利用小动物成像技术在体非创评估小鼠胰岛B细胞容量  

In vivo non-invasive measurement of mouse pancreatic B cell mass through bioluminescence imaging

作　　者：赵瑜[1] 林紫薇[1] 孙赟[1] 慕开达[1] 王琛[1] 贾伟平[1] 

机构地区：[1]上海交通大学附属第六人民医院内分泌代谢科,上海市糖尿病研究所,上海市糖尿病重点实验室,上海200233

出　　处：《上海医学》2016年第1期10-15,I0001,共7页Shanghai Medical Journal

基　　金：国家自然科学基金资助项目(31340072)

摘　　要：目的利用在小鼠胰岛素2启动子(MIP)后携带有串联三联报告基因(TF,其中包含荧光素酶报告基因)的转基因(MIP-TF)小鼠,通过活体成像系统对其生理和病理状态下的胰岛B细胞容量进行在体评估,从而建立基于该小鼠的对胰岛B细胞进行在体追踪的小动物活体成像平台。方法 MIP-TF小鼠购自美国Jackson实验室。葡萄糖耐量实验(GTT)和葡萄糖刺激的胰岛素分泌实验分别采用小鼠腹腔内注射1.5和3.0g/kg葡萄糖进行。45%高脂饮食(HFD)喂养MIP-TF小鼠制备饮食诱导的肥胖小鼠,200mg/kg链脲佐菌素(STZ)腹腔内注射制备小鼠糖尿病模型。胰腺胰岛素免疫荧光染色用于胰岛B细胞组织学检测,小动物活体成像检测MIP-TF小鼠胰岛B细胞容量。结果普食或HFD喂养的MIP-TF小鼠与同窝野生型小鼠(WT小鼠)间体重和空腹血糖水平的差异均无统计学意义(P值均>0.05)。GTT和葡萄糖刺激的胰岛素分泌实验结果显示,HFD喂养12周的MIP-TF小鼠与WT小鼠间各时间点的血糖和胰岛素水平的差异均无统计学意义(P值均>0.05)。MIP-TF小鼠在葡萄糖刺激后,反映胰岛B细胞容量的腹部荧光强度较刺激前增加约1倍,差异有统计学意义(刺激前为74 378.80±23 370.9,刺激后为143 617.50±20 968.65,P<0.05)。HFD喂养MIP-TF小鼠10周后,荧光强度显著增强至普食喂养的MIP-TF小鼠的4倍,差异有统计学意义(普食喂养为479 846.60±103 619.63,HFD喂养为2 014 852.80±345 009.72,P<0.01);胰岛素免疫荧光染色显示,HFD喂养的MIP-TF小鼠的胰岛素阳性面积显著大于普食喂养的MIP-TF小鼠。普食喂养的MIP-TF小鼠注射大剂量STZ(200mg/kg)后,荧光强度明显减弱,甚至完全消失,与注射前的差异有统计学意义(注射前为490 482.20±215 546.18,注射后为0.00±0.00,P<0.01);胰岛素免疫荧光染色也显示,STZ给药的小鼠胰岛素阳性区域几乎完全消失。结论 MIP-TF小鼠胰岛B细胞中特异性表达荧光素酶不影响小鼠Objective To evaluate pancreatic B-cell mass under physiological and pathological conditions and to establish an in vivo tracking platform for pancreatic B-cell mass through non-invasive bioluminescence imaging and mouse insulinⅡ promoter（MIP）-mice expressing trifusion（TF）proteins of three imaging reporters（including luciferase）driven by MIP.Methods MIP-TF mice were purchased from Jackson Lab（USA）.Glucose tolerance test（GTT）and glucose-stimulated insulin secretion test were conducted through glucose intraperitoneal injection（1.5g/kg for GTTs,3.0g/kg for insulin secretion tests）.MIP-TF mice were fed with a high-fat diet（HFD,45% kcal fat）.Diabetic mice were produced by intraperitoneal injection of streptozotocin（STZ,200mg/kg）.Immunofluorescence histology was conducted to detect insulin staining in pancreas.Pancreatic B-cell mass was determined by non-invasive bioluminescence imaging.Results There were no significant differences in body weight and fasting plasma glucose between wide type（WT）and MIP-TF mice fed with either a chow food or HFD（all P〈0.05）.The GTT and glucose-stimulated insulin secretion tests showed that there were no significant differences in plasma glucose or insulin levels at each time point between WT and MIP-TF mice after a 12-week HFD（all P〉0.05）.In MIP-TF mice,the fluorescence intensity reflecting the pancreatic B-cell mass after glucose stimulation was statistically higher than that before glucose stimulation（74 378.80±23 370.9 vs.143 617.50±20 968.65,P〈0.05）.Pancreatic B-cell mass represented by bioluminescence intensity was four-fold higher in 12-week HFD-fed MIP-TF mice than in the chow-diet fed mice（479 846.60±103 619.63 vs.2 014 852.80±345 009.72,P〈0.01）.Insulin immunofluorescence staining assay showed that pancreatic insulin-positive area in HFD MIP-TF mice was significantly greater than that in chow diet fed mice.Furthermore,the fluorescence intensity disappeared completely after STZ injection（490 482.20±215 546.18 vs.

关 键 词：胰岛素抵抗 糖尿病 B细胞 小动物活体成像 

分 类 号：R587.1[医药卫生—内分泌]