目的基于酪氨酸激酶/信号传导及转录激活蛋白(Janus kinase/signal transducer and activator of transcription,JAK/STAT)信号通路与免疫缺陷性疾病中CD4^(+)T细胞占比减少的相关性,探讨免疫缺陷大鼠CD4^(+)T淋巴细胞分化的机制。方法...目的基于酪氨酸激酶/信号传导及转录激活蛋白(Janus kinase/signal transducer and activator of transcription,JAK/STAT)信号通路与免疫缺陷性疾病中CD4^(+)T细胞占比减少的相关性,探讨免疫缺陷大鼠CD4^(+)T淋巴细胞分化的机制。方法将SPF级SD大鼠48只,随机分为正常大鼠(24只)和模型大鼠(24只),采用环孢素制备免疫缺陷模型。每组随机选6只验证造模效果,将剩余36只大鼠分为正常组、低鲁组、高鲁组、模型组、模型低鲁组、模型高鲁组,每组6只。低鲁组和模型低鲁组分别注射1.75 mg·kg^(-1)鲁索利替尼,高鲁组和模型高鲁组分别注射3.5 mg·kg^(-1)鲁索利替尼,正常组和模型组注射1.75 mg·kg^(-1)生理盐水,隔日1次,共注射6次。采用流式细胞术检测CD4^(+)T和CD8^(+)T淋巴细胞百分比,计算大鼠脾脏和胸腺指数,HE染色法观察脾脏和胸腺病理改变,ELISA检测白细胞介素-2(interleukin-2,IL-2)、γ-干扰素(interferon-γ,IFN-γ)、白细胞介素-12(interleukin-12,IL-12)、白细胞介素-4(interleukin-4,IL-4)、白细胞介素-6(interleukin-6,IL-6)、白细胞介素-10(interleukin-10,IL-10)细胞因子表达量,Western blot检测脾脏组织中酪氨酸激酶2(Janus kinase 2,JAK2)、T盒家族转录因子表达蛋白(T-box family transcription factor expression protein,T-bet)、信号传导及转录激活蛋白4(signal transducer and activator of transcription 4,STAT4)、信号传导及转录激活蛋白6(signal transducer and activator of transcription 6,STAT6)和GATA结合蛋白-3(GATA-binding protein-3,GATA3)蛋白表达量。结果模型组大鼠CD4^(+)T淋巴细胞百分比、胸腺指数较正常组明显下降(P<0.05)。与正常组比较,模型组CD4^(+)T淋巴细胞百分比减少(P<0.05),IL-2、IFN-γ和IL-12表达量下降(P<0.01),IL-10表达量升高(P<0.05);与模型组相比,模型高鲁组CD4^(+)T淋巴细胞百分比、胸腺和脾脏指数、IL-2、IFN-γ显著下降(P<0.05),而GATA3、STAT6蛋白表达量升高(P<0.05),IL-6、IL-10表达量明显增加(P<0.05,P<0.01)。结论免疫缺陷疾病以CD4^(+)T淋巴细胞减少为主要特征,CD4^(+)T细胞亚群失调与JAK/STAT信号通路表达失衡有关,其机制可能与IL-12/STAT4通路表达下调和IL-4/STAT6通路表达上调有关,CD4^(+)T淋巴细胞分化由辅助性T细胞1(helper T cell 1,Th1)向辅助性T细胞2(helper T cell 2,Th2)漂移,造成Th1/Th2失衡,引发免疫缺陷。展开更多
AIM To investigate the abundance and potential functions of LAP^+CD4^+ T cells in colorectal cancer(CRC). METHODS Proportions of LAP^+CD4^+ T cells were examined in peripheral blood and tumor/paratumor tissues of CRC ...AIM To investigate the abundance and potential functions of LAP^+CD4^+ T cells in colorectal cancer(CRC). METHODS Proportions of LAP^+CD4^+ T cells were examined in peripheral blood and tumor/paratumor tissues of CRC patients and healthy controls using flow cytometry. Expression of phenotypic markers such as forkhead box(Fox)p3, cytotoxic T-lymphocyte-associated protein(CTLA)-4, chemokine CC receptor (CCR)4 and CCR5 was measured using flow cytometry. LAP^-CD4^+ and LAP^+CD4^+ T cells were isolated using a magnetic cellsorting system and cell purity was analyzed by flow cytometry. Real-time quantitative polymerase chain reaction was used to measure expression of cytokines interleukin (IL)-10 and transforming growth factor(TGF)-β.RESULTS The proportion of LAP^+CD4^+ T cells was significantly higher in peripheral blood from patients (9.44% ± 3.18%) than healthy controls (1.49% ± 1.00%, P < 0.001). Among patients, the proportion of LAP^+CD4^+ T cells was significantly higher in tumor tissues(11.76% ± 3.74%) compared with paratumor tissues (3.87% ± 1.64%, P < 0.001). We also observed positive correlations between the proportion of LAP^+CD4^+ T cells and TNM stage(P < 0.001), distant metastasis(P < 0.001) and serum level of carcinoembryonic antigen(P < 0.05). Magnetic-activated cell sorting gave an overall enrichment of LAP^+CD4^+ T cells (95.02% ± 2.87%), which was similar for LAP^-CD4^+ T cells(94.75% ± 2.76%). In contrast to LAP^-CD4^+ T cells, LAP^+CD4^+ T cells showed lower Foxp3 expression but significantly higher levels of CTLA-4, CCR4 and CCR5(P < 0.01). LAP^+CD4^+ T cells expressed significantly larger amounts of IL-10 and TGF-β but lower levels of IL-2, IL-4, IL-17 and interferon-γ, compared with LAPCD4+ T cells.CONCLUSION LAP^+CD4^+ T cells accumulated in the tumor microenvironment of CRC patients and were involved in immune evasion mediated by IL-10 and TGF-β.展开更多
