3K信号转导蛋白的表达，进而降低磷酸化PKB／Akt、PDX-1蛋白的表达引起胰岛细胞自身的胰岛素抵抗，这种损伤作用随葡萄糖浓度增高而加强。在同一浓度水平上饱和脂肪酸(软脂酸)对PI3K信号转导蛋白的抑制明显大于不饱和脂肪酸(油酸)，提示对β细胞“脂毒性”而言饱和脂肪酸大于不饱和脂肪酸。高浓度软脂酸可通过抑制PI3K信号转导蛋白的表达，使之抗凋亡作用减弱，进而导致促凋亡基因的表达上调，抗凋亡基因表达下调，从而发生胰岛细胞凋亡，并随葡萄糖浓度升高凋亡数量增加。更多还原

【Abstract】 ObjectiveGlucose is the primary factor for insulin secreted by pancreaticβ-cells.Short-term exposure ofβ-cells to high concentration of glucose triggers the synthesis and secretion of insulin. But,long-term effection of high concentration of glucose would causeβ-cells dysfunction.In typeⅡdiabetes mellitus,the concentration of free fatty acid (FFA) and triglycerine(TG) is generally high, especially in obesity.Long-term exposure ofβ-cells to high concentration of FFA will induceβ-cell ’ s apoptosis.Many studies have shown that glucose and FFA effect synthesis and secretion of insulin stimulated by the insulin secreted byβ-cells trigged by themselves.AS similar of surrounding target tissues of insulin,there are insulin recrptors and the series of proteins of insulin sinaling cascade inβ-cells,of which the phosphatidylinositol-3kinase (PI-3K)pathway is mainly involved in insulin gene transcription and secretion.Insulin up-regulates itself’s gene transcription by activation of transcription factor such as PKB/Akt、PDX-1 in PI3K pathway.Inhibition of PI3K activation deregulate activation of transactive factor of insulin gene which cause decreasing of insulin gene expression stimulated by insulin.We have a hypothsis that the mechanism of glutoxic and lipotocxic effection is related to decrease of PI3K activation and to deregulation of PDX-1 activation,which caused reducing of insulin synthesis,secretion and promoted apoptosis.Our study will observe the expression model ofβ-cells dysfunction by evalue ofβ-cells function after exposed to high concentration of FFA synergize with elevated glucose.Then we analyse dysfunction by morphology technology of apoptosis, insulin secretion detection and molecular biological technology to reveal molecular mechanism ofβ-cells dysfunction caused by high concentration of FFA synergize with elevated glucose.MethodWistar rat islets isolation and primary culture and establishment ofβ-cells dysfunctional model.Islets were isol;ated and purified according to modified Minnesota program.After intraductal infusion digestion,the islet were purifed by discontinous Ficoll density gradient (25%,23%,20.5%,11%).The degree of purity was defined as dithizone (DTZ) staining.Cells were cultured overnight and were divided into nine groups: A: 5.5mM Glucose; B: 5.5mM Glucose, 0.4mM PA; C: 5.5mM Glucose, 0.4mM OA; D: 11mM Glucose; E: 11mM Glucose, 0.4mM PA; F: 11mM Glucose,0.4mM OA; G: 25mM Glucose;H: 25mM Glucose,0.4mM PA;I: 25mM Glucose, 0.4mM OA.After 24h and 48h, to detect insulin level in the supernatant after sitimulated 1h by 16.7mM glucose with RIA method and to assay insulin mRNA level with semi-quantity RT-PCR(n=4)Protein levels of p85 subunit of PI3K、phosphorylation of PKB was determined in total lysates by western immunoblot. To determined mRNA levels of bax、myc and PDX-1 by RT-PCR at the end of 24h,48h cell culture. Western biot analysis was used to assay the protein expression levels of PDX-1 in different time.Indirect immunofluorescence and immunocytochemistry staining was processed to observe the translocation of PDX-1 in selets treated with 16.7mM glucose after cultured by high concentration of FFA synergize with elevated glucose.Result438±27 islets were procured from one rat.the degree of purity was 87.68±3.2%. Assessment of islets viability by Trypan Blue Dye Exclusion Test which results is 92.3±2.3%.Insulin secretin response to glucose challenge in vitro showed the mean value of insulin in the low-glucose medium was 39.74±2.73mM, while that of high-glucose medium was 128.94±8.72mM, the SI was 3.25±0.26, that means the islets functioned well.The releasing insulin levels of the nine groups responding to 16.7mM glucose at the end of 24h and 48h: At the end of the first 24h, the controls (A, D, G)secretion were similar in two phases, while the G group has increasing tendency;As for oleate groups, the base secretion have no difference,while the stimulated secretion was descending;As palmic acid as concerned,both base and sitimulated secretion was descending(p＜0.01) accompany with the elevated glucose. At the end of 48h, there was no difference in the