徐華林　趙娟娟　崔盼盼　郭萌萌　陶弋婧　陳　超　徐　林

( 遵義醫(yī)學(xué)院免疫學(xué)教研室暨貴州省生物治療人才基地，遵義563099)

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MicroRNA-7在CD4+T細(xì)胞體外活化中的表達(dá)及意義①

徐華林趙娟娟崔盼盼郭萌萌陶弋婧陳超徐林

( 遵義醫(yī)學(xué)院免疫學(xué)教研室暨貴州省生物治療人才基地，遵義563099)

目的：觀察microRNA-7(miR-7)在體外活化CD4+T細(xì)胞中表達(dá)的變化，初步探討其意義。方法：采用免疫磁珠分選法(MACS)獲得FVB小鼠脾臟中CD4+CD62L+T細(xì)胞，經(jīng)抗CD3/CD28抗體刺激后，Real-time PCR檢測(cè)miR-7表達(dá)水平，F(xiàn)ACS檢測(cè)活化膜分子CD69的表達(dá)變化；進(jìn)一步觀察刺激不同時(shí)間點(diǎn)CD4+T細(xì)胞中miR-7表達(dá)的變化；觀察ERK抑制劑 PD98059 處理后，CD4+T細(xì)胞活化下miR-7表達(dá)的變化，同時(shí)CCK8法檢測(cè)細(xì)胞增殖變化，F(xiàn)ACS檢測(cè)CD69和CD62L分子的表達(dá)變化；最后， Real-time PCR檢測(cè)各組細(xì)胞中IL-6、IL-10和IFN-γ等細(xì)胞因子表達(dá)水平變化。結(jié)果：與對(duì)照組相比，抗CD3/CD28抗體刺激后，CD4+CD62L+T細(xì)胞中miR-7表達(dá)水平顯著上調(diào),CD69分子的表達(dá)明顯增加(P<0.05)；與刺激0 h組和24 h組相比，48 h組和72 h組miR-7的表達(dá)水平顯著上調(diào)(P<0.05)；PD98059處理組中，CD4+T細(xì)胞的miR-7表達(dá)水平顯著下降(P<0.05)，同時(shí)，活化膜分子CD69的表達(dá)比例明顯降低(P<0.05)；最后，細(xì)胞表達(dá)IL-6、IL-10和IFN-γ的水平均顯著減弱(P<0.05)。結(jié)論：miR-7在活化的CD4+T細(xì)胞中明顯上調(diào)，并與ERK信號(hào)相關(guān)，為后續(xù)探討miR-7在CD4+T細(xì)胞功能中的作用提供了前期實(shí)驗(yàn)基礎(chǔ)。

miR-7；CD4+T細(xì)胞；ERK信號(hào)途徑；細(xì)胞因子

微小RNA (microRNA，miRNA)是一類主要在轉(zhuǎn)錄后水平降解靶mRNA 或抑制蛋白質(zhì)翻譯來(lái)調(diào)控目的基因表達(dá)的非編碼小分子RNA[1]。大量研究顯示，miRNAs參與了機(jī)體組織細(xì)胞的發(fā)育、分化及腫瘤的發(fā)生、發(fā)展等多種重要的生物學(xué)過(guò)程[2,3]。miR-7作為miRNA分子家族成員之一，在肺癌、肝癌、乳腺癌等多種腫瘤的發(fā)生發(fā)展中有重要作用[4-6]。我們前期研究中也發(fā)現(xiàn)其可有效調(diào)控人肺癌細(xì)胞的體內(nèi)、外生長(zhǎng)和侵襲[7,8]。有意思的是，我們新近利用海綿體(Sponge)技術(shù)構(gòu)建了miR-7基因敲減(Knock down，KD)小鼠模型[9,10]，且發(fā)現(xiàn)在miR-7KD小鼠的腸系膜淋巴結(jié)αβCD4+T細(xì)胞的比例和細(xì)胞絕對(duì)數(shù)發(fā)生了顯著變化，且其活化相關(guān)膜分子CD62L和CD69的表達(dá)也發(fā)生明顯改變[11]，提示miR-7可能參與了CD4+T細(xì)胞活化等生物學(xué)功能的調(diào)控。然而， miR-7在CD4+T細(xì)胞的活化及相關(guān)功能中的潛在作用及分子機(jī)制至今仍未有研究報(bào)道。因此，本研究中我們擬初步觀察CD4+T細(xì)胞體外活化前后miR-7表達(dá)的變化，并初步探討其表達(dá)變化的可能機(jī)制，以期為后續(xù)深入探討miR-7在CD4+T細(xì)胞活化及功能中的作用提供前期實(shí)驗(yàn)依據(jù)。

