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卡介苗对1型糖尿病小鼠的免疫治疗作用及其机制.pdf
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1、解放军医学杂志2023年7月28日第48卷第7期768卡介苗对1型糖尿病小鼠的免疫治疗作用及其机制结核病的预防、诊断与治疗专题研究II基金项目国家自然科学基金(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)作者简介康亚莉,硕士研究生,主要从事免疫与炎症机制方面的研究通信作者柏银兰,E-mail:;张琳娜,E-mail:论著康亚莉1,2,扈启宽1,宁唤唤2,周洁3,任瑞2,康健2,路延之2,韦垠1,2,高晓菁1,2,张琳娜1*,柏银兰2*1宁夏医科大
2、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中图分类号R587.1文献标志码ADOI10.11855/j.issn.0577-7402.1521.2023.0221声明本文所有作者声明无利益冲突引用本文康亚莉,扈启宽,宁唤唤,等.卡介苗对1型糖尿病小鼠的免疫治疗作用及其机制J.解放军医学杂志,2023,48(7):768-775.收稿日期2022-07-12录用日期2022-11-05上线日期2023-02-21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
3、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 1106 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
4、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(P0.001),而BCG组血糖水平虽高于对照组(P0.01),但明显低于T1DM组(P0.01)。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,T1D
5、M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(P0.05)。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(P0.0001),进食量明显增加(P0.0001)。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(P0.0001),进食量明显增加(P0.0001);与T1DM组比较,BCG组小鼠体重增长百分比增高(P0.001),进食量减少(P0.05)。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
6、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(P0.01),而BCG组胰岛素阳性面积虽然少于对照组(P0.05),但明显多于T1DM组(P0.05)。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(P0.001,P0.05),但BCG组血清C-肽含量明显高于T1DM组(P0.05);BCG组白细胞介素-2(IL-2)、IL-10、转化生长因子-(TGF-)mRNA水平与对照组和T1DM组比较均明显升高(P0.05)。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(P0.05);
7、与T1DM组比较,BCG组小鼠Treg细胞百分比明显增高(P0.05)。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。关键词卡介苗;1型糖尿病;免疫治疗Immunotherapeutic effect of BCG on type 1 diabetes mice and its mechanismKang Ya-Li1,2,Hu Qi-Kuan1,Ning Huan-Huan2,Zhou Jie3,Ren Rui2,Kang Jian2,Lu Yan-Zhi2,Wei Yin1,2,Gao
8、Xiao-Med J Chin PLA,Vol.48,No.7,July 28,2023769Jing1,2,Zhang Lin-Na1*,Bai Yin-Lan2*1School of Basic Medicine,Ningxia Medical University,Yinchuan,Ningxia 750001,China2Department of Microbiology and Pathogen Biology,Basic Medical School,Air Force Medical University,Xian,Shaanxi 710032,China3Department
9、 of Endocrinology,Xijing Hospital,Air Force Medical University,Xian,Shaanxi 710032,China*Corresponding author.Bai Yin-Lan,E-mail:;Zhang Lin-Na,E-mail:This work was supported by the National Natural Science Foundation of China(81971560),the National Major Special Projects of 13th Five-Year Plan of Ch
10、ina(2018ZX10302302002004),the Provincial Natural Science Foundation of Shaanxi Province(2022ZDLSF01-07),and the Natural Science Foundation of Ningxia Hui Autonomous Region(2021AAC03124)AbstractObjectiveTo evaluate the immunotherapeutic effect of Bacille Calmette-Gurin(BCG)in type 1 diabetes mellitus
11、(T1DM)mice and explore its mechanism.MethodsFifteen 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
12、 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 1106 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(P
13、BS).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 u
14、sed 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
15、 of cytokines in mice spleen.The proportion of regulatory T cells(Treg)in splenocytes was detected by flow cytometry.ResultsAfter twice immunization with BCG(13-week),the blood glucose level increased significantly of mice in T1DM group than that in control group(P0.001);while in BCG group was still
16、 higher than that in control group,but was significantly lower than that in T1DM group(P0.01).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 18
17、0 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(P0.05).At initial immunization(5th-week),compared with control group,the percentage of wei
18、ght gain decreased in T1DM group and BCG group(P0.0001),and the food consumption increased significantly(P0.0001);After twice immunization with BCG(13th-week),compared with control group,the percentage of weight gain in T1DM group was still lower significantly(P0.0001),and the food consumption was s
19、ignificantly higher(P0.001).Compared with T1DM group,the percentage of weight gain in BCG group increased(P0.001),and the food consumption was lower(P0.05).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
20、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 significan
21、tly in T1DM group(P0.01),and although the insulin positive area in BCG group was lower than that in control group(P0.05),but was still significantly higher than that in T1DM group(P0.05).ELISA test showed that the contents of serum C-peptide in T1DM group and BCG group decreased obviously than that
22、in control group(P0.001,P0.05),however,the content of serum C-peptide in BCG group was significantly higher than that in T1DM group(P0.05);Compared with T1DM group and control group,the mRNA levels of IL-2,IL-10 and transforming growth factor-(TGF-)were significantly increased in BCG group(P0.05).Th
23、e results of flow cytometry showed that compared with control group,the percentage of Treg cells in T1DM group decreased(P0.05),while compared with T1DM group,the percentage of Treg cells in BCG group increased obviously(P0.05).ConclusionBCG immunotherapy could induce the increase of Treg cells,regu
24、late 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.Key wordsBacille Calmette-Gurin(BCG);diabetes mellitus,type 1;immunotherapy解放军医学杂志2023年7月28日第48卷第7期770糖尿病(diabetes m
25、ellitus,DM)是一组因胰岛素分泌不足或胰岛素抵抗导致的、以高血糖为特征的代谢性疾病。根据国际糖尿病联盟的最新报告,2021年全球约5.37亿成年人(2079岁)患有糖尿病,预计到2045年将增加到7.83亿1。临床上将糖尿病分为1型糖尿病(type 1 diabetes mellitus,T1DM)、2型糖尿病(type 2 diabetes mellitus,T2DM)及妊娠糖尿病等。T1DM是由于宿主免疫系统对胰岛自身抗原的反应性增强,引起胰岛炎症,破坏胰岛细胞,使胰岛素缺乏或分泌不足所致2,因此被认为是一种自身免疫性疾病3。目前T1DM的治疗依然以使用外源性胰岛素为主,但由于不能
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