江志超,唐發(fā)清,田道法,范靖瑩
(1.湖南省第二人民醫(yī)院,湖南長(zhǎng)沙410007;2.珠海市人民醫(yī)院,
廣東珠海519000;3.湖南中醫(yī)藥大學(xué)中西醫(yī)結(jié)合學(xué)院,湖南長(zhǎng)沙410007)
中晚期鼻咽癌患者CD4+CD25+T調(diào)節(jié)細(xì)胞和Th17細(xì)胞的免疫病理意義
江志超1,唐發(fā)清2,田道法3,范靖瑩3
(1.湖南省第二人民醫(yī)院,湖南長(zhǎng)沙410007;2.珠海市人民醫(yī)院,
廣東珠海519000;3.湖南中醫(yī)藥大學(xué)中西醫(yī)結(jié)合學(xué)院,湖南長(zhǎng)沙410007)
目的探討中晚期鼻咽癌患者外周血CD4+CD25+T調(diào)節(jié)細(xì)胞和Th17細(xì)胞的免疫病理意義。方法采用流式細(xì)胞術(shù)、RT-PCR方法檢測(cè)15例中晚期鼻咽癌患者外周血CD4+CD25+T調(diào)節(jié)細(xì)胞、Th17細(xì)胞比例和Foxp3、ROR-γt轉(zhuǎn)錄活性,并與10例健康體檢者相應(yīng)指標(biāo)進(jìn)行比較,分析其免疫病理意義。結(jié)果中晚期鼻咽癌患者外周血CD4+CD25+T調(diào)節(jié)細(xì)胞比例及Foxp3轉(zhuǎn)錄活性明顯高于健康體檢者(P<0.05);而Th17細(xì)胞比例及ROR-γt轉(zhuǎn)錄活性低于健康體檢者(P<0.05)。結(jié)論CD4+CD25+T調(diào)節(jié)細(xì)胞、轉(zhuǎn)錄因子Foxp3和Th17細(xì)胞比例、轉(zhuǎn)錄因子ROR-γt共同構(gòu)成一個(gè)平衡反饋體系,參與中晚期鼻咽癌患者腫瘤微環(huán)境中免疫耐受現(xiàn)象的發(fā)展及免疫逃逸的發(fā)生過(guò)程。
CD4+CD25+T調(diào)節(jié)細(xì)胞;Foxp3;Th17細(xì)胞;ROR-γt;鼻咽癌
腫瘤的發(fā)生、發(fā)展與機(jī)體的免疫細(xì)胞是否處于平衡狀態(tài)密切相關(guān),而作為細(xì)胞免疫的主體細(xì)胞T淋巴細(xì)胞及其亞群在腫瘤免疫中分化和作用不斷地被認(rèn)識(shí)。CD4+CD25+T調(diào)節(jié)細(xì)胞和Th17細(xì)胞是CD4+T細(xì)胞在不同的細(xì)胞因子刺激下所產(chǎn)生的亞群,CD4+CD25+T調(diào)節(jié)細(xì)胞作為對(duì)正向免疫應(yīng)答起負(fù)向調(diào)控作用的T淋巴細(xì)胞亞群,在肝癌、肺癌、胃癌等多種實(shí)體瘤中數(shù)量增多,且與這些腫瘤的發(fā)生、發(fā)展密切相關(guān)[1];而Th17細(xì)胞在腫瘤中的作用仍存在較大爭(zhēng)議,可能同時(shí)具有促進(jìn)腫瘤和抑制腫瘤的雙重作用[2]。作者擬通過(guò)檢測(cè)中晚期鼻咽癌患者外周血 CD4+CD25+T調(diào)節(jié)細(xì)胞、Th17細(xì)胞數(shù)量及其與相關(guān)轉(zhuǎn)錄因子Foxp3、ROR-γt水平,探討CD4+CD25+T調(diào)節(jié)細(xì)胞和Th17細(xì)胞在中晚期鼻咽癌患者免疫耐受病理中的意義。
1.1 臨床資料收集湖南省第二人民醫(yī)院、湖南中醫(yī)藥大學(xué)第一附屬醫(yī)院、湖南省腫瘤醫(yī)院經(jīng)病理檢查確診的中晚期鼻咽癌患者15例,年齡21~65歲,其中男11例,女4例;鱗癌9例,腺癌4例,未分化癌2例。所有患者均未接受過(guò)放、化療等干預(yù)因素處理。并設(shè)立同期健康體檢者對(duì)照組10例,年齡20~60歲。患者及健康體檢者均簽署了知情同意書。
1.2 試劑及儀器Human Regulatory TC ell Staining Kit、Human Th17 Flow TM Kit購(gòu)于美國(guó)eBioscience公司,RNA提取試劑盒、反轉(zhuǎn)錄試劑盒、UltraSYBR Mixture購(gòu)自北京康為世紀(jì)生物科技有限公司,PCR引物由上海生工合成。Cytomics TM FC500型流式細(xì)胞儀為美國(guó)Beckman Coulter公司產(chǎn)品;熒光定量PCR儀為美國(guó)Thermo公司產(chǎn)品。
1.3 標(biāo)本采集與處理符合納入標(biāo)準(zhǔn)的15例中晚期鼻咽癌患者與10名健康體檢者,分別于清晨空腹采集肝素鈉抗凝的外周靜脈血4 mL,用淋巴細(xì)胞分離液梯度離心法分離外周血單個(gè)核細(xì)胞,-70℃保存?zhèn)溆?,用于流式?xì)胞術(shù)分析和RT-PCR檢測(cè)。