1　材料與方法

1.1材料FVB 背景的SPF 級(jí)雌性野生型小鼠；鼠源CD4+CD62L+T細(xì)胞磁珠分選試劑盒(德國(guó)Miltenyibiotec公司)；ERK信號(hào)抑制劑PD98059(TOCRIS公司)；TRIZOL試劑、CDNA單鏈合成試劑盒以及SYBR Premix Ex Taq Ⅱ(2×)均購(gòu)自TaKaRa；R-Phycoerythrin(PE) 標(biāo)記的CD62L抗鼠的單克隆抗體，Percp-Cy5.5 標(biāo)記的CD4抗鼠單克隆抗體，Allophycocyain(APC)標(biāo)記的CD69 抗鼠單克隆抗體， Fixation/Perme-abilization Diluent (Ebioscie-nce)；CCK-8試劑(博士德公司)；顯微鏡(Olymp-us)；流式細(xì)胞儀(Beckman Coulter)；SW-CJ-2FD型超凈工作臺(tái)(蘇州凈化設(shè)備廠)；二氧化碳培養(yǎng)箱(THERMO公司)；TD5AWS臺(tái)式低速離心機(jī)(湘儀儀器)；C1000TMThermalcycler 熒光定量PCR儀。

1.2方法

1.2.1免疫磁珠分選CD4+CD62L+T細(xì)胞收集與培養(yǎng)將8 周齡WT 小鼠斷頸處死，酒精浸泡5 min，常規(guī)提取脾臟細(xì)胞后做成單細(xì)胞懸液，預(yù)留一部分脾細(xì)胞，剩余細(xì)胞按德國(guó)Miltenyibiotec公司磁珠分選試劑盒說(shuō)明書(shū)先陰性分選獲得CD4+T細(xì)胞，后經(jīng)陽(yáng)性分選獲得CD4+CD62L+T細(xì)胞和 CD4+CD62L-T細(xì)胞，計(jì)數(shù)，并預(yù)留一部分CD4+CD62L+T細(xì)胞，剩余CD4+CD62L+T細(xì)胞并按1.6×105孔密度鋪于預(yù)先用CD3抗體包被的96孔板，同時(shí)加入4 μg/ml的CD28和20 ng/ml的IL-2。48 h后，實(shí)驗(yàn)組加入ERK信號(hào)抑制劑(PD98059)，使終濃度為990 nmol/L，而對(duì)照組則加入相同劑量的無(wú)菌PBS。小鼠的CD4+CD62L+T細(xì)胞分為對(duì)照組和PD98059組。

1.2.2CCK-8法檢測(cè)CD4+CD62L+T細(xì)胞增殖情況Cont組和PD98059組在分別加入PBS和抑制劑培育24 h時(shí)，分別加入20 μl CCK8，輕柔混勻避免產(chǎn)生氣泡，繼續(xù)培養(yǎng)2 h后，利用酶標(biāo)儀檢測(cè)各孔的吸光度(OD)值(波長(zhǎng)450 nm)。