controls (A, D, G)secretion;As for oleate groups, both base and sitimulated secretion was descending(p＜0.05) accompany with the elevated glucose;As palmic acid as concerned,the descending tendency was sharply (p＜0.01).In the same concentration of glucose, the oleate group has no statistical sense (p＞0.05) in the end of 24h; while the palmic acid group shew decreasing tendency (p＜0.05).At the end of 48h, both the oleate group and the palmic acid groups were decreased.Semi-quantitative RT-PCR revealed the insulin mRNA levels of the control groups were significant difference neither in the first 24h nor in the end of 48h; The oleate groups shew no significant difference during the first 24h (p＞0.05), while there was statistical sense between C、F groups at the end of 48h (p＜0.05);the palmic acid groups were decreased early in 24h (p＜0.05),and decreased sharply after 48h (p＜0.01).In situ end labelling technique (TUUNEL) for apoptosis: There was no statistical sense in the control groups (p＞0.05),apoptosis cell population was increasing in oleate groups in 24h,and had distinction between C、H and I (p＜0.05)after 48h; while obviously apoptosis happened in the palmic acid groups (p＜0.01)in 24h,and even more after 48h(p＜0.01).The mRNA expression of Apoptosis related gene bax、myc in control groups and experiment groups: the control groups were significant difference neither in the first 24h nor in the end of 48h; The oleate groups shew no significant difference during the first 24h (p＞0.05), while there was statistical sense between C、F groups at the end of 48h (p＜0.05);the palmic acid groups were decreased early in 24h (p＜0.05),and decreased sharply after 48h (p＜0.01).the expression of protein PI3K、phosphorylated-PKB、PDX-1: The control groups were significant difference neither in the first 24h nor in the end of 48h; The oleate groups shew decreasing tendency with the eleveated glucose and time extending, difference during the first 24h (p＞0.05), while there was significant statistical sense between C group and I group at the end of 48h (p＜0.05);the palmic acid groups were decreased early in 24h (p＜0.05),and decreased sharply after 48h (p＜0.01).The mRNA expression of PDX-1 in different groups: There was no significant difference in control groups neither in the first 24h nor in the end of 48h; The oleate groups shew no statistics difference during the first 24h (p＞0.05), while there was statistical sense between C and I groups at the end of 48h (p＜0.05);While B、E、H groups were decreased early in 24h (p＜0.05),and decreased sharply after 48h (p＜0.01).Nuclear translocation of protein PDX-1 in different groups: Indirect immunofluorescence showed PDX-1 was mainly located in cytosolic slightly in the basical station, while translocated to nucleoplasmic greatly followed by stimulated with 16.7mM glucose. With the elevated glucose and time extending,the expression was lower and the translocating rates was decreased,which was Hgroup＜Egroup＜Bgroup,(p＜0.01 or 0.05).ConclusionShort-term exposure ofβ-cells to high concentration of glucose triggers the synthesis and secretion of insulin.But co-cultivated with FFA will damageβ-cells function, which shew that the synthesis and secretion ability of insulin descended both in base phase and stimulated phase. High concentration of FFA induced insulin mRNA level decreased, which lower accompany with the elevated glucose and time prolongation.High concentration of FFA induced insulin synthesis dysfunction before secretion impairment. In the same concentration level, impairment caused by saturated fatty acid exceeded those caused by unsaturated fatty acid, which increased accompany with the elevated glucose and time prolongation.High concentration of FFA inhibited the expression of phosphorylated-PKB protein and PDX-1protein by inhibiting the expression of PI3K signal transducer, which strengthened accompany with the elevated glucose and time prolongation. In the same concentration level, impairment caused by saturated fatty acid exceeded those caused by unsaturated fatty acid, which indicated that "lipotoxic"caused by saturated fatty acid was more harmful.High concentration of FFA up-regulated expression of apoptosis gene,which would promoteβ-cells apoptosis by inhibiting the expression of PI3K signal transducer, which strengthened accompany with the elevated glucose and time prolongation.更多还原