调节性T细胞参与调控新生小鼠心肌再生  

Regulatory T Cells Regulate Myocardial Regeneration in Newborn Mice

作　　者：赵文静[1] 冯杰 聂宇[2] 王玉瑶[1] 郭睿[1] ZHAO Wen-Jing;FENG Jie;NIE Yu;WANG Yu-Yao;GUO Rui(Department of Biochemistry and Molecular Biology,Shanxi Medical University,Taiyuan 030000,China;State Key Laboratory of Cardiovascular Disease,Fuwai Hospital,National Center for Cardiovascular Disease Chinese Academy of Medical Sciences and Peking Union Medical College,Beifing 100037,China)

机构地区：[1]山西医科大学生物化学与分子生物学教研室,太原030000 [2]北京协和医学院国家心血管病中心阜外医院心血管疾病国家重点实验室,北京100037

出　　处：《中国生物化学与分子生物学报》2018年第11期1211-1220,共10页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.81500364);山西省自然科学基金(No.201601D102071)资助~~

摘　　要：为探究调节性T(regulatory T,T_(reg))细胞在新生小鼠心肌损伤后再生中的作用,首先建立新生小鼠心肌再生模型。C57BL/6J(C57)新生1 d小鼠20只随机分成2组。实验组进行心尖切除(apex resection,AR),假手术(Sham,SH)组只进行开胸。术后7 d取心脏组织,利用在细胞核表达的增殖标志物磷酸化组蛋白H3(phospho-histone H3,pH3)和Ki67分别与在心肌细胞胞质特异表达的α-辅肌动蛋白(alpha-actinin cytoskeletal isoform,α-actinin),进行免疫共染检测心肌细胞增殖。结果显示,与SH组相比,AR组pH3+及Ki67+的心肌细胞明显增多。而且Masson三色染色结果显示,术后21 d被切除的心肌组织完全再生。为研究T_(reg)细胞是否参与调控新生小鼠心肌损伤后的再生,Western印迹检测T_(reg)细胞特异转录因子叉头/翼状螺旋转录因子3(forkhead box P3,Foxp3)蛋白表达水平。结果显示,术后7 d、14 d,AR组心和脾中Foxp3与SH组相比显著升高(P <0. 05)。同时,免疫组化染Foxp3结果显示,术后7 d、14 d,AR组与SH组相比,心尖处有大量的T_(reg)细胞富集。为更直观地检测AR后T_(reg)细胞的数目变化,利用流式细胞仪检测术后7 d T_(reg)细胞数目。结果显示,AR组心和脾中T_(reg)细胞数目与SH组相比显著增多(P <0. 01)。为研究T_(reg)细胞对AR后心肌再生的影响,引入注射白喉毒素(diphtheria toxin,DT)的Foxp3^(DTR)小鼠,可特异性敲除T_(reg)细胞。实时定量PCR结果显示,AR+DT组与AR+PBS组相比,抑炎因子白介素IL(interleukin,IL)-10、IL-13与转化生长因子TGF(transforming growth factor,TGF)-β表达均降低(P<0. 05,P <0. 01,P <0. 01)。而促炎因子IL-6、IL-1β和肿瘤坏死因子-α(tumor necrosis factor,TNF-α)表达均升高(P <0. 01,P <0. 001,P <0. 01)。免疫荧光染色检测结果显示,AR+DT组与AR+PBS组相比,术后7 d pH3+及Ki67+的心肌细胞明显减少;并且Masson三色染色结果显示,术后21 d AR+DT组被切除的心肌组织不能再生。综上所述,敲除To investigate the role of regulatory T（ Treg） cells in heart regeneration of newborn mice,a model of myocardial regeneration of C57 BL/6 J newborn mice was established and the mice were randomlydivided into two groups： the experimental group underwent apex resection（ AR）,and the sham（ SH）operated group only took the thoracic surgery. Heart tissues were harvested at seven days post surgery.The immunofluorescence co-staining of phospho-histone H3（ pH3） and Ki67 with cardiomyocyte cytoplasma antigen α-actinin was used to observe cardiomyocyte proliferation. The results showed that the number of pH3＋cardiomyocytes and Ki67＋cardiomyocytes in the AR group was significantly increased.Masson staining showed that the myocardium fully regenerated at 21 days post resection（ dpr）. To study whether Tregcells participate in the regulation of myocardial regeneration after AR in newborn mice,the protein levels of forkhead box P3（ Foxp3,the specific transcription factor of Tregcells） were detected by Western blotting. The results showed that the expression of Foxp3 in the heart and spleen of the AR group was significantly higher than that in the SH group at 7 and 14 days post surgery（ P〈 0. 05）.Immunohistochemical staining was also applied for Foxp3. The results revealed that a large amount of Treg cells were enriched in the apex of the AR group compared with the SH group at 7 and 14 days post surgery. The number of Tregcells measured by flow cytometer showed that Tregcells were increased in the AR than the SH group（ P 〈0. 01）,indicating that Tregcells might be involved in newborn heart regeneration. Importantly,to investigate the mechanism of Tregcells regulating myocardial regeneration,Foxp3 DTRmice were injected with diphtheria toxin（ DT） to delete Tregcells. Real-time quantitative PCR revealed that the expression of anti-inflammatory factors（ interleukin-10,interleukin-13 and transforming growth factor β） were reduced（ P 〈0. 05,P 〈0. 01,P 〈0. 01）,while the express

关 键 词：心尖切除 心肌再生 TREG细胞 Foxp3DTR 

分 类 号：R392[医药卫生—免疫学]