中华结核和呼吸杂志
中華結覈和呼吸雜誌
중화결핵화호흡잡지
Chinese Journal of Tuberculosis and Respiratory Diseases
2013年
8期
587-591
,共5页
黄燕%路聪哲%王彩彩%姜毅%王晓阳%段蕴铀
黃燕%路聰哲%王綵綵%薑毅%王曉暘%段蘊鈾
황연%로총철%왕채채%강의%왕효양%단온유
结节病%肉芽肿,浆细胞,肺%模型,动物
結節病%肉芽腫,漿細胞,肺%模型,動物
결절병%육아종,장세포,폐%모형,동물
Sarcoidosis%Granuloma,plasma cell,pulmonary%Model,animal
目的 建立并鉴定分枝杆菌过氧化物歧化酶多肽(SodA)诱导的C57BL/6小鼠结节病样肉芽肿模型.方法 将30只雌性C57BL/6小鼠按数字随机表法分为5组:复合组(SodA+琼脂糖)、SodA组,不完全弗氏佐剂(IFA)组、琼脂糖组及空白对照组,每组6只.第1天,复合组、SodA组给予50 μg SodA与0.25 ml IFA,混合后背部皮下注射；IFA组、琼脂糖组分别给予IFA 0.25 ml及PBS0.25 ml背部皮下注射；空白对照组未予任何处理.第14天,复合组给予50 μg SodA与6000粒琼脂糖4B珠(溶于0.5ml PBS)共价偶联后尾静脉注射；SodA组给予50 μg SodA(溶于PBS 0.5 ml)尾静脉注射；IFA组、琼脂糖组给予6000粒琼脂糖4B珠(溶于0.5 ml PBS)尾静脉注射；空白对照组未予任何处理.第22天,解剖小鼠,观察淋巴结、肺部大体标本及组织学改变；免疫组织化学检测肉芽肿内巨噬细胞和CD4+ T细胞；检测BALF中细胞分类；流式细胞术检测BALF中CD4+/CD8+及肺组织内干扰素-γ与IL-12水平.结果 复合组肺部与淋巴结均可见结节病样肉芽肿,SodA组淋巴结可见结节病样肉芽肿,但肺部未见肉芽肿,IFA组、琼脂糖组及空白对照组淋巴结及肺部均未见结节病样肉芽肿；肉芽肿中心巨噬细胞特异性抗原Mac-2表达阳性,周围CD4+ T细胞表达阳性；复合组、SodA组BALF中淋巴细胞百分比(分别为19.4％±6.5％、22.3％±8.5％)与IFA组、琼脂糖组及空白对照组(分别为8.5％±4.3％、7.7％±3.4％、0.8％±0.6％)相比均明显增高(均P ＜0.05)；复合组、SodA 组BALF中CD;/CD8+(分别为3.5±1.4、3.2±1.1)与IFA组、琼脂糖组(分别为1.2±0.5、1.0±0.4)相比均明显增高(均P＜ 0.05)；复合组、SodA组肺组织内干扰素-γ、IL-12水平[干扰素-γ:(32.9 ±9.7)ng/L、(26.4 ±7.2)ng/L;IL-12:(29.6±9.4)ng/L、(26.1 ±8.9) ng/L]与IFA组、琼脂糖组及空白对照组[干扰素-γ:(16.5 ±6.8)ng/L、(12.2±5.0) ng/L和(9.0±2.6) ng/L；IL-12:(16.7±4.6) ng/L、(13.6±4.4) ng/L和9.6 ±5.3 ng/L]相比均明显增高(均P＜0.05)；复合组、SodA组之间以上指标无明显差异,IFA组、琼脂糖组及空白对照组之间以上指标无明显差别(均P＞0.05).结论 SodA可以诱导建立C57BL/6小鼠结节病样肉芽肿模型,免疫学特点与人类结节病相似.
