【作者】 汤云昭；

【导师】 于德民； 李代清；

【作者基本信息】 天津医科大学 ， 内科学， 2010， 博士

【摘要】 目的早期研究显示在小鼠胰岛B细胞中有一种与P糖蛋白(也称为多药耐药蛋白1, Multidrug Resistantance Protein 1, MDR1)相似的65kDa大小的膜蛋白,且借助膜片钳技术发现此蛋白有类似磺脲类受体的功能,可促进胰岛素颗粒释放。本课题目的首先探测大鼠胰岛β细胞中mini-P糖蛋白存在的可能性,并使用拮抗剂及RNA干扰技术进行该蛋白的功能研究,分析其在胰岛素两相分泌中的作用。方法(1)经胆管不离体灌注胶原酶V消化分离Wistar大鼠胰岛；INS-1细胞培养。分别提取大鼠胰岛、胰腺及INS-1细胞中的总RNA,针对MDR1特异基因序列abcblb的3’末端及5’末端设计多对引物,进行RT-PCR;萃取大鼠胰岛及INS-1细胞蛋白,以MDR1特异性抗体(C219)进行免疫印迹实验厂探测目的蛋白的存在。(2)针对P糖蛋白特异基因序列(abcblb)设计寡核苷酸序列,使用siRNA干扰技术孵育胰岛；转染效率的观察分别采用荧光显微镜观察,real-time PCR法测定目的基因的表达量及Western Blot去测定蛋白表达量的变化；进行葡萄糖刺激的胰岛素释放实验,采用放免法测定RNA干扰后大鼠胰岛的胰岛素两相分泌的变化。(3)建立高糖培养大鼠胰岛凋亡的模型,采用real-time PCR法测定siRNA干扰后,凋亡基因表达的变化,评价RNA干扰对于胰岛凋亡的影响,进一步分析目的蛋白在胰岛素两相分泌中的作用。(4)使用MDR1特异性拮抗剂(环孢菌素A)干预大鼠胰岛,放免法测定胰岛素的两相分泌。结果(1)RT-PCR实验提取的大鼠胰岛、胰腺及INS-1细胞中均扩增出了相应的目的片段,且在胰岛中的表达量较胰腺及INS-1细胞中表达量大。免疫印迹实验中,使用C219在胰岛匀浆蛋白中探测到65kDa大小的蛋白,在INS-1细胞蛋白萃取液中探测到了160kDa大小的蛋白。(2)在RNA干扰实验中,转染后48小时的胰岛于荧光显微镜下观察可见90%的胰岛均呈现红色荧光,提示转染成功；Western Blot实验结果提示在空白组、实验组及阴性对照组,使用C219均探测到了65kDa大小的蛋白,且实验组较空白组及阴性对照组蛋白表达量下调,而空白组及阴性对照无明显变化；siRNA干扰后abcblb基因的表达量实验组的较阴性及空白组减少,阴性组及空白组两组无变化；与abcblb基因相似的abcbla基因的表达量在三组中无变化,胰岛素合成相关基因insuin1、insulin2及胰高血糖素合成基因glucagon表达量无变化；siRNA干扰后,三组胰岛素分泌均呈现两相分泌,实验组的胰岛素一相及二相分泌较空白组及阴性对照组均受损。(3)高糖孵育后高糖组casp3及bax基因的表达量较正常组上调,高糖组Bcl-2基因的表达量较正常组下调,而Bcl-xl基因在两组中表达无差异；siRNA干扰后casp3 bax Bcl-2及Bcl-xl基因表达量在实验组、阴性对照组及空白组中的表达量均无变化。(4)在体外胰岛刺激实验中,MDR1拮抗剂环孢菌素A,抑制了胰岛素第二时相分泌,但胰岛素第一时相分泌无明显变化。结论(1)在INS-1细胞中有全长的P糖蛋白的表达,在大鼠胰岛中有mini-P糖蛋白的表达。P糖蛋白的表达不同,提示着二者可能具有不同的生物学功能。在INS-1细胞中,P糖蛋白可能主要与耐药相关,而在胰岛细胞中,mini的P糖蛋白可能主要与胰岛素的分泌释放相关。(2)siRNA干扰后,目的基因abcb16表达量下调,胰岛素的两相分泌均受损,而凋亡相关基因及胰岛素合成相关基因的表达无改变,提示siRNA干扰后未直接影响胰岛素的合成,且胰岛素的两相分泌受损并非通过凋亡途径,进一步支持了前述观点,P糖蛋白或mini-P糖蛋白影响胰岛素颗粒的酸化成熟,调节胰岛素的两相分泌。(3)使用环孢菌素A干预胰岛后,胰岛素的一相分泌未受影响,而胰岛素的二相分泌明显受抑制,推测其拮抗P糖蛋白或mini-P糖蛋白,从而影响胰岛素颗粒的酸化成熟,表现为胰岛素的二相分泌受损。(4)深入研究mini-P糖蛋白的结构及功能,可为新的降糖药物的研发提供理论基础,为糖尿病的治疗提供新思路。更多还原

