CAJ | 학술논문

目的：观察胰岛素基因突变所致的青少年型糖尿病（MIDY）其突变型胰岛素原对非折叠蛋白反应（UPR）信号通路的影响，探讨错误折叠的胰岛素原导致内质网应激（ESR）、β细胞功能衰竭的分子机制。方法用含有小鼠野生型胰岛素原（正常对照组）和3种突变型胰岛素原cDNA的重组质粒C（A7）Y、G（B8）S、G（C28）R分别转染293T细胞，转染72 h后收集细胞分为两组，一组细胞提取总RNA，采用SQRT-PCR法检测剪切型和非剪切型X-盒结合蛋白（XBP-1）mRNA的表达。另一组细胞提取蛋白，采用Western blotting技术检测细胞中真核细胞转录起始因子α（eIF2α）和磷酸化eIF2α（p-eIF2α）的蛋白表达。采用单因素方差分析及t检验进行统计学分析。结果与正常对照组相比，C （A7）Y组和G（B8）S组剪切型/非剪切型XBP-1 mRNA比值均升高，差异均有统计学意义[C（A7）Y组（0.84±0.17），G（B8）S组（0.70±0.16），与正常对照组（0.52±0.16）相比，t=3.17、2.35，均P<0.01]；G （C28）R组与正常对照组相比差异无统计学意义[G（C28）R组（0.55±0.14），t=0.30，P>0.05]。计算各组p-eIF2α/eIF2α比值，C（A7）Y组和G（B8）S组与正常对照组相比均升高，差异均有统计学意义[正常对照组（0.10±0.01）,C（A7）Y组（0.35±0.03）,G（B8）S组（0.27±0.02），t=13.57、9.37，均P<0.01]，G（C28）R组与正常对照组相比差异无统计学意义[G（C28）R组（0.08±0.01），t=1.09，P>0.05]。结论与MIDY相关的突变型胰岛素原C（A7）Y和G（B8）S可引发细胞UPR和ERS，错误折叠的突变型胰岛素原可能是通过激活IRE1-XBP1和PERK-eIF2α-ATF4信号传导通路来诱发UPR的。
목적：관찰이도소기인돌변소치적청소년형당뇨병（MIDY）기돌변형이도소원대비절첩단백반응（UPR）신호통로적영향，탐토착오절첩적이도소원도치내질망응격（ESR）、β세포공능쇠갈적분자궤제。방법용함유소서야생형이도소원（정상대조조）화3충돌변형이도소원cDNA적중조질립C（A7）Y、G（B8）S、G（C28）R분별전염293T세포，전염72 h후수집세포분위량조，일조세포제취총RNA，채용SQRT-PCR법검측전절형화비전절형X-합결합단백（XBP-1）mRNA적표체。령일조세포제취단백，채용Western blotting기술검측세포중진핵세포전록기시인자α（eIF2α）화린산화eIF2α（p-eIF2α）적단백표체。채용단인소방차분석급t검험진행통계학분석。결과여정상대조조상비，C （A7）Y조화G（B8）S조전절형/비전절형XBP-1 mRNA비치균승고，차이균유통계학의의[C（A7）Y조（0.84±0.17），G（B8）S조（0.70±0.16），여정상대조조（0.52±0.16）상비，t=3.17、2.35，균P<0.01]；G （C28）R조여정상대조조상비차이무통계학의의[G（C28）R조（0.55±0.14），t=0.30，P>0.05]。계산각조p-eIF2α/eIF2α비치，C（A7）Y조화G（B8）S조여정상대조조상비균승고，차이균유통계학의의[정상대조조（0.10±0.01）,C（A7）Y조（0.35±0.03）,G（B8）S조（0.27±0.02），t=13.57、9.37，균P<0.01]，G（C28）R조여정상대조조상비차이무통계학의의[G（C28）R조（0.08±0.01），t=1.09，P>0.05]。결론여MIDY상관적돌변형이도소원C（A7）Y화G（B8）S가인발세포UPR화ERS，착오절첩적돌변형이도소원가능시통과격활IRE1-XBP1화PERK-eIF2α-ATF4신호전도통로래유발UPR적。
Objective To investigate the molecular pathogenesis of misfolded proinsulin inducedβ-cell failure. Methods 293T cells were transfected with vector expressing preproinsulin wild type (normal control group)or preproinsulin missense mutants C(A7)Y,G(B8)S,G(C28)R. At 72 h post-transfection,cells were collected and divided into 2 groups. In one group,the expression of spliced XBP-1 mRNA and unspliced XBP-1 mRNA in cell were detected with semi-quantitive RT-PCR. In another group, Western blotting chest protein BiP,eukaryotic initiation factor 2α(eIF2α)and phosphorylation eIF2α(p-eIF2α). Data were analyzed with one-way ANOVA and t test. Results Compared with WT-mouse proinsulin,C(A7),G(B8)S mutant increased XBP-1s/ XBP-1u in 293T cell,and the difference was statistically significant(0.52 ± 0.16 vs 0.84 ± 0.17,0.52 ± 0.16 vs 0.70 ± 0.16,t=3.17,2.35,both P<0.01). While mutant G(C28)S didn′t up-regulate the level of XBP-1s/XBP-1u(0.52 ±0.16 vs 0.55 ±0.14,t=0.30, P>0.05). Compared with wild type,mutant C(A7)Y and G(B8)S increased p-eIF2α/eIF2α(0.10 ± 0.01 vs 0.35 ± 0.03,0.10 ± 0.01 vs 0.27 ± 0.02,t=13.57,9.37,both P<0.01). Mutant proinsulin G(C28)R didn′t up-regulate the level of p-eIF2α/eIF2α(0.10±0.01 vs 0.08±0.01,t=1.09,P>0.05). Conclusion Expression of MIDY mutants C(A7)Y and G(B8)S could lead endoplasmic reticulum stress and unfolded protein response,which may be through the activation of IRE1-XBP1 and PERK-eIF2α-ATF4 pathway.