CD4 T helper （Th） cells play critical roles in adaptive immune responses. They recruit and activate other immune cells including B cells, CD8 T cells, macrophages, mast cells, neutrophils, eosinophils and basophils....CD4 T helper （Th） cells play critical roles in adaptive immune responses. They recruit and activate other immune cells including B cells, CD8 T cells, macrophages, mast cells, neutrophils, eosinophils and basophils. Based on their functions, their pattern of cytokine secretion and their expression of specific transcription factors, Th cells, differentiated from naive CD4 T cells, are classified into four major lineages, Thl, Th2, Th17 and T regulatory （Treg） cells, although other Th lineages may exist. Subsets of the same lineage may express different effector cytokines, reside at different locations or give rise to cells with different fates, whereas cells from different lineages may secrete common cytokines, such as IL-2, IL-9 and IL-10, resulting in massive heterogeneity of the Th cell population. In addition, the pattern of cytokine secretion may switch from that of one lineage toward another under certain circumstances, suggesting that Th cells are plastic. Tregs are also more heterogeneous and plastic than were originally thought. In this review, we summarize recent reports on heterogeneity and plasticity of Th cells, and discuss potential mechanisms and implications of such features that Th cells display.展开更多
目的:探讨人染色质解旋酶DNA结合蛋白4(recombinant chromodomain helicase DNA binding protein 4,CHD4)基因表达对急性T细胞淋巴细胞白血病细胞增殖和凋亡的影响及其启动子鉴定。方法:使用siRNA-CHD4瞬时转染T淋巴细胞白血病细胞(Jurk...目的:探讨人染色质解旋酶DNA结合蛋白4(recombinant chromodomain helicase DNA binding protein 4,CHD4)基因表达对急性T细胞淋巴细胞白血病细胞增殖和凋亡的影响及其启动子鉴定。方法:使用siRNA-CHD4瞬时转染T淋巴细胞白血病细胞(Jurkat)敲低CHD4基因表达,实时荧光定量q RT-PCR和Western blot检测细胞转染后CHD4表达量,流式细胞术测定细胞凋亡率及细胞周期变化;CCK-8分析测定CHD4基因对Jurkat细胞增殖的影响。根据生物信息学分析,以Jurkat细胞提取的全基因组DNA为模板,PCR扩增CHD4基因候选启动子区2 091 bp片段,以pGL3-Basic为载体,克隆含有CHD4基因候选启动子区的序列,制备重组质粒,并且构建一系列含CHD4基因候选启动子5′侧翼区截短序列质粒。将含CHD4启动子序列的质粒及截短序列质粒转染至Jurkat细胞和人胚胎肾T细胞(HEK293T),双荧光素酶报告基因检测各片段的启动子活性,确定CHD4基因启动子最小活性区域,并通过生物信息学方法分析该区域转录因子结合位点,构建结合位点突变的质粒,通过双荧光素酶报告基因检测,分析结合位点对CHD4转录的影响。结果:流式细胞检测结果发现,与对照组比较,CHD4抑制Jurkat细胞凋亡,转染siRNA-CHD4的Jurkat细胞G0/G1期比例显著升高,而S期比例下降(P<0.01);CCK-8检测CHD4基因对Jurkat细胞的增殖有促进作用(P<0.05);成功构建含有CHD4基因候选启动子序列的质粒及其截短序列质粒,与pGL3-Basic空载体相比,含有CHD4基因候选启动子序列的质粒活性明显增加(P<0.05)。CHD4基因最小活性区域位于转录起始位点-233~-13 bp,其中包含NF-κB、MZF1等转录因子结合位点;NF-κB对CHD4启动子活性具有正向调控作用。结论:CHD4基因对Jurkat细胞凋亡有抑制作用,并且促进其增殖。CHD4最小活性区域位于转录起始位点-233~-13 bp,转录因子NF-κB对CHD4基因启动子活性具有正向调控作用。展开更多
