【作者】 项靖楠；

【导师】 杨立勇；

【作者基本信息】 福建医科大学 ， 内科学， 2012， 博士

【摘要】 目的用不同浓度棕榈酸（PA）干预小鼠胰岛细胞瘤系βTC6细胞不同时长，并加用c-jun氨基末端激酶（JNK）特异性抑制剂SP600125，观察JNK信号转导通路关键蛋白JNK及其下游底物c-jun磷酸化情况，以及胰腺衍生因子（PANDER）的表达变化，探讨PA对β细胞PANDER表达的影响及JNK信号转导通路在其中的作用；建立小干扰RNA（siRNA）沉默PANDER表达，观察βTC6细胞PANDER沉默前后脂性凋亡的变化以及凋亡关键分子caspase-3的活性变化，探讨PA作用下PANDER上调对β细胞脂性凋亡的影响及可能机制；加用胰高血糖素样肽-1（GLP-1）受体长效激动剂Exendin-4处理细胞，观察Exendin-4对PA作用下βTC6细胞凋亡及PANDER表达的影响，以及在这一过程中JNK信号通路和caspase-3的活性变化，探讨Exendin-4拮抗β细胞脂性凋亡的可能机制，为临床新药开发提供一定的实验和理论依据。第一部分棕榈酸对βTC6细胞PANDER表达的影响方法（1）分别以不同浓度PA（0，0.125，0.25，0.5或1.0mmol/L）干预βTC6细胞不同时长（0，6，12，24或48h），用实时荧光RT-PCR法及Westernblot法检测PANDER mRNA及蛋白的表达变化；（2）以0.5mmol/L的PA干预βTC6细胞0，0.1，0.25，0.5，1，6，12和24h，应用Western blot法，使用能特异识别JNK、c-jun的磷酸化位点的抗体来检测JNK, c-jun的磷酸化水平，从而说明这两种蛋白的活化程度；（3）将βTC6细胞分为四组，分别为对照组、PA组（0.5mmol/L的PA干预24h）、SP600125组（25umol/L SP600125处理细胞24h）和PA+SP600125组（25umol/L SP600125预处理细胞1h，再予25umol/LSP600125+0.5mmol/L PA处理细胞24h），应用Western blot法检测PANDER、p-JNK、JNK、c-jun、p-c-jun的变化。结果（1）PA可上调βTC6细胞PANDER表达，在mRNA水平，PA的作用高峰为0.5mmol/L，干预12h；而在蛋白水平，PA的作用高峰出现在0.5mmol/L，干预24h。（2）PA可致JNK通路活化，且有两个高峰，分别出现在PA干预6min及12h。（3）应用SP600125可有效抑制PA诱导的JNK及其下游c-jun磷酸化，且PA诱导的PANDER表达随之降低。结论PA通过活化JNK信号转导通路上调β细胞PANDER表达。第二部分PANDER在PA所致的βTC6细胞脂性凋亡中的作用方法（1）根据小鼠PANDER基因（GenBank Accession Number:52793）的序列，选择6个位点，于体外设计并合成siRNA。评估转染效率后，以Lipofectamine2000作为载体进行瞬时转染。同时设置空白对照组（Blank Control，BC），阴性对照组（Negative Control，NC）以及转染试剂组暴露组（mock）。应用实时荧光PT-PCR及Western blot法检测各组PANDER mRNA及蛋白表达情况。（2）将βTC6细胞分NC组，siRNA组，NC+PA组及siRNA+PA组进行干预。PA的干预浓度和时间根据第一部分实验结果选择，为0.5mmol/L，24h。用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法（terminaldexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling，TUNEL）和Annexin V-FITC-PI双染，流式细胞术检测细胞凋亡。用Western blot法检测caspase-3蛋白水解后的两个亚单位，评估caspase3活化程度。结果（1）应用序列Fam3b-mus-208可抑制PANDER基因表达，与NC组相比，在mRNA水平，抑制率达78%，在蛋白水平，抑制率约为60%，可用于后续实验（；2）PA干预后，NC组细胞caspase-3活化，凋亡显著增加；转染PANDERsiRNA的细胞caspase-3活化减少，细胞凋亡率有所降低。结论PA可能通过上调PANDER表达进而活化caspase-3诱导胰岛β细胞凋亡。第三部分Exendin-4对βTC6细胞脂性凋亡的保护作用方法（1）以浓度逐渐升高的Exendin-4（12.5，25，50或100nmol/L）预处理βTC6细胞24h，再予相应浓度的Exendin-4联合0.5mmol/L PA共同干预βTC6细胞24h，用Western blot法检测细胞PANDER蛋白表达。（2）将βTC6细胞分对照组、Exendin-4组、PA组及Exendin-4+PA组进行干预。PA的干预浓度和时间根据第一部分实验结果选择，为0.5mmol/L，24h。Exendin-4的干预浓度根据本部分第一小节实验结果选择，为50nmol/L，预孵育细胞24h后再与0.5mmol/L PA共同干预24h。用TUNEL法和Annexin V-FITC-PI双染，流式细胞术检测细胞凋亡。用Western blot法检测细胞PANDER、p-JNK、JNK、c-jun、p-c-jun及cleaved caspase-3表达的变化。结果（1）Exendin-4浓度依赖性降低PA诱导的βTC6细胞PANDER表达。（2）0.5mmol/L PA处理βTC6细胞24h后，p-JNK、PANDER表达增多，caspase-3活化，细胞凋亡明显增多；加用Exendin-4可一定程度抑制PA诱导的JNK活化，进而下调PANDER表达，减少caspase-3活化，拮抗脂性凋亡。结论Exendin-4可能部分通过抑制JNK-PANDER-caspase-3通路，拮抗β细胞脂性凋亡更多还原

