急性冠脉综合征患者调节性T细胞稳态变化研究
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摘要
目的:在本课题组前期研究发现急性冠脉综合征(ACS)患者外周血调节性T细胞(Treg)数目存在异常的基础上进一步分析该群细胞的组成和凋亡变化。
方法:本研究纳入研究对象共109例,其中包括急性ST段抬高心肌梗死(STEAMI)患者18例、非ST段抬高急性冠脉综合征(NSTACS,包括NSTEAMI和UA)患者64例、慢性稳定性心绞痛(CSA)患者12例和胸痛综合征(CPS)患者15例,留取外周血标本,密度梯度离心法分离获取淋巴细胞,流式细胞术检测Treg亚群CD4~+CD25~+CD127-CD45RO~+CD45RA-T细胞(memory Treg, mTreg),CD4~+CD25~+CD127-CD45RO-CD45RA~+T细胞(naive Treg, nTreg)和CD4~+CD25~+CD127-CD45RO-CD45RA~+TCD31~+细胞(recent thymic emigrants-naive Treg, RTE-nTreg)占CD4~+CD25~+CD127-Treg细胞的比例及AnnexinⅤ~+7-AAD-- -CD4~+CD25~+CD127-的凋亡细胞占CD4~+CD25~+CD127-Treg的比例;进一步纳入ACS,CSA及CPS患者各12例,磁珠分选(MACS)CD4~+CD25highTreg细胞, real-time PCR技术比较三组人群Treg细胞中T细胞受体重排删除环(TREC)拷贝数的差异。
结果:STEAMI, NSTACS,CSA和CPS四组病人间的mTreg/Treg,nTreg/Treg的比例无统计学差异,但RTE-nTreg占nTreg的比例在STEAMI组(16.91±7.90)和NSTACS组(20.39±10.31)则较CSA组(29.78±9.86)和CPS组(31.54±10.52)显著性降低(p<0.01)。凋亡分析提示STEAMI患者(18.42±10.93)和NSTACS患者(16.24±9.67)处于早期凋亡的Treg细胞占总Treg细胞的比例较CSA患者(12.76±6.52)和CPS患者(11.38±4.85)有升高趋势,但尚未达统计学差异(STEAMI vs CSA, p=0.122; STEAMI vs CPS , p=0.068; NSTACS vs CSA , p=0.235 ; NSTACS vs CPS, p=0.170)。ACS患者(1.06±0.48)的Treg细胞的TREC拷贝数较CSA患者(1.98±0.69)与CPS患者(2.26±0.98)显著降低(p<0.05)。
结论:ACS患者中Treg亚群中的RTE-nTreg及Treg细胞中TREC的拷贝数较CSA患者及CPS患者明显减低,提示ACS患者Treg细胞数目改变的同时存在内部稳态异常。
Objective: To investigate the subsets and apoptosis of regulatory T cells (Treg) in patients with acute coronary syndrome based on our previous studies.
Methods: A total of 109 subjects were enrolled in our study, including 18 patients with st-elevation myocardial infarction (STEAMI), 64 patients with NSTACS (including NSTEAMI and UA), 12 patients with chronic stable angina (CSA)and 15 patients with chest pain syndrome (CPS ). The peripherial lymphocytes were isolated using gradient-density confriguration. The ration of CD4~+CD25~+CD127-CD45RO~+ -CD45RA- memory Treg (mTreg), CD4~+CD25~+CD127-CD45RO-CD45RA~+ naive Treg (nTreg) , CD4~+CD25~+CD127-CD45RO-CD45RA~+TCD31~+ recent thymic emigrants-naive Treg (RTE-nTreg)and AnnexinⅤ~+7-AAD-CD4~+CD25~+CD127- in CD4~+CD25~+CD127-total Treg were analyzed using flow cytometry. 12 cases with ACS, 12 cases with CSA and 12 cases with CPS were also included and CD4~+CD25high Treg were purified by magnetic activated cell sorting (MACS), and T-cell receptor excision circles (TREC) in the purifed Treg were determined by realtime-PCR.
Results: The frequencies of mTreg and nTreg in total Treg were comparable among the STEAMI, NSTACS, CSA and CPS group. In contrast, the frequency of RTE-nTreg in total nTreg significantly decreased in both STEAMI group (16.91±7.90) and NSTACS group (20.39±10.31) compared to CSA group (29.78±9.86) and CPS group (31.54±10.52) (p<0.01). The frequency of apoptotic Treg in total Treg increased in the group of STEAMI group (18.42±10.93) and NSTACS group (16.24±9.67) increased compared with CSA group (12.76±6.52) and CPS group (11.38±4.85) , but the differences were not significant (STEAMI vs CSA, p=0.122; STEAMI vs CPS, p=0.068; NSTACS vs CSA, p=0.235; NSTACS vs CPS, p=0.170). The copies of TREC were also markedly lower in the ACS group (1.06±0.48) than in the CSA group (1.98±0.69) and CPS group (2.26±0.98) (p<0.05).
Conclusion: Our data demonstrated that the frequency of RTE-nTreg in total Treg and the the copies of TREC in purified Treg significantly decreased in STEAMI and NSTACS patients compared to CSA and CPS patients, suggesting that Treg homeostatis was disturbed in patients with ACS.