Background T cell factor 4 (TCF 4) plays an important role in development and carcinogenesis Recently, the role of TCF 4 has been described in colon cancer and other cancers However, whether TCF 4 plays a similar role...Background T cell factor 4 (TCF 4) plays an important role in development and carcinogenesis Recently, the role of TCF 4 has been described in colon cancer and other cancers However, whether TCF 4 plays a similar role in lung cancer is unknown To answer this question, we studied the expression of TCF 4 protein and mRNA in nonsmallcell lung cancer (NSCLC) and the relation of TCF 4 expression pattern to histological type and cell differentiation Methods Tissue samples from sixty cases of pathologically diagnosed NSCLC and eight normal tissue samples were obtained between September 2001 and March 2003 Immunohistochemistry was used to investigate the distribution of TCF 4 protein The staining patterns of the tumors were divided into 4 categories: nuclear staining alone or nuclear staining greater than cytoplasmic staining; cytoplasmic staining or cytoplasmic staining greater than nuclear staining; equal nuclear and cytoplasmic staining; no nuclear staining or cytoplasmic staining The integrated optical density (OD) values of all sections were analyzed by UIC MetaMorph image analysis software The expression of TCF 4 mRNA was detected by onestep reverse transcriptionpolymerase chain reaction (RTPCR) The integrated density values of the PCR products were analyzed semiquantitatively Results Immunohistochemistry showed that there was no expression of TCF 4 in normal tissue However, TCF 4 was expressed in 86.7% (52/60) of NSCLC samples, mainly in the nuclei of tumor cells Furthermore, there was a significant difference in TCF 4 localization patterns between squamous cell carcinomas and adenocarcinomas (P<005) The integrated OD values of TCF 4 expression was significantly higher in tumors with moderatepoor cell differentiation than in well differentiated tumors (5163±667 vs 4613±1231, P<001) There was no TCF 4 mRNA expression in normal tissue However, 63.9% (23/36) of carcinoma samples expressed TCF 4 mRNA TCF 4 mRNA expression was significantly higher in tumors with moderatepoor cell differentiation than in well differentiated tumors (P<005) There were no significant differences in mRNA expression in comparison with histological type Conclusions The subcellular distribution of TCF 4 may correlate with NSCLC histological type High expression of TCF 4 mRNA and protein may be associated with the degree of cell differentiation in NSCLC展开更多
Circulating tumour cells(CTCs)were enriched in the peripheral blood of four patients with Stage I non-small cell lung cancer(NSCLC).Octamer-binding transcription factor-4 positive(OCT4+)and negative(OCT4−)CTCs were id...Circulating tumour cells(CTCs)were enriched in the peripheral blood of four patients with Stage I non-small cell lung cancer(NSCLC).Octamer-binding transcription factor-4 positive(OCT4+)and negative(OCT4−)CTCs were identified and captured by interphase fluorescence in situ hybridisation(iFISH).Single cell whole exome sequencing(WES)was performed and the corresponding bioinformatics data were analysed.OCT4+cells were successfully detected in peripheral blood collected from all four Stage I lung cancer patients.Moreover,the tumour mutational burden(TMB)values observed for OCT4+samples from the same patients were slightly smaller than those of the OCT4−samples;the difference was not statistically significant(P>0.05).Thirteen and six characteristic mutations were found in negative samples and positive samples,respectively.The findings indicate that this methodology provides a potential diagnostic index for the early detection of NSCLC.展开更多