【Abstract】 Objective The mouse insulinoma cell line βTC6cells were treated withdifferent concentration of palmitic acid (PA) for different times, and the c-junNH2-terminal kinase (JNK)-specific inhibitor, SP600125was added to observe theexpression of PANDER as well as the level of phosphorylated JNK and its substrateprotein c-jun, to explore the effects of PA on PANDER expression in β cells and therole of JNK signaling pathway plays on this process. A small interfering (si)RNA wasconstructed to interfere with the PANDER expression, the PA induced β-cell apoptosisand the activation of a major apoptotic molecule, cysteine containing aspartatespecific protease(caspase)-3, were measured to investigate the relationship betweenPANDER and PA mediated β-cell apoptosis. βTC6cells were pretreated withExendin-4and then treated with combination of PA and Exendin-4to observe theeffects of Exendin-4on PA-induced apoptosis and PANDER expression, to determinethe possible mechanism of Exendin-4on protecting β cells from lipoapoptosis.Part One The effects of PA on the expression of PANDER in βTC6cellsMethods (1) βTC6cells were treated with different concentration of PA (0，0.125，0.25，0.5或1.0mmol/L) for different times (0，6，12，24或48h), the expressionof PANDER mRNA and protein were detected by quantitative Real Time RT-PCR andWestern blotting respectively.(2) βTC6cells were treated with0.5mmol/L of PA for0，0.1，0.25，0.5，1，6，12and24h, the expression of JNK, p-JNK, c-jun, p-c-junwas detected using Western blotting.(3) βTC6cells were divided into four groups:control (treated with fresh medium), PA (treated with0.5mmol/L of PA for24h),SP600125(treated with25umol/L of SP600125for24h) and PA+SP600125 (pretreated with25umol/L of SP600125for1h, and then treated with thecombination of PA and SP600125for24h). The expression of PANDER, JNK, p-JNK,c-jun and p-c-jun was detected using Western blotting.Results (1) PANDER expression could be enhanced when the cells werestimulated with higher concentration of PA for increasing lengths of time. The peakvalue of PA-induced PANDER mRNA expression appeared at0.5mmol/L with12hincubation period as well as0.5mmol/L with24h incubation period at the proteinlevels of PANDER expression.(2) JNK could be actived by PA, and the peak valueappeared at6min and12h.(3)24-h exposure to PA significantly increased thephosphorylation of JNK, pretreatment of βTc6cells with SP600125reducedPA-induced PANDER expression.Conclusions PA up-regulates PANDER expression in β-cells through theactivation of JNK signaling pathway.Part Two The contribution of PANDER to PA-induced apoptosis in βTC6cellsMethods (1) Six siRNAs were designed and synthesized in vitro according tothe sequence of mouse PANDER gene(GenBank Accession Number:52793). ThesiRNAs were transfected respectively into βTC6cells using Lipofectamine2000. Cellscultured in medium (Blank Control, BC) or transfected with Negative siRNA(Negative Control, NC) or exposured to Lipofectamine2000(mock) served as thecontrol. The expression of PANDER mRNA and protein were detected by quantitativeReal Time RT-PCR and Western blotting respectively.(2) βTC6cells were dividedinto four groups: NC, siRNA, NC+PA and siRNA+PA. The concentration and theintervention time of PA were chosen according to the results of the part-oneexperiment of this study (0.5mmol/L of PA for24h). The apoptosis of βTC6cellswere detected by the terminal dexynucleotidyl transferase (TdT)-mediated dUTP nickend labeling(TUNEL) and the flow cytometric analysis.The cleaved caspase-3wasdetected by Western blotting to assess the activated level of caspase-3.Results (1) βTC6cells transfected with PANDER siRNA(Fam3b-mus-208) caused about72%decrease in the expression of PANDER mRNA as well as about60%decrease at the protein level of PANDER when compared with NC.(2) The24-hexposure to PA significantly increased the level of cleaved caspase-3as well as thepercentage of apoptotic cells in NC, whereas after24-h exposure to PA, the level ofcleaved caspase-3and apoptosis rate in PANDER siRNA transfected cells was lessthan that in NC transfected cells.Conclusions PANDER may play a role in β-cell lipoapoptosis throughactivating caspase-3.Part three The protective role of Exendin-4against βTC6-cell lipoapoptosisMethods (1) βTC6cells were pretreated with increasing concentration ofExendin-4(0,12.5,25,50and100nmol/L) for24h, and then treated withcombination of PA and Exendin-4for24h, the expression of PANDER wasdetected by Western blotting.(2) βTC6cells were divided into four groups: control(treated with fresh medium), Exendin-4(treated with50nmol/L of Exendin-4for24h)PA(treated with0.5mmol/L of PA for24h), and Exendin-4+PA (pretreated with50nmol/L of Exendin-4for24h, and then treated with the combination of PA andExendin-4for24h). The concentration and the intervention time of Exendin-4werechosen according to the results of the previous experiment of this part. The apoptosisof βTC6cells were detected by TUNEL and the flow cytometric analysis. Theexpression of PANDER, JNK, p-JNK, c-jun, p-c-jun and cleaved caspase-3wasdetected using Western blotting.Results (1) Exendin-4decreased PA-induced PANDER expression in adose-dependent manner in βTC6cells.(2) The PA-induced toxicity could be partlyprevented in the presence of Exendin-4, and24-h exposure to PA significantlyincreased the expression of p-JNK, p-c-jun, PANDER and cleaved caspase-3, whereasthese enhancements could be partly weakened by the addition of Exendin-4in βTC6cells.Conclusions The reverse role of Exendin-4on lipapoptosis of β-cells may bepartly related with interfering JNK-PANDER-caspase-3pathway.更多还原