1.4 流式細(xì)胞儀檢測(cè)外周血CD4+CD25+T調(diào)節(jié)細(xì)胞密度梯度離心法分離獲得外周血單個(gè)核細(xì)胞。按Human Regulatory T Cell Staining Kit說(shuō)明書進(jìn)行操作,應(yīng)用CD4、CD25、Foxp3抗體做細(xì)胞表面三色染色,采用FC500型流式細(xì)胞儀進(jìn)行檢測(cè)。測(cè)定10 000個(gè)細(xì)胞,激發(fā)波長(zhǎng)為488 nm,發(fā)射波長(zhǎng)為535 nm。分析時(shí)設(shè)門以除去細(xì)胞碎片和細(xì)胞團(tuán)塊,排除對(duì)測(cè)定過(guò)程的干擾。以同型抗體染色作為陰性對(duì)照。
1.5 流式細(xì)胞儀檢測(cè)外周血Th17細(xì)胞將外周血單個(gè)核細(xì)胞在體積分?jǐn)?shù)10%胎牛血清的RPMI 1640培養(yǎng)基中調(diào)整細(xì)胞密度至2×106細(xì)胞/mL,加入50 μg·L-1佛波醇乙酯、離子霉素和莫能霉素共刺激培養(yǎng)5 h(37℃,體積分?jǐn)?shù)5%CO2);用三色標(biāo)記的CD3、CD4、IL-17抗體對(duì)IL-17分子進(jìn)行細(xì)胞因子胞內(nèi)染色標(biāo)記,按Human Th17 Flow TM Kit說(shuō)明書進(jìn)行操作。采用FC500型流式細(xì)胞儀進(jìn)行檢測(cè),測(cè)定10 000個(gè)細(xì)胞,激發(fā)波長(zhǎng)為488 nm,發(fā)射波長(zhǎng)為535 nm,分析時(shí)設(shè)門以除去細(xì)胞碎片和細(xì)胞團(tuán)塊對(duì)測(cè)定的干擾。以同型抗體染色作為陰性對(duì)照。
1.6 RT-PCR檢測(cè)Foxp3 mRNA和ROR-γt mRNA參照有關(guān)文獻(xiàn),運(yùn)用Primer5.0軟件設(shè)計(jì)引物序列,由上海生物工程技術(shù)有限公司合成ROR-γt引物。其中 Foxp3引物 Forward Primer為 5'-CACAACATGCGACCCCCTTTCACC-3',Reverse Primer為5'-AGGTTGTGGCGGATGGCGTTCTTC-3';ROR-γt引物Forward Primer為 5'-CCTGGGCTCCTCGCCTGACC-3',Reverse Primer為5'-TCTCTCTGCCCTCAGCCTTGCC-3';β-actin引物 Forward Primer為 5'-CATCCTGCGTCTGGACCTGG-3',Reverse Primer為5'-TAATGTCACGCACGATTTCC-3'。Trizol法提取外周血單個(gè)核細(xì)胞總RNA,瓊脂糖凝膠電泳鑒定RNA的濃度、純度,顯示提取的各樣本總RNA具有很好的濃度和純度后,繼續(xù)后續(xù)的RT-PCR實(shí)驗(yàn)。用熒光定量PCR儀進(jìn)行ROR-γt mRNA擴(kuò)增,采用2-△△ct法計(jì)算Foxp3 mRNA相對(duì)表達(dá)量。
1.7 統(tǒng)計(jì)學(xué)處理采用SPSS 21.0處理數(shù)據(jù),計(jì)量資料用s表示,數(shù)據(jù)符合正態(tài)分布,2組比較采用成組設(shè)計(jì)t檢驗(yàn),檢驗(yàn)水準(zhǔn)α=0.05。
2.1 CD4+CD25+T調(diào)節(jié)細(xì)胞和Th17細(xì)胞比例比較見(jiàn)表1,圖1、2。
表1 CD4+CD25+T細(xì)胞、Th17細(xì)胞占CD4+T細(xì)胞比例比較%
2.2 Foxp3和ROR-γt mRNA水平比較中晚期鼻咽癌患者外周血 Foxp3 mRNA表達(dá)水平(3.699± 0.309)明顯高于健康體檢者(1.019±0.146),而ROR-γt mRNA表達(dá)水平(0.303±0.115)明顯低于健康體檢者(1.007±0.039),差異有均統(tǒng)計(jì)學(xué)意義(P均<0.05)。見(jiàn)圖3。
圖1 健康體檢者(A)和中晚期鼻咽癌患者(B)外周血CD4+CD25+T調(diào)節(jié)細(xì)胞流式細(xì)胞圖
圖2 健康體檢者(A)和中晚期鼻咽癌患者(B)外周血Th17細(xì)胞流式細(xì)胞圖
圖3 Foxp3、ROR-γt基因表達(dá)水平比較結(jié)果
