慢性间歇性低氧和高脂饲养对兔胰岛细胞凋亡的影响研究
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摘要
目的观察并评价慢性间歇性低氧(CIH)和高脂饲养对胰岛细胞凋亡的影响及可能机制,同时分析慢性间歇性低氧与高脂饲养因素之间是否存在交互作用而影响胰岛细胞凋亡,为研究阻塞性睡眠呼吸暂停低通气综合征(OSAHS)与2型糖尿病的病理生理联系提供新的思路。
方法采用前瞻性动物实验,随机对照研究和CIH、高脂饲养二因素二水平析因设计。24只4月龄新西兰兔随机分为对照组(C组)、慢性间歇性低氧组(H组)、高脂饲养组(F组)、慢性间歇性低氧并高脂饲养组(H+F组),每组6只。H组、H+F组置于间歇低氧舱中每天5小时,每周期8分钟,舱内最低氧浓度8%;F组、H+F组饲以高脂食物;CIH和高脂饲养干预实验持续8周。分别于0、4、8周时给实验兔称体重,采血化验总胆固醇(TC)、低密度脂蛋白(LDL-CH)、游离脂肪酸(FFA)。干预8周后,经兔耳缘静脉注射空气处死。迅速解剖取出胰腺组织,一部分石蜡包埋后切片用TUNEL法检测胰岛细胞凋亡;一部分立即冻入液氮中保存,用RT-PCR法检测Caspase-3基因的mRNA水平改变,Western印迹法(Western blot)检测Caspase-3蛋白表达的变化,并进行半定量分析。组间两两比较采用LSD-t检验,用2×2析因分析评价CIH、高脂饲养两因素对TUNEL检测的胰岛细胞凋亡率、RT-PCR检测的Caspase-3基因mRNA表达和Western blot检测的Caspase-3蛋白表达各指标影响的主效应及交互作用。
结果1、四组动物血脂各指标两两比较:H+F组血FFA水平第8周为(0.75±0.35)mmol/L,高于0周(0.20±0.14)mmol/L和4周(0.27±0.13)mmol/L,差异有统计学意义。(P＜0.05)。
2、2×2析因分析显示CIH对TUNEL检测的胰岛细胞凋亡率、RT-PCR检测的Caspase-3基因mRNA表达和Western blot检测的Caspase-3蛋白表达各指标无显著影响,P值分别为0.22、0.90、0.27;高脂饲养对TUNEL检测的胰岛细胞凋亡率、RT-PCR检测的Caspase-3基因mRNA表达和Western blot检测的Caspase-3蛋白表达各指标均有影响,P值分别为0.01、0.03、0.03;慢性间歇性低氧与高脂饲养对上述3个指标不存在交互作用,P值分别为0.84、0.97、0.80。
结论
1、高脂饲养促进胰岛细胞发生凋亡,并可能通过Caspase-3信号通路促进胰岛细胞凋亡。
2、未发现慢性间歇性低氧及其与高脂饲养的联合作用促进胰岛细胞凋亡。
Objective:To investigate the effects of chronic intermittent hypoxia (CIH)and high-fat-feeding on the apoptosis of islet cells and related mechanism,and analyse the synergistic effect between CIH and high-fat-feeding.To present a new view for the pathophysiologic relationship of OSAHS and T2DM.
Methods:Using prospective animal experiment,randomized controlled study and factorial experiment,24 New-Zealand white rabbits(4 months old) were divided into the following 4 groups:control group(C),chronic intermittent hypoxia group(H),high-fat-feeding group(F)and hypoxia & high-fat-feeding group(H+F).Each group had 6 rabbits.Rabbits in group H and H+F were placed in a cabin into which nitrogen and air were periodically infused every 8 minutes for 5h/day.The lowest level of oxygen concentration was at 8%.Group F and H+F were fed with fat-rich-stoyer..The intervention experiment lasted for 8 weeks.Blood samples for measurements of total cholesterol(TC),low density lipoprotein cholesterol(LDL-CH)and free fatty acids(FFA)were collected and body weight was measured at 0,4W and 8W.After intervention of eight weeks, each rabbit was put to death by injecting air into ear-lip vein.Then the rabbits were dissected and the pancreata were picked quickly.One part was fixed in formaldehyde for the detection of the apoptosis of islet cells by TUNEL method;the other was preserved in liquid nitrogen immediately for Caspase-3 detection using RT-PCR and Western blot.All results of Caspase-3 expression were used for semi-quantitative analysis. Comparisons among groups were analysed by LSD-test.The effects analysis of two factors of CIH and high-fat-feeding on the apoptosis of islet cells, expression changes of Caspase-3 at mRNA and protein level were carried out using factor analysis.
Results:
1.Multiple comparisons of lipids.FFA level in group H+F at 8W was (0.75±0.35)mmol/L,which was the highest among the three time points (P＜0.05),vs(0.20±0.14)mmol/L(0W)and(0.27±0.13)mmol/L(4W).
2.The 2×2 factorial analysis showed no significant diffrence in the apoptosis of islet cells and Caspase-3 expression by CIH,as the P value of the apoptosis rate of islet cells,the expression level of Caspase-3 mRNA and the level of Caspase-3 protein were 0.22,0.90,and 0.27.The main effect of high-fat-feeding had statistic significance,as the P value of the apoptosis rate of islet cells,the expression level of Caspase-3 mRNA,the level of Caspase-3 protein were 0.01,0.03 and 0.03.The two factores had no significant synergistic effects on the apoptosis of islet cells,as the P value were 0.84,0.97 and 0.80.
Conclusion:
1.This study confirms that high-fat-feeding can induce the apoptosis of islet cells.It seems that the apoptosis may be by Caspase-3 pathway.