文摘目的基于酪氨酸激酶/信号传导及转录激活蛋白(Janus kinase/signal transducer and activator of transcription,JAK/STAT)信号通路与免疫缺陷性疾病中CD4^(+)T细胞占比减少的相关性,探讨免疫缺陷大鼠CD4^(+)T淋巴细胞分化的机制。方法将SPF级SD大鼠48只,随机分为正常大鼠(24只)和模型大鼠(24只),采用环孢素制备免疫缺陷模型。每组随机选6只验证造模效果,将剩余36只大鼠分为正常组、低鲁组、高鲁组、模型组、模型低鲁组、模型高鲁组,每组6只。低鲁组和模型低鲁组分别注射1.75 mg·kg^(-1)鲁索利替尼,高鲁组和模型高鲁组分别注射3.5 mg·kg^(-1)鲁索利替尼,正常组和模型组注射1.75 mg·kg^(-1)生理盐水,隔日1次,共注射6次。采用流式细胞术检测CD4^(+)T和CD8^(+)T淋巴细胞百分比,计算大鼠脾脏和胸腺指数,HE染色法观察脾脏和胸腺病理改变,ELISA检测白细胞介素-2(interleukin-2,IL-2)、γ-干扰素(interferon-γ,IFN-γ)、白细胞介素-12(interleukin-12,IL-12)、白细胞介素-4(interleukin-4,IL-4)、白细胞介素-6(interleukin-6,IL-6)、白细胞介素-10(interleukin-10,IL-10)细胞因子表达量,Western blot检测脾脏组织中酪氨酸激酶2(Janus kinase 2,JAK2)、T盒家族转录因子表达蛋白(T-box family transcription factor expression protein,T-bet)、信号传导及转录激活蛋白4(signal transducer and activator of transcription 4,STAT4)、信号传导及转录激活蛋白6(signal transducer and activator of transcription 6,STAT6)和GATA结合蛋白-3(GATA-binding protein-3,GATA3)蛋白表达量。结果模型组大鼠CD4^(+)T淋巴细胞百分比、胸腺指数较正常组明显下降(P<0.05)。与正常组比较,模型组CD4^(+)T淋巴细胞百分比减少(P<0.05),IL-2、IFN-γ和IL-12表达量下降(P<0.01),IL-10表达量升高(P<0.05);与模型组相比,模型高鲁组CD4^(+)T淋巴细胞百分比、胸腺和脾脏指数、IL-2、IFN-γ显著下降(P<0.05),而GATA3、STAT6蛋白表达量升高(P<0.05),IL-6、IL-10表达量明显增加(P<0.05,P<0.01)。结论免疫缺陷疾病以CD4^(+)T淋巴细胞减少为主要特征,CD4^(+)T细胞亚群失调与JAK/STAT信号通路表达失衡有关,其机制可能与IL-12/STAT4通路表达下调和IL-4/STAT6通路表达上调有关,CD4^(+)T淋巴细胞分化由辅助性T细胞1(helper T cell 1,Th1)向辅助性T细胞2(helper T cell 2,Th2)漂移,造成Th1/Th2失衡,引发免疫缺陷。
基金Supported by the National Natural Science Foundation of China,No.81260316
文摘AIM To investigate the abundance and potential functions of LAP^+CD4^+ T cells in colorectal cancer(CRC). METHODS Proportions of LAP^+CD4^+ T cells were examined in peripheral blood and tumor/paratumor tissues of CRC patients and healthy controls using flow cytometry. Expression of phenotypic markers such as forkhead box(Fox)p3, cytotoxic T-lymphocyte-associated protein(CTLA)-4, chemokine CC receptor (CCR)4 and CCR5 was measured using flow cytometry. LAP^-CD4^+ and LAP^+CD4^+ T cells were isolated using a magnetic cellsorting system and cell purity was analyzed by flow cytometry. Real-time quantitative polymerase chain reaction was used to measure expression of cytokines interleukin (IL)-10 and transforming growth factor(TGF)-β.RESULTS The proportion of LAP^+CD4^+ T cells was significantly higher in peripheral blood from patients (9.44% ± 3.18%) than healthy controls (1.49% ± 1.00%, P < 0.001). Among patients, the proportion of LAP^+CD4^+ T cells was significantly higher in tumor tissues(11.76% ± 3.74%) compared with paratumor tissues (3.87% ± 1.64%, P < 0.001). We also observed positive correlations between the proportion of LAP^+CD4^+ T cells and TNM stage(P < 0.001), distant metastasis(P < 0.001) and serum level of carcinoembryonic antigen(P < 0.05). Magnetic-activated cell sorting gave an overall enrichment of LAP^+CD4^+ T cells (95.02% ± 2.87%), which was similar for LAP^-CD4^+ T cells(94.75% ± 2.76%). In contrast to LAP^-CD4^+ T cells, LAP^+CD4^+ T cells showed lower Foxp3 expression but significantly higher levels of CTLA-4, CCR4 and CCR5(P < 0.01). LAP^+CD4^+ T cells expressed significantly larger amounts of IL-10 and TGF-β but lower levels of IL-2, IL-4, IL-17 and interferon-γ, compared with LAPCD4+ T cells.CONCLUSION LAP^+CD4^+ T cells accumulated in the tumor microenvironment of CRC patients and were involved in immune evasion mediated by IL-10 and TGF-β.