腫瘤的發(fā)生、發(fā)展涉及免疫監(jiān)視、免疫耐受、免疫逃逸、免疫無(wú)能等與機(jī)體免疫紊亂有關(guān)的問(wèn)題。免疫耐受現(xiàn)象的持續(xù)存在,可引起突變細(xì)胞對(duì)宿主體內(nèi)免疫監(jiān)視功能的無(wú)反應(yīng)性,出現(xiàn)腫瘤細(xì)胞的免疫逃逸,其中起主導(dǎo)作用的細(xì)胞主要是CD4+CD25+T調(diào)節(jié)細(xì)胞[3-4]。諸多研究[5-6]表明,CD4+CD25+T調(diào)節(jié)細(xì)胞在肝癌、肺癌、胃癌、結(jié)腸癌等惡性腫瘤患者外周血和病變組織中均有不同程度的升高,且與腫瘤的預(yù)后關(guān)系密切。本研究結(jié)果顯示,中晚期鼻咽癌患者外周血單個(gè)核細(xì)胞中,CD4+CD25+T調(diào)節(jié)細(xì)胞占CD4+T淋巴細(xì)胞的比例明顯高于健康體檢者,提示中晚期鼻咽癌患者CD4+CD25+T調(diào)節(jié)細(xì)胞水平的升高,應(yīng)該是該類患者病情進(jìn)展的重要相關(guān)因素之一。同時(shí),中晚期鼻咽癌患者外周血單個(gè)核細(xì)胞中Foxp3 mRNA表達(dá)水平也明顯高于健康體檢者,與CD4+CD25+T調(diào)節(jié)細(xì)胞數(shù)量比例變化趨勢(shì)相一致,進(jìn)一步顯示了Foxp3 mRNA對(duì)CD4+CD25+T調(diào)節(jié)細(xì)胞分化與增殖活性的影響及其在腫瘤免疫耐受現(xiàn)象中重要作用。T淋巴細(xì)胞在不同的細(xì)胞因子的影響下,可以分化為不同的CD4+T淋巴細(xì)胞亞型,在TGF-β的影響下,可以分化為CD4+CD25+T調(diào)節(jié)細(xì)胞,而在同時(shí)有IL-6存在的情況下,則分化為Th17細(xì)胞。也就是說(shuō)CD4+CD25+T細(xì)胞與Thl7細(xì)胞的誘導(dǎo)分化過(guò)程存在微妙的相互關(guān)系,影響著機(jī)體的免疫穩(wěn)態(tài),涉及腫瘤、自身免疫性疾病的發(fā)生[7]。
有關(guān)Th17細(xì)胞在腫瘤中的作用存在2種不同觀點(diǎn)[8-10]:有研究認(rèn)為Th17細(xì)胞能夠促進(jìn)腫瘤生長(zhǎng),也有研究認(rèn)為這類細(xì)胞能夠抑制腫瘤,甚至還有學(xué)者提出Th17細(xì)胞只是起著一個(gè)“旁觀者”的作用。本研究發(fā)現(xiàn),中晚期鼻咽癌患者外周血中,Th17細(xì)胞占CD4+T淋巴細(xì)胞的比例明顯低于健康體檢者,且ROR-γt mRNA表達(dá)水平也明顯低于健康體檢者。提示在中晚期鼻咽癌患者,Th17細(xì)胞及其轉(zhuǎn)錄因子ROR-γt應(yīng)該具有積極的抗腫瘤作用。Horlock等[11]也發(fā)現(xiàn),與健康體檢者相比,乳腺癌患者外周血Th17細(xì)胞水平明顯偏低,這同我們的觀察結(jié)果是相一致的。
基于上述研究發(fā)現(xiàn),中晚期鼻咽癌患者在CD4+CD25+T淋巴細(xì)胞數(shù)量及其轉(zhuǎn)錄水平升高的同時(shí),伴隨Th17細(xì)胞數(shù)量比例及其轉(zhuǎn)錄水平明顯下降,表明這類患者體內(nèi)存在CD4+CD25+T淋巴細(xì)胞與Th17細(xì)胞失平衡現(xiàn)象,有可能是促進(jìn)中晚期鼻咽癌免疫耐受存在的重要原因之一。且這種不平衡可能還與病情進(jìn)展過(guò)程有關(guān),到了腫瘤的中晚期,Th17細(xì)胞減低趨勢(shì)日益明顯,而CD4+CD25+T淋巴細(xì)胞數(shù)量則更趨增多,免疫耐受現(xiàn)象更為突出。
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Immunopathological Significance of CD4+CD25+Regulatory T Cells and Th17 Cells in Patients with Intermiddle and Advanced Stage Nasopharyngeal Carcinoma
Jiang Zhichao1,Tang Faqing2,Tian Daofa3,F(xiàn)an Jingying3
(1.The Second People's Hospital of Hunan Province,Changsha 410007,China;
2.The People's Hospital of Zhuhai City,Zhuhai 519000,China;3.College of Integrated
