Treg—Th17荧光双标纯合子小鼠的培育及其在内毒素血症研究中的初步应用  

Breeding of homozygous mice of Treg-Thl7 fluorescence double labeling and their preliminary use in endotoxemia study

作　　者：杜娟[1] 王海燕[1] 高文婷[1] 杨策[1] 顾玮[1] 曾灵[1] 严军[2] 蒋建新[1] 

机构地区：[1]第三军医大学附属大坪医院野战外科研究所第四研究室,创伤、烧伤与复合伤国家重点实验室,全军交通医学研究所,重庆400042 [2]第三军医大学附属大坪医院野战外科研究所第一研究室,重庆400042

出　　处：《中华创伤杂志》2012年第9期842-848,共7页Chinese Journal of Trauma

基　　金：国家自然科学基金资助项目，重庆市自然科学基金资助项目，人力资源和社会保障部留学人员科技活动资助项目

摘　　要：目的培育Treg—Th17荧光双标纯合子小鼠并探讨内毒素血症发生过程中Treg—Th17平衡的作用规律。方法从美国哈佛大学医学院引进Treg和Th17荧光单标小鼠，依照遗传学规则进行配对繁殖，提取子2代离乳仔鼠尾部组织DNA，通过PCR方法进行基因分型，选取荧光双标纯合子小鼠连续自交3代并检测其基因型和生活状态；制备该小鼠内毒素血症模型，流式细胞检测内毒素血症发生24h和48hTreg—Th17平衡的变化。结果第一批7只子2代小鼠中有3只小鼠（261早）为所需纯合子，自交3代连续基因分型结果均为Foxp3RFPKI—homo—IL-17AGFPKI，而且小鼠生长状态正常；内毒素重要成分脂多糖（1ipopolysacchafide，LPS）处理能诱导明显的红色荧光蛋白（redfluorescenceprotein，RFP）和绿色荧光蛋白（greenfluorescencepro—tein，GFP）信号表达；LPS处理24h组总体淋巴细胞中CD8a＋Foxp3＋、CD8a＋IL-17A＋、CD4＋Foxp3＋和CD4＋IL-17A＋的数量百分比明显上升（P〈0．01或0．05），但对照组与LPS处理48h组之间的差异无统计学意义（P〉0．05）。结论本实验培育的Foxp3RFPKI—homo—IL-17AGFPKI小鼠具有相同的基因表型和稳定的遗传学特征，是一种能够有效实时监测机体Treg—Th17平衡变化的模式动物和全新在体研究平台；Treg—Th17平衡作用在LPS刺激48h内表现显著；CD8a＋Foxp3和CD8a＋IL-17A＋在Treg—Th17平衡中的作用值得进一步关注。Objective To breed homozygous mice of Treg-Th17 fluorescence double labeling and to investigate the role of Treg-Thl7 balance in endotoxemia. Methods Mice of Treg and Thl7 fluo- rescence single labeling were brought from Harvard Medical School, USA and were mated and bred based on the genetic rules. The genomic DNA was extracted from the tails of second generation weaning mice for gen- otyping by polymerase chain reaction （PCR）. Thereafter, the homozygous mice of fluorescence double labe- ling were selected to have successive inbreeding for three generations and their genotypes and growth status were measured. The endotoxemia model in vivo was duplicated and changes of Treg-Thl7 balance were de- tected by flow cytometry analysis at 24 h and 48 h after endotoxemia. Results Among the seven second generation mice in the first batch, only three （two males and one female） were the wanted homozygotes.Meanwhile, the genotypes of their offsprings for the next three generations were all Foxp3 RFPKl-homo IL- 17A GFPKI with normal growing status. As important component of endotoxin, lipopolysaccharide （LPS） could induce significant signal expressions of red fluorescence protein （RFP） and green fluorescence pro- tein （GFP）. The ratio of CD8a ＋ Foxp3 ＋ , CD8a ＋ IL-17A ＋, CD4 ＋ Foxp3 ＋ and CD4 ＋ IL-17A ＋ in the lymphocytes went up significantly in the group of 24 hours of LPS treatment, as compared with the control group （ P 〈 0.01 or P 〈 O. 05 ） , while the difference between the control group and the group of 48 hours of LPS treatment had no statistical significance （ P 〉 0.05 ）. Conclusion Foxp3 RFPKI-homo- IL-17A GFPKI mice have identical genotypes and stable genetic characteristics and is a model animal and novel research platform that can provide real-time monitoring of the changes of Treg-Thl7 balance in vivo. The role of T reg-Thl7 balance works significantly at 48 hous after the subject is exposed to LPS stimula- tion, and the roles of CD8a ＋ Foxp3 and CD8a ＋ IL-17

关 键 词：荧光 纯合子 内毒素血症 Treg—Th17 

分 类 号：R641[医药卫生—外科学]