2.The combination effect of CIH and high-fat-feeding doesn't induce the apoptosis of islet cells.
方法采用前瞻性动物实验,随机对照研究和CIH、高脂饲养二因素二水平析因设计。24只4月龄新西兰兔随机分为对照组(C组)、慢性间歇性低氧组(H组)、高脂饲养组(F组)、慢性间歇性低氧并高脂饲养组(H+F组),每组6只。H组、H+F组置于间歇低氧舱中每天5小时,每周期8分钟,舱内最低氧浓度8%;F组、H+F组饲以高脂食物;CIH和高脂饲养干预实验持续8周。分别于0、4、8周时给实验兔称体重,采血化验总胆固醇(TC)、低密度脂蛋白(LDL-CH)、游离脂肪酸(FFA)。干预8周后,经兔耳缘静脉注射空气处死。迅速解剖取出胰腺组织,一部分石蜡包埋后切片用TUNEL法检测胰岛细胞凋亡;一部分立即冻入液氮中保存,用RT-PCR法检测Caspase-3基因的mRNA水平改变,Western印迹法(Western blot)检测Caspase-3蛋白表达的变化,并进行半定量分析。组间两两比较采用LSD-t检验,用2×2析因分析评价CIH、高脂饲养两因素对TUNEL检测的胰岛细胞凋亡率、RT-PCR检测的Caspase-3基因mRNA表达和Western blot检测的Caspase-3蛋白表达各指标影响的主效应及交互作用。
结果1、四组动物血脂各指标两两比较:H+F组血FFA水平第8周为(0.75±0.35)mmol/L,高于0周(0.20±0.14)mmol/L和4周(0.27±0.13)mmol/L,差异有统计学意义。(P＜0.05)。
2、2×2析因分析显示CIH对TUNEL检测的胰岛细胞凋亡率、RT-PCR检测的Caspase-3基因mRNA表达和Western blot检测的Caspase-3蛋白表达各指标无显著影响,P值分别为0.22、0.90、0.27;高脂饲养对TUNEL检测的胰岛细胞凋亡率、RT-PCR检测的Caspase-3基因mRNA表达和Western blot检测的Caspase-3蛋白表达各指标均有影响,P值分别为0.01、0.03、0.03;慢性间歇性低氧与高脂饲养对上述3个指标不存在交互作用,P值分别为0.84、0.97、0.80。
结论
1、高脂饲养促进胰岛细胞发生凋亡,并可能通过Caspase-3信号通路促进胰岛细胞凋亡。
2、未发现慢性间歇性低氧及其与高脂饲养的联合作用促进胰岛细胞凋亡。
Objective:To investigate the effects of chronic intermittent hypoxia (CIH)and high-fat-feeding on the apoptosis of islet cells and related mechanism,and analyse the synergistic effect between CIH and high-fat-feeding.To present a new view for the pathophysiologic relationship of OSAHS and T2DM.
Methods:Using prospective animal experiment,randomized controlled study and factorial experiment,24 New-Zealand white rabbits(4 months old) were divided into the following 4 groups:control group(C),chronic intermittent hypoxia group(H),high-fat-feeding group(F)and hypoxia & high-fat-feeding group(H+F).Each group had 6 rabbits.Rabbits in group H and H+F were placed in a cabin into which nitrogen and air were periodically infused every 8 minutes for 5h/day.The lowest level of oxygen concentration was at 8%.Group F and H+F were fed with fat-rich-stoyer..The intervention experiment lasted for 8 weeks.Blood samples for measurements of total cholesterol(TC),low density lipoprotein cholesterol(LDL-CH)and free fatty acids(FFA)were collected and body weight was measured at 0,4W and 8W.After intervention of eight weeks, each rabbit was put to death by injecting air into ear-lip vein.Then the rabbits were dissected and the pancreata were picked quickly.One part was fixed in formaldehyde for the detection of the apoptosis of islet cells by TUNEL method;the other was preserved in liquid nitrogen immediately for Caspase-3 detection using RT-PCR and Western blot.All results of Caspase-3 expression were used for semi-quantitative analysis. Comparisons among groups were analysed by LSD-test.The effects analysis of two factors of CIH and high-fat-feeding on the apoptosis of islet cells, expression changes of Caspase-3 at mRNA and protein level were carried out using factor analysis.
Results:
1.Multiple comparisons of lipids.FFA level in group H+F at 8W was (0.75±0.35)mmol/L,which was the highest among the three time points (P＜0.05),vs(0.20±0.14)mmol/L(0W)and(0.27±0.13)mmol/L(4W).
2.The 2×2 factorial analysis showed no significant diffrence in the apoptosis of islet cells and Caspase-3 expression by CIH,as the P value of the apoptosis rate of islet cells,the expression level of Caspase-3 mRNA and the level of Caspase-3 protein were 0.22,0.90,and 0.27.The main effect of high-fat-feeding had statistic significance,as the P value of the apoptosis rate of islet cells,the expression level of Caspase-3 mRNA,the level of Caspase-3 protein were 0.01,0.03 and 0.03.The two factores had no significant synergistic effects on the apoptosis of islet cells,as the P value were 0.84,0.97 and 0.80.
Conclusion:
1.This study confirms that high-fat-feeding can induce the apoptosis of islet cells.It seems that the apoptosis may be by Caspase-3 pathway.
2.The combination effect of CIH and high-fat-feeding doesn't induce the apoptosis of islet cells.
引文
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[2]Catskadon MA,Dement WC.Respiration during sleep in the aged human[J].J Gerontol,1981,36(4):420-423.
[3]刘建红,韦彩周,黄陆颖,等.广西地区打鼾及阻塞性睡眠呼吸暂停低通气综合征的流行病学调查[J].中华流行病学杂志,2007,28(2):115-118.
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