胰淀素受体在大鼠胰岛β细胞中的表达及其参与胰淀素细胞毒作用的研究  

Coexpression of amylin receptors in rat pancreatic β cells and its participation in cytotoxicity of amylin

作　　者：李晓通[1] 朱铁虹[1] 李常颖[2] 张婷[2] 畅继武[2] 

机构地区：[1]天津医科大学总医院内分泌科,300052 [2]天津医科大学第二附属医院天津市泌尿外科研究所

出　　处：《中华糖尿病杂志》2015年第4期250-256,共7页CHINESE JOURNAL OF DIABETES MELLITUS

基　　金：国家自然科学基金，天津市应用基础与前沿技术研究计划

摘　　要：目的：鉴定三种胰淀素受体在大鼠胰岛β细胞中的表达，探讨其在人胰淀素对β细胞的毒性作用中的影响。方法采用逆转录聚合酶链反应，免疫荧光方法检测大鼠胰岛素瘤细胞系INS-1和正常大鼠胰岛β细胞中胰淀素受体表达。人胰淀素孵育INS-1细胞24 h，实时定量逆转录聚合酶链反应及免疫荧光法检测胰淀素孵育对受体活性修饰蛋白（RAMP）mRNA转录及细胞表面表达的影响。在细胞毒性试验中，首先用胰淀素、胰淀素受体拮抗剂AC253单独或二者联合孵育INS-1细胞24～48 h，再用不同浓度RAMP1抗体与胰淀素联合孵育24 h，检测细胞存活率变化。组间数据比较采用t检验。结果逆转录聚合酶链反应、免疫荧光染色均证实三种胰淀素受体在大鼠胰岛β细胞中的表达。胰淀素孵育24 h使INS-1细胞RAMP2和RAMP3的mRNA分别下降至0.77±0.10（t=2.546，P〈0.05）和0.66±0.09（t=4.559，P〈0.05），而RAMP1的mRNA有轻度升高趋势，升至1.25±0.17（t=-3.084， P〈0.05），并且RAMP1在细胞表面分布增加。细胞毒性实验中，人胰淀素孵育24 h组细胞存活率为70%±13%，较对照组（100%±14%）明显下降（t=4.409，P〈0.05），胰淀素受体拮抗剂AC253与人胰淀素同时作用24 h组细胞存活率为94%±16%，较胰淀素单独孵育24 h组明显升高（t=-3.341，P〈0.05）。但对胰淀素作用48 h诱导的细胞死亡未有明显缓解。RAMP1受体在1∶500和1∶1000稀释度下与人胰淀素同时孵育24 h组的细胞存活率分别为94%±12%和96%±11%，较胰淀素单独作用组也有明显升高（t=-4.904、-5.565，P〈0.05），也可抑制人胰淀素的毒性作用，而同型IgG对人胰淀素作用无影响。结论本研究证实了三种胰淀素受体均在大鼠胰岛β细胞中表达，并发现胰淀素受体尤其是RAMP1蛋白可能参与人胰淀素对β细胞的毒性作用。Objective To determine the expression of three types of amylin receptors in rat isletβcells and explore their possible roles in cytotoxic effect of human amylin. Methods Reverse transcription-polymerase chain reaction（RT-PCR）, immunofluorescent staining were used to determine expression of amylin receptors in rat islet β cell line INS-1 and normal β cells. The influence of amylin incubation on mRNA transcription of three receptor activity-modifying proteins （RAMPs） was observed by real time RT-PCR, and change of RAMP1 distribution on cell membrane was observed by immunofluorescent staining. In cytotoxicity experiment, INS-1 cells were first incubated with amylin or amylin receptor antagonist AC253 alone or the two reagents together for 24 hours or 48 hours, and survival rate of the cell was measured by MTT test. RAMP1 antibody at different concentrations together with amylin were also applied to incubate cells for 24 hours and then MTT test was carried out. Group data were compared by using t test with the P value set at 0.05. Results RT-PCR and immunofluorescent staining demonstrated that three types of amylin receptor presented in rat islet βcells. RAMP2 and RAMP3 mRNA transcription decreased to 0.77 ± 0.10 （t=2.546, P〈0.05） and 0.66 ± 0.09 （t= 4.559, P〈0.05） respectively, while, RAMP1 mRNA was slightly increased to 1.25±0.17（t=-3.084, P〈0.05） after 24 hours′treatment with human amylin. Cell membrane distribution of RAMP1 was also slightly increased. Treatment with human amylin for 24 h significantly evoked cell death with cell viability decreased to 70%±13%（t=4.409, P〈0.05）. Application of amylin receptor antagonist AC253 significantly promoted cell viability to 94%± 16%compared with cells that incubated with human amylin alone （t=-3.341, P〈0.05）. While for cells treated for 48 hours, no obvious increase of cell survival was detected. Application of RAMP1 antibody diluted at 1∶500 and 1∶1 000 with human amylin also significantly promoted cell viability to 94

关 键 词：胰淀素 胰淀素受体 胰岛β细胞 细胞存活 

分 类 号：R587.1[医药卫生—内分泌]