文摘CD4 T helper （Th） cells play critical roles in adaptive immune responses. They recruit and activate other immune cells including B cells, CD8 T cells, macrophages, mast cells, neutrophils, eosinophils and basophils. Based on their functions, their pattern of cytokine secretion and their expression of specific transcription factors, Th cells, differentiated from naive CD4 T cells, are classified into four major lineages, Thl, Th2, Th17 and T regulatory （Treg） cells, although other Th lineages may exist. Subsets of the same lineage may express different effector cytokines, reside at different locations or give rise to cells with different fates, whereas cells from different lineages may secrete common cytokines, such as IL-2, IL-9 and IL-10, resulting in massive heterogeneity of the Th cell population. In addition, the pattern of cytokine secretion may switch from that of one lineage toward another under certain circumstances, suggesting that Th cells are plastic. Tregs are also more heterogeneous and plastic than were originally thought. In this review, we summarize recent reports on heterogeneity and plasticity of Th cells, and discuss potential mechanisms and implications of such features that Th cells display.
文摘Background T cell factor 4 (TCF 4) plays an important role in development and carcinogenesis Recently, the role of TCF 4 has been described in colon cancer and other cancers However, whether TCF 4 plays a similar role in lung cancer is unknown To answer this question, we studied the expression of TCF 4 protein and mRNA in nonsmallcell lung cancer (NSCLC) and the relation of TCF 4 expression pattern to histological type and cell differentiation Methods Tissue samples from sixty cases of pathologically diagnosed NSCLC and eight normal tissue samples were obtained between September 2001 and March 2003 Immunohistochemistry was used to investigate the distribution of TCF 4 protein The staining patterns of the tumors were divided into 4 categories: nuclear staining alone or nuclear staining greater than cytoplasmic staining; cytoplasmic staining or cytoplasmic staining greater than nuclear staining; equal nuclear and cytoplasmic staining; no nuclear staining or cytoplasmic staining The integrated optical density (OD) values of all sections were analyzed by UIC MetaMorph image analysis software The expression of TCF 4 mRNA was detected by onestep reverse transcriptionpolymerase chain reaction (RTPCR) The integrated density values of the PCR products were analyzed semiquantitatively Results Immunohistochemistry showed that there was no expression of TCF 4 in normal tissue However, TCF 4 was expressed in 86.7% (52/60) of NSCLC samples, mainly in the nuclei of tumor cells Furthermore, there was a significant difference in TCF 4 localization patterns between squamous cell carcinomas and adenocarcinomas (P<005) The integrated OD values of TCF 4 expression was significantly higher in tumors with moderatepoor cell differentiation than in well differentiated tumors (5163±667 vs 4613±1231, P<001) There was no TCF 4 mRNA expression in normal tissue However, 63.9% (23/36) of carcinoma samples expressed TCF 4 mRNA TCF 4 mRNA expression was significantly higher in tumors with moderatepoor cell differentiation than in well differentiated tumors (P<005) There were no significant differences in mRNA expression in comparison with histological type Conclusions The subcellular distribution of TCF 4 may correlate with NSCLC histological type High expression of TCF 4 mRNA and protein may be associated with the degree of cell differentiation in NSCLC
基金the National Natural Science Foundation of China(No.81773273)。
文摘Circulating tumour cells(CTCs)were enriched in the peripheral blood of four patients with Stage I non-small cell lung cancer(NSCLC).Octamer-binding transcription factor-4 positive(OCT4+)and negative(OCT4−)CTCs were identified and captured by interphase fluorescence in situ hybridisation(iFISH).Single cell whole exome sequencing(WES)was performed and the corresponding bioinformatics data were analysed.OCT4+cells were successfully detected in peripheral blood collected from all four Stage I lung cancer patients.Moreover,the tumour mutational burden(TMB)values observed for OCT4+samples from the same patients were slightly smaller than those of the OCT4−samples;the difference was not statistically significant(P>0.05).Thirteen and six characteristic mutations were found in negative samples and positive samples,respectively.The findings indicate that this methodology provides a potential diagnostic index for the early detection of NSCLC.