【Abstract】 Objective A 65-kda mdrl (multi-drug resistance protein 1, P-glycoprotein)-like protein has been suggested to be a regulatory protein to chloride channel protein 3 (C1C-3) mediating insulin granules acidification and release in mouse beta cells. But the protein has not been deeply investigated. In this study, we identified existence of the 65-kda protein in rat islets and preliminarily explored its biological functions.Methods (1) Rat pancreas were digested by collagenase V through bile-duct injection and islets were isolated manually. A insulinoma cells (INS-1) were cultured. Total RNAs of the rat kidneys served as positive controls, pancreata, islets and INS-1 cells were extracted for reverse transcript PCR (RT-PCR), respectively. The cDNAs were run with specific primers selected from the mRNA of abcb1b encoding P-glycoprotein. All PCR products were visualized in agarose gel electrophoresis and sequenced. Homogenates of rat islets and INS-1 cells were applied to SDS-PAGE. P-glycoprotein was detected by a specific monoclonal antibody (C219). (2) Rat Islets were incubated with designed siRNAs to down-regulate expression of the abcb1b gene. Transfection efficiency was evaluated by fluorescence microscope, quantitative PCR and western blot. The transfected islets were incubated with high glucose (16.7mM) buffer and biphasic insulin release was measured by radioimmunology (RIA) assay. Meanwile the expression of ins1, ins2 and glucagon were also determined by quantitative PCR (3) Rat islets were incubated in RPMI1640 (with 33.3mM glucose) for 48 hours to induce apoptosis. The apoptosis were assessed both in the silenced islets and in the islets incubated with the 33.3mM glucose media by quantitative PCR. (4) Rat islets were treated by cyclosporine which is a specific inhibitor to P-glycoprotein. Comparison of insuling release between the treated islets and non-treated islets was carried out by RIA.Results (1) Compared with the positive control, expression of the P-glycoprotein mRNA segments were detected in the islets, INS-1 cells and pancreas. Sequence analysis confirmed the PCR products were matched with mRNA of P-glycoprotein. In western blot, a 65-kda protein was recognized by the antibody in the islets homogenate but not in that of INS-1 cells. Instead, the homogenate of INS-1 cells contained a 160-kda protein recognized by the antibody. (2)90% islets showed red-fluorescence 48 hours after the oligonucleotides (the silencers to the abc1b1 and the control oligonucleotides) transfection. The expression of 65kDa protein and the mRNA of abcb1b were down-regulated in the silenced islets. All of them suggested the RNAi was successful. Insulin secretion of rat islets stimulated by high glucose (16.7mM) showed biphasic curve during 60-min incubation. After the RNAi, both the first and the second phase of insulin secretion was reduced significantly. The expression of insl, ins2 and glucagon were not interfered with the RNAi. (3) Several key genes mediating apoptosis did not show any differences between pre and post-RNAi in the rat islets, whereas the apoptosis genes were changed significantly by the high glucose incubation accordingly. (4) After incubation with cyclosporine A, the second phase of insulin secretion was reduced significantly but the first phase was not influenced.Conclusion The 65-kda protein expressed in rat islets is most likely a mini-P-glycoprotein. It may play a key role regulating biphasic insulin release. The regulation may not induce by apoptosis or insulin synthesis. It might regulate the insulin granule acidification and the priming. Characterization of the structures and functions of the mini-P-glycoprotein may provide potentials to develop new anti-diabetic drugs.更多还原