卡介苗对1型糖尿病小鼠的免疫治疗作用及其机制  

Immunotherapeutic effect of BCG on type 1 diabetes mice and its mechanism

作　　者：康亚莉 扈启宽[1] 宁唤唤 周洁 任瑞 康健 路延之 韦垠 高晓菁 张琳娜[1] 柏银兰 Kang Ya-Li;Hu Qi-Kuan;Ning Huan-Huan;Zhou Jie;Ren Rui;Kang Jian;Lu Yan-Zhi;Wei Yin;Gao Xiao-Jing;Zhang Lin-Na;Bai Yin-Lan(School of Basic Medicine,Ningxia Medical University,Yinchuan,Ningxia 750001,China;Department of Microbiology and Pathogen Biology,Basic Medical School,Air Force Medical University,Xi’an,Shaanxi 710032,China;Department of Endocrinology,Xijing Hospital,Air Force Medical University,Xi’an,Shaanxi 710032,China)

机构地区：[1]宁夏医科大学基础医学院,宁夏银川750001 [2]空军军医大学基础医学院微生物与病原生物学教研室,陕西西安710032 [3]空军军医大学西京医院内分泌科,陕西西安710032

出　　处：《解放军医学杂志》2023年第7期768-775,共8页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(81971560);国家“十三五”重大传染病专项课题(2018ZX10302302002004);陕西省重点研发项目(2022ZDLSF01-07);宁夏自然科学基金(2021AAC03124)。

摘　　要：目的评价卡介苗(BCG)对1型糖尿病(T1DM)小鼠的免疫治疗效果,并探索其作用机制。方法将15只SPF级雄性C57BL/6小鼠随机分为造模组(n=10,腹腔注射50 mg/kg链尿佐菌素建立T1DM模型)与对照组(n=5,腹腔注射等量柠檬酸缓冲液);取造模成功即随机血糖≥16.7 mmol/L的10只小鼠分为T1DM组(n=5)与卡介苗(BCG)组(n=5)。随后BCG组小鼠经皮下注射BCG 1×10^(6) cfu/只进行免疫治疗,4周后加强免疫治疗1次,T1DM组与对照组注射相同剂量磷酸盐缓冲液(PBS)。在实验周期(13周)内,每周监测小鼠体重和进食量变化,尾静脉采血检测各组小鼠血糖水平,采用口服葡萄糖耐量实验(OGTT)检测各组小鼠葡萄糖负荷后的血糖调节能力。采用HE染色观察各组小鼠胰腺组织病理变化,免疫组化染色检测胰腺胰岛素水平,酶联免疫吸附法(ELISA)检测各组小鼠血清C-肽含量,实时荧光定量PCR(qRT-PCR)检测各组小鼠脾组织细胞因子mRNA水平,流式细胞术检测各组小鼠脾细胞中调节性T细胞(Treg)比例。结果BCG免疫治疗2次后(第13周),T1DM组小鼠血糖水平明显高于对照组(P<0.001),而BCG组血糖水平虽高于对照组(P<0.01),但明显低于T1DM组(P<0.01)。OGTT结果显示,3组小鼠在葡萄糖灌胃后血糖水平迅速升高,120 min时开始下降,到180 min时,对照组小鼠血糖水平降至正常水平;与对照组比较,T1DM组及BCG组小鼠在120 min后血糖水平虽有下降趋势,但在180 min时仍明显高于对照组(P<0.001),而BCG组与T1DM组间差异无统计学意义(P>0.05)。在初次免疫时(第5周),与对照组比较,T1DM组和BCG组小鼠体重增长百分比明显降低(P<0.0001),进食量明显增加(P<0.0001)。BCG免疫治疗2次后(第13周),与对照组比较,T1DM组小鼠体重增长百分比仍明显降低(P<0.0001),进食量明显增加(P<0.0001);与T1DM组比较,BCG组小鼠体重增长百分比增高(P<0.001),进食量减少(P<0.001),且与对照组比较差异无统计学意义(P>0.05)。HE�Objective To evaluate the immunotherapeutic effect of Bacille Calmette-Guérin(BCG)in type 1 diabetes mellitus(T1DM)mice and explore its mechanism.Methods Fifteen SPF grade male C57BL/6 mice were randomly divided into molding group(n=10,established T1DM model by intraperitoneal injection of streptozotocin 50 mg/kg)and control group(n=5,intraperitoneal injection of equivalent amount of citric acid buffer).Ten mice with random blood glucose≥16.7 mmol/L were divided into T1DM group and BCG group(5 each).Mice in BCG group were then subcutaneously injected with 1×10^(6) cfu/mouse of BCG at an interval of 4 weeks for 2 times,and the other two groups were injected with the same dose of phosphate buffer solution(PBS).During the experimental period(13 weeks),the body weight and food consumption of mice were monitored weekly,and blood glucose levels were measured by tail vein sampling.Oral glucose tolerance test(OGTT)was used to detect the glucose regulation ability of mice after glucose load.HE staining was used to observe the pathological changes of pancreatic tissue.Pancreatic insulin level was detected by immunohistochemistry(IHC).Serum C-peptide levels were detected by enzyme linked immunosorbent assay(ELISA).Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the transcription levels of cytokines in mice spleen.The proportion of regulatory T cells(Treg)in splenocytes was detected by flow cytometry.Results After twice immunization with BCG(13-week),the blood glucose level increased significantly of mice in T1DM group than that in control group(P<0.001);while in BCG group was still higher than that in control group,but was significantly lower than that in T1DM group(P<0.01).The oral glucose tolerance test(OGTT)showed that,the blood glucose levels of mice in the three groups increased rapidly after glucose load,and began to decline at 120 min,and decreased to normal level at 180 min in control group;Compared with control group,the blood glucose levels of mice in T1DM group and BCG group also decreased

关 键 词：卡介苗 1型糖尿病 免疫治疗 

分 类 号：R587.1[医药卫生—内分泌]