方法:本研究纳入研究对象共109例,其中包括急性ST段抬高心肌梗死(STEAMI)患者18例、非ST段抬高急性冠脉综合征(NSTACS,包括NSTEAMI和UA)患者64例、慢性稳定性心绞痛(CSA)患者12例和胸痛综合征(CPS)患者15例,留取外周血标本,密度梯度离心法分离获取淋巴细胞,流式细胞术检测Treg亚群CD4~+CD25~+CD127-CD45RO~+CD45RA-T细胞(memory Treg, mTreg),CD4~+CD25~+CD127-CD45RO-CD45RA~+T细胞(naive Treg, nTreg)和CD4~+CD25~+CD127-CD45RO-CD45RA~+TCD31~+细胞(recent thymic emigrants-naive Treg, RTE-nTreg)占CD4~+CD25~+CD127-Treg细胞的比例及AnnexinⅤ~+7-AAD-- -CD4~+CD25~+CD127-的凋亡细胞占CD4~+CD25~+CD127-Treg的比例;进一步纳入ACS,CSA及CPS患者各12例,磁珠分选(MACS)CD4~+CD25highTreg细胞, real-time PCR技术比较三组人群Treg细胞中T细胞受体重排删除环(TREC)拷贝数的差异。
结果:STEAMI, NSTACS,CSA和CPS四组病人间的mTreg/Treg,nTreg/Treg的比例无统计学差异,但RTE-nTreg占nTreg的比例在STEAMI组(16.91±7.90)和NSTACS组(20.39±10.31)则较CSA组(29.78±9.86)和CPS组(31.54±10.52)显著性降低(p<0.01)。凋亡分析提示STEAMI患者(18.42±10.93)和NSTACS患者(16.24±9.67)处于早期凋亡的Treg细胞占总Treg细胞的比例较CSA患者(12.76±6.52)和CPS患者(11.38±4.85)有升高趋势,但尚未达统计学差异(STEAMI vs CSA, p=0.122; STEAMI vs CPS , p=0.068; NSTACS vs CSA , p=0.235 ; NSTACS vs CPS, p=0.170)。ACS患者(1.06±0.48)的Treg细胞的TREC拷贝数较CSA患者(1.98±0.69)与CPS患者(2.26±0.98)显著降低(p<0.05)。
结论:ACS患者中Treg亚群中的RTE-nTreg及Treg细胞中TREC的拷贝数较CSA患者及CPS患者明显减低,提示ACS患者Treg细胞数目改变的同时存在内部稳态异常。
Objective: To investigate the subsets and apoptosis of regulatory T cells (Treg) in patients with acute coronary syndrome based on our previous studies.
Methods: A total of 109 subjects were enrolled in our study, including 18 patients with st-elevation myocardial infarction (STEAMI), 64 patients with NSTACS (including NSTEAMI and UA), 12 patients with chronic stable angina (CSA)and 15 patients with chest pain syndrome (CPS ). The peripherial lymphocytes were isolated using gradient-density confriguration. The ration of CD4~+CD25~+CD127-CD45RO~+ -CD45RA- memory Treg (mTreg), CD4~+CD25~+CD127-CD45RO-CD45RA~+ naive Treg (nTreg) , CD4~+CD25~+CD127-CD45RO-CD45RA~+TCD31~+ recent thymic emigrants-naive Treg (RTE-nTreg)and AnnexinⅤ~+7-AAD-CD4~+CD25~+CD127- in CD4~+CD25~+CD127-total Treg were analyzed using flow cytometry. 12 cases with ACS, 12 cases with CSA and 12 cases with CPS were also included and CD4~+CD25high Treg were purified by magnetic activated cell sorting (MACS), and T-cell receptor excision circles (TREC) in the purifed Treg were determined by realtime-PCR.
Results: The frequencies of mTreg and nTreg in total Treg were comparable among the STEAMI, NSTACS, CSA and CPS group. In contrast, the frequency of RTE-nTreg in total nTreg significantly decreased in both STEAMI group (16.91±7.90) and NSTACS group (20.39±10.31) compared to CSA group (29.78±9.86) and CPS group (31.54±10.52) (p<0.01). The frequency of apoptotic Treg in total Treg increased in the group of STEAMI group (18.42±10.93) and NSTACS group (16.24±9.67) increased compared with CSA group (12.76±6.52) and CPS group (11.38±4.85) , but the differences were not significant (STEAMI vs CSA, p=0.122; STEAMI vs CPS, p=0.068; NSTACS vs CSA, p=0.235; NSTACS vs CPS, p=0.170). The copies of TREC were also markedly lower in the ACS group (1.06±0.48) than in the CSA group (1.98±0.69) and CPS group (2.26±0.98) (p<0.05).
Conclusion: Our data demonstrated that the frequency of RTE-nTreg in total Treg and the the copies of TREC in purified Treg significantly decreased in STEAMI and NSTACS patients compared to CSA and CPS patients, suggesting that Treg homeostatis was disturbed in patients with ACS.
引文
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