Traditional Chinese and Western Medicine,Hunan University of Chinese Medicine,Changsha 410007,China)
ObjectiveTo investigate the immunopathological significance of CD4+CD25+regulatory T cells and Th17 cells in peripheral blood of the patients with intermiddle and advanced stage nasopharyngeal carcinoma.Methods In this study,15 cases of intermiddle and advanced stage nasopharyngeal carcinoma of the observation group and 10 healthy persons of the control group were included to detect their ratio of CD4+CD25+T cells and Th17 cells in the peripheral blood by flow cytometry technique,transcriptional activity of Foxp3 and ROR-γt by RT-PCR procedure.Then,a comparative analysis was made to explore their implications in the immunopathological correlation of nasopharyngeal carcinoma.ResultsThe ratio of CD4+CD25+T cells to total CD4+T cells and transcriptional activity of Foxp3 in the peripheral blood among the patients with intermiddle and advanced stage nasopharyngeal carcinoma were significantly higher than those in the healthy persons(P<0.05).Meanwhile,the ratio of Th17 cells to total CD4+T cells and transcriptional activity of ROR-γt in the peripheral blood among the patients with intermiddle and advanced stage nasopharyngeal carcinoma was significantly lower than those in the healthy persons(P<0.05).ConclusionIt may be true that CD4+CD25+T cells,transcriptiona regulatory factor Foxp3,and Th17 cells as well as ROR-γt altogether compose of a regulating system in a balance feedback way to evolve the developing process ofimmune tolerance phenomena in the microtumor enviroment and the implication of immune escape among patients with intermiddle and advanced stage nasopharyngeal carcinoma.
CD4+CD25+regulatory T cells;Foxp3;Th17 cells,ROR-γt;nasopharyngeal carcinoma
10.3969/j.issn.1673-5412.2014.02.004
R739.63;R730.23
A
1673-5412(2014)02-0105-04
2013-07-01)
國(guó)家自然科學(xué)基金資助項(xiàng)目(編號(hào):30973856);湖南省中醫(yī)藥管理局科研重點(diǎn)項(xiàng)目(編號(hào):2010007)
江志超(1977-),男,博士,主治醫(yī)師,主要從事頭頸腫瘤防治研究。E-mail:jiang.zhichao@126.com
田道法(1951-),男,教授,主要從事頭頸腫瘤防治研究。E-mail:tiandaofa@163.com