胰岛β细胞Metrnl基因敲除小鼠的鉴定及初步表型分析

Identification and preliminary phenotypic analysis of mice with Metrnl knockout in isletβcells

目的:基于Cre-LoxP重组酶系统,构建胰岛β细胞Metrnl基因敲除小鼠(Metrnl-KO),为进一步探究Metrnl基因在糖尿病疾病中的发病机制提供动物模型。方法:将雌雄基因型均为Metrnlfloxp/+的小鼠合笼杂交,筛选出基因型为Metrnl^(floxp/floxp)的小鼠,将该基因型小鼠与胰岛β细胞特异性表达Cre重组酶(Ins-2)工具鼠进行杂交繁育,获得基因型为Metrnl^(floxp/+;Cre+)的小鼠;再将Metrnl^(floxp/+;Cre+)小鼠与Metrnl^(floxp/floxp)小鼠杂交获得Metrnl^(floxp/floxp;Cre+)小鼠,基因型为Metrnl^(floxp/floxp;Cre+)的小鼠即为目的鼠。采用RT-q PCR检测Metrnl和胰岛素(Ins-1和Ins-2)的m RNA水平;免疫组化和免疫荧光染色观察胰岛组织Metrnl和胰岛素的蛋白表达情况;记录小鼠体重,同时测定小鼠糖耐量和胰岛素耐量。结果:RT-q PCR和免疫组化结果均显示Metrnl-KO小鼠胰岛组织中Metrnl的m RNA和蛋白水平显著低于对照鼠(P<0.01);Metrnl-KO小鼠与对照鼠相比,体重无明显变化,但糖耐量受损且对胰岛素敏感性降低(P<0.05);RTq PCR结果显示胰岛组织中Ins-1和Ins-2的m RNA水平显著下调(P<0.01),且免疫荧光结果提示胰岛组织中胰岛素表达低于对照鼠(P<0.05);葡萄糖刺激的胰岛素分泌实验结果显示,Metrnl敲除可引起小鼠胰岛素分泌减少(P<0.05)。结论:我们构建并验证了条件性胰岛β细胞Metrnl基因敲除小鼠模型,还发现Metrnl的特异性敲除影响了小鼠的糖耐量和胰岛素耐受性,同时Metrnl敲除引起小鼠胰岛素分泌能力受损,为进一步研究Metrnl在糖尿病发生发展中的具体机制提供了研究基础。

AIM:Based on the Cre-LoxP recombinase system,Metrnl gene inβcells of islet was knockout(Metrnl-KO)to provide an animal model for further investigating the pathogenesis of Metrnl gene in diabetic diseases.METHODS:Male mice and female Metrnlfloxp/+mice were hybridized to obtain Metrnl^(floxp/floxp) mice,which were then crossed with mice specifically expressingβcell Cre recombinase(Ins-2)to obtain Metrnl^(floxp/+;Cre+)mice.Finally,the target mice with the genotype Metrnl^(floxp/floxp;Cre+)were obtained from the crosses between Metrnl^(floxp/+;Cre+)mice and Metrnl^(floxp/floxp) mice.The mRNA levels of Metrnl and insulin(Ins-1 and Ins-2)were measured using RT-qPCR.The protein expression of Metrnl and insulin in the islets was observed using immunohistochemistry and immunofluorescence.The body weight,and the glucose and insulin tolerance of the mice were recorded.RESULTS:Both RT-qPCR and immunohistochemistry showed that the Metrnl mRNA and protein levels in the islets of Metrnl-KO mice were significantly lower than those of control mice(P<0.01).The body weight of Metrnl-KO mice did not show a significant change compared with control mice,but Metrnl-KO mice exhibited impaired glucose tolerance and reduced sensitivity to insulin(P<0.01).The mRNA levels of Ins-1 and Ins-2 in the islets of Metrnl-KO mice significantly decreased according to RT-qPCR(P<0.01),and the insulin protein expres-sion in the islets was lower than that in control mice according to immunofluorescence(P<0.05).The glucose-stimulated insulin secretion experiment showed that Metrnl knockout decreased insulin secretion in mice(P<0.05).CONCLU⁃SION:We successfully generated and validated an Metrnl gene knockout mouse model inβcells of the islets.Our findings demonstrate that Metrnl knockout inβcells affects the glucose and insulin tolerance,and impairs the insulin secretion in mice.This model provides a basis for further investigating the mechanism of Metrnl in diabetes.