目的 建立併鑒定分枝桿菌過氧化物歧化酶多肽(SodA)誘導的C57BL/6小鼠結節病樣肉芽腫模型.方法 將30隻雌性C57BL/6小鼠按數字隨機錶法分為5組:複閤組(SodA+瓊脂糖)、SodA組,不完全弗氏佐劑(IFA)組、瓊脂糖組及空白對照組,每組6隻.第1天,複閤組、SodA組給予50 μg SodA與0.25 ml IFA,混閤後揹部皮下註射；IFA組、瓊脂糖組分彆給予IFA 0.25 ml及PBS0.25 ml揹部皮下註射；空白對照組未予任何處理.第14天,複閤組給予50 μg SodA與6000粒瓊脂糖4B珠(溶于0.5ml PBS)共價偶聯後尾靜脈註射；SodA組給予50 μg SodA(溶于PBS 0.5 ml)尾靜脈註射；IFA組、瓊脂糖組給予6000粒瓊脂糖4B珠(溶于0.5 ml PBS)尾靜脈註射；空白對照組未予任何處理.第22天,解剖小鼠,觀察淋巴結、肺部大體標本及組織學改變；免疫組織化學檢測肉芽腫內巨噬細胞和CD4+ T細胞；檢測BALF中細胞分類；流式細胞術檢測BALF中CD4+/CD8+及肺組織內榦擾素-γ與IL-12水平.結果 複閤組肺部與淋巴結均可見結節病樣肉芽腫,SodA組淋巴結可見結節病樣肉芽腫,但肺部未見肉芽腫,IFA組、瓊脂糖組及空白對照組淋巴結及肺部均未見結節病樣肉芽腫；肉芽腫中心巨噬細胞特異性抗原Mac-2錶達暘性,週圍CD4+ T細胞錶達暘性；複閤組、SodA組BALF中淋巴細胞百分比(分彆為19.4％±6.5％、22.3％±8.5％)與IFA組、瓊脂糖組及空白對照組(分彆為8.5％±4.3％、7.7％±3.4％、0.8％±0.6％)相比均明顯增高(均P ＜0.05)；複閤組、SodA 組BALF中CD;/CD8+(分彆為3.5±1.4、3.2±1.1)與IFA組、瓊脂糖組(分彆為1.2±0.5、1.0±0.4)相比均明顯增高(均P＜ 0.05)；複閤組、SodA組肺組織內榦擾素-γ、IL-12水平[榦擾素-γ:(32.9 ±9.7)ng/L、(26.4 ±7.2)ng/L;IL-12:(29.6±9.4)ng/L、(26.1 ±8.9) ng/L]與IFA組、瓊脂糖組及空白對照組[榦擾素-γ:(16.5 ±6.8)ng/L、(12.2±5.0) ng/L和(9.0±2.6) ng/L；IL-12:(16.7±4.6) ng/L、(13.6±4.4) ng/L和9.6 ±5.3 ng/L]相比均明顯增高(均P＜0.05)；複閤組、SodA組之間以上指標無明顯差異,IFA組、瓊脂糖組及空白對照組之間以上指標無明顯差彆(均P＞0.05).結論 SodA可以誘導建立C57BL/6小鼠結節病樣肉芽腫模型,免疫學特點與人類結節病相似.
목적 건립병감정분지간균과양화물기화매다태(SodA)유도적C57BL/6소서결절병양육아종모형.방법 장30지자성C57BL/6소서안수자수궤표법분위5조:복합조(SodA+경지당)、SodA조,불완전불씨좌제(IFA)조、경지당조급공백대조조,매조6지.제1천,복합조、SodA조급여50 μg SodA여0.25 ml IFA,혼합후배부피하주사；IFA조、경지당조분별급여IFA 0.25 ml급PBS0.25 ml배부피하주사；공백대조조미여임하처리.제14천,복합조급여50 μg SodA여6000립경지당4B주(용우0.5ml PBS)공개우련후미정맥주사；SodA조급여50 μg SodA(용우PBS 0.5 ml)미정맥주사；IFA조、경지당조급여6000립경지당4B주(용우0.5 ml PBS)미정맥주사；공백대조조미여임하처리.제22천,해부소서,관찰림파결、폐부대체표본급조직학개변；면역조직화학검측육아종내거서세포화CD4+ T세포；검측BALF중세포분류；류식세포술검측BALF중CD4+/CD8+급폐조직내간우소-γ여IL-12수평.결과 복합조폐부여림파결균가견결절병양육아종,SodA조림파결가견결절병양육아종,단폐부미견육아종,IFA조、경지당조급공백대조조림파결급폐부균미견결절병양육아종；육아종중심거서세포특이성항원Mac-2표체양성,주위CD4+ T세포표체양성；복합조、SodA조BALF중림파세포백분비(분별위19.4％±6.5％、22.3％±8.5％)여IFA조、경지당조급공백대조조(분별위8.5％±4.3％、7.7％±3.4％、0.8％±0.6％)상비균명현증고(균P ＜0.05)；복합조、SodA 조BALF중CD;/CD8+(분별위3.5±1.4、3.2±1.1)여IFA조、경지당조(분별위1.2±0.5、1.0±0.4)상비균명현증고(균P＜ 0.05)；복합조、SodA조폐조직내간우소-γ、IL-12수평[간우소-γ:(32.9 ±9.7)ng/L、(26.4 ±7.2)ng/L;IL-12:(29.6±9.4)ng/L、(26.1 ±8.9) ng/L]여IFA조、경지당조급공백대조조[간우소-γ:(16.5 ±6.8)ng/L、(12.2±5.0) ng/L화(9.0±2.6) ng/L；IL-12:(16.7±4.6) ng/L、(13.6±4.4) ng/L화9.6 ±5.3 ng/L]상비균명현증고(균P＜0.05)；복합조、SodA조지간이상지표무명현차이,IFA조、경지당조급공백대조조지간이상지표무명현차별(균P＞0.05).결론 SodA가이유도건립C57BL/6소서결절병양육아종모형,면역학특점여인류결절병상사.
