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染色结果显示,与对照组比较,T1DM组小鼠胰岛发生明显的萎缩,而BCG组小鼠胰岛形态与对照组接近,且胰岛内细胞数目明显多于T1DM组。免疫组化染色结果显示,与对照组比较,T1DM组小鼠胰腺中胰岛素阳性面积明显减少(P<0.01),而BCG组胰岛素阳性面积虽然少于对照组(P<0.05),但明显多于T1DM组(P<0.05)。ELISA检测结果显示,与对照组比较,T1DM组、BCG组血清C-肽含量明显降低(P<0.001,P<0.05),但BCG组血清C-肽含量明显高于T1DM组(P<0.05)。qRT-PCR结果显示,与对照组比较,T1DM组脾组织细胞因子mRNA水平无明显变化(P>0.05);BCG组白细胞介素-2(IL-2)、IL-10、转化生长因子-β(TGF-β)mRNA水平与对照组和T1DM组比较均明显升高(P<0.0001),而γ干扰素(IFN-γ)mRNA水平无明显变化(P>0.05)。流式细胞术检测结果显示,与对照组比较,T1DM组小鼠体内Treg细胞百分比降低(P<0.05);与T1DM组比较,BCG组小鼠Treg细胞百分比明显增高(P<0.05),且与对照组比较差异无统计学意义(P>0.05)。结论BCG免疫治疗可诱导Treg细胞增多,调节自身免疫应答,并通过减轻胰腺病理损伤、恢复胰岛细胞功能以促进胰岛素分泌,从而降低T1DM小鼠的血糖水平。
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 after 120 min,but were still higher significantly than in control group at 180 min(P<0.001),while the blood glucose level in BCG group was lower than that in T1DM group,but the difference was not statistically significant(P>0.05).At initial immunization(5th-week),compared with control group,the percentage of weight gain decreased in T1DM group and BCG group(P<0.0001),and the food consumption increased significantly(P<0.0001);After twice immunization with BCG(13th-week),compared with control group,the percentage of weight gain in T1DM group was still lower significantly(P<0.0001),and the food consumption was significantly higher(P<0.001).Compared with T1DM group,the percentage of weight gain in BCG group increased(P<0.001),and the food consumption was lower(P<0.001),and there was no significant difference compared with control group(P>0.05).The results of HE staining showed that,compared with control group,the islets of mice in T1DM group had obvious atrophy,while the morphology of islets of mice in BCG group was similar to that in control group,and the number of cells in islets was significantly higher than that in T1DM group.The insulin immunohistochemistry(IHC)staining showed that,compared with control group,the insulin-positive area in pancreas of mice decreased significantly in T1DM group(P<0.01),and although the insulin positive area in BCG group was lower than that in control group(P<0.05),but was still significantly higher than that in T1DM group(P<0.05).ELISA test showed that the contents of serum C-peptide in T1DM group and BCG group decreased obviously than that in control group(P<0.001,P<0.05),however,the content of serum C-peptide in BCG group was significantly higher than that in T1DM group(P<0.05).qRT-PCR showed that,compared with control group,the level of cytokine mRNA in T1DM group did not change(P>0.05);Compared with T1DM group and control group,the mRNA levels of IL-2,IL-10 and transforming growth factor-β(TGF-β)were significantly increased in BCG group(P<0.0001),but there was no change in mRNA level ofγ-interferon(IFN-γ)(P>0.05).The results of flow cytometry showed that compared with control group,the percentage of Treg cells in T1DM group decreased(P<0.05),while compared with T1DM group,the percentage of Treg cells in BCG group increased obviously(P<0.05),and there was no statistical difference compared with control group(P>0.05).Conclusion BCG immunotherapy could induce the increase of Treg cells,regulate autoimmune response,and promotes insulin secretion by reducing pancreatic pathological damage and restoring islet cell function,thus reducing the blood glucose level of T1DM mice.
作者
康亚莉
扈启宽
宁唤唤
周洁
任瑞
康健
路延之
韦垠
高晓菁
张琳娜
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)
出处
《解放军医学杂志》
CAS
CSCD
北大核心
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)。
关键词
卡介苗
1型糖尿病
免疫治疗
Bacille Calmette-Guérin(BCG)
diabetes mellitus,type 1
immunotherapy