Objective To establish a C57BL/6 mouse sarcoidosis granuloma model elicited by mycobacterial superoxide dismutase A peptide (SodA).Methods Thirty female C57BL/6 mice were randomly divided equally into 5 groups:a combination (SodA + Sepharose) group,a SodA group,a IFA (incomplete Freund' s adjuvant)group,a sepharose group and a blank control group.On the first day,the combination group and the SodA group were sensitized by subcutaneous injection of 50 μg SodA incorporated into IFA 0.25 ml.The IFA group and the Sepharose group were treated with subcutaneous injection of IFA 0.25 ml and PBS 0.25 ml respectively,while the blank control group was not given any treatment.On the 14th day,the combination group was challenged by tail vein injection of 50 μg SodA covalently coupled to 6000 agarose 4B beads(in PBS 0.5 ml).The SodA group was challenged by tail-vein injection of 50 μg SodA (in PBS 0.5 ml).The IFA group and the Sepharose group were treated by tail-vein injection of 6000 agarose 4B beads(in PBS 0.5 ml),while the blank control group was not given any treatment.On the 22th day,the mice were dissected and the gross and pathological changes of lymph nodes and lungs were observed.Immunohistochemisty was used to identify Mac-2 and CD4+T in granuloma.Counts and differentials of BALF cells were measured.CD4+/CD8+ in BALF and cytokines (IFN-γ and IL-12) levels in the lungs were detected by flow cytometry.Results Enlargement of peripheral and pulmonary hilar lymph nodes were found in the combination group and the SodA group,and sarcoidosis granuloma was found in the lymph nodes and lungs of the combination group.Sarcoidosis granuloma was also found in the lymph nodes but not in the lungs of the SodA group.No sarcoidosis granuloma was observed in the lungs and lymph nodes of the IFA group,the Sepharose group and the blank control group.Macrophage specific antigen Mac-2 and CD4+ T were positive in the core and rim of the granuloma respectively.The lymphocyte percentages in the BALF of the combination group and the SodA group [(19.4 ± 6.5) ％ and (22.3 ± 8.5) ％] were significantly higher than that in the IFA group,the Sepharose group and the blank control group [(8.5 ±4.3) ％,(7.7 ± 3.4) ％,(0.8 ± 0.6％)] (P ＜ 0.05).CD4+/CD8+ in the BALF of the combination group and the SodA group (3.5 ± 1.4,3.2 ± 1.1) were significantly higher than that in the IFA group and the Sepharose group(1.2 ± 0.5,1.0 ± 0.4) (P ＜ 0.05).IFN-γ and IL-12 in the lungs of the combination group and the SodA group [IFN-γ:(32.9 ± 9.7) ng/L,(26.4 ± 7.2) ng/L ; IL-12:(29.6 ± 9.4) ng/L,(26.1 ± 8.9) ng/L] were significantly higher than those of the IFA group,the Sepharose group and the blank control group [IFN-γ:(16.5±6.8)ng/L,(12.2 ±5.0) ng/L,(9.0±2.6) ng/L;IL-12:(16.7 ±4.6)ng/L,(13.6 ±4.4) ng/L,(9.6 ±5.3) ng/L] (P ＜0.05).But these indexes were not significantly different between the combination group and the SodA group,and among the IFA group,the Sepharose group and the blank control group (P ＞ 0.05).Conclusion SodA can elicit sarcoidosis granuloma in C57BL/6mice,and the immunological features of the model were similar to those in human sarcoidosis.