1.2.3流式細(xì)胞術(shù)檢測(cè)CD4+T細(xì)胞膜表面分子CD62L、CD69表達(dá)變化 獲得的未活化的CD4+CD62L+T細(xì)胞(對(duì)照組)和活化48 h后CD4+CD62L+T細(xì)胞(aCD3/CD28組)并制成單細(xì)胞懸液，PBS洗滌1遍，1 200 r/min離心5 min，吸掉上清，加入200 μl PBS重懸細(xì)胞，加入抗鼠熒光標(biāo)記抗體APC-CD69各1 μl，4℃避光孵育30 min，PBS洗滌2遍，加入PBS重懸細(xì)胞后用Beckman流式細(xì)胞儀檢測(cè)。后收集已加入無(wú)菌PBS共陪育24 h的對(duì)照組和加入ERK抑制劑共培育24 h的PD98059組細(xì)胞并分別制成單細(xì)胞懸液，PBS洗滌1遍，1 200 r/min離心5 min，棄去上清，加入200 μl PBS重懸細(xì)胞，加入抗鼠熒光標(biāo)記抗體Percp-Cy5.5-CD4、PE-CD62L、APC-CD69各1 μl，4℃避光孵育30 min，PBS洗滌2遍，加入PBS重懸細(xì)胞后用Beckman流式細(xì)胞儀檢測(cè)。

1.2.4miR-7表達(dá)水平的檢測(cè)將1.2.1的預(yù)留的CD4+T細(xì)胞、CD4+CD62L-T細(xì)胞、CD4+CD62L+T細(xì)胞(0 h的CD4+CD62L+T細(xì)胞)、經(jīng)抗CD3/CD28抗體活化24 h的CD4+CD62L+T細(xì)胞、經(jīng)抗CD3/CD28抗體活化48 h的CD4+CD62L+T細(xì)胞、加入PD98059抑制劑后共孵育24 h的PD98059組細(xì)胞、加入無(wú)菌PBS后共孵育24 h的對(duì)照組分別經(jīng)TRIZOL法提取RNA，使用U6和miR-7反轉(zhuǎn)錄引物，對(duì)提取的RNA進(jìn)行轉(zhuǎn)錄，最后經(jīng)基于SYBR GreenⅠ的Real-time PCR莖環(huán)法檢測(cè)各組miR-7和U6的表達(dá)水平[12]。

1.2.5CD4+T細(xì)胞相關(guān)細(xì)胞因子表達(dá)水平的檢測(cè)利用Real-time PCR，使用SYBR Premix Ex Taq Ⅱ(×2)檢測(cè)以下基因。見(jiàn)表1。

2　結(jié)果

2.1CD4+T細(xì)胞活化前后miR-7表達(dá)水平變化MACS常規(guī)分選小鼠CD4+CD62L+T細(xì)胞和CD4+CD62L-T細(xì)胞。我們首先檢測(cè)了CD4+CD62L+T細(xì)胞中miR-7的表達(dá)情況。結(jié)果顯示，與CD4+CD62L-T細(xì)胞相比，CD4+CD62L+T細(xì)胞低表達(dá)miR-7(圖1A，P<0.05)。重要的是，我們發(fā)現(xiàn)經(jīng)抗CD3/CD28抗體刺激48 h后，CD4+CD62L+T細(xì)胞中miR-7表達(dá)水平明顯增加，同時(shí)細(xì)胞膜分子CD69的表達(dá)水平顯著增加(圖1B、C，P<0.05)。

2.2在CD4+T細(xì)胞活化的不同時(shí)間點(diǎn) miR-7表達(dá)情況顯微鏡下觀察顯示，活化后的CD4+T細(xì)胞克隆數(shù)量明顯增多(圖2A)。Real-time PCR 結(jié)果檢測(cè)顯示CD4+T細(xì)胞中miR-7的表達(dá)水平在活化后12 h降低，然后逐漸增加，并到72 h達(dá)到高峰，呈現(xiàn)先下降后升高的趨勢(shì)(圖2B，P<0.05)。

2.3ERK抑制劑對(duì)CD4+CD62L+T細(xì)胞活化中miR-7表達(dá)的影響為了進(jìn)一步探討CD4+T細(xì)胞活化過(guò)程中miR-7表達(dá)上調(diào)的可能信號(hào)途徑機(jī)制，我們觀察了ERK抑制劑PD98059處理對(duì)miR-7表達(dá)的可能影響。結(jié)果顯示，與對(duì)照組相比，PD98059處理組中細(xì)胞增殖明顯減弱(圖3A、B，P<0.05)，更重要的是，在抗CD3/CD28抗體刺激48 h后加入抑制劑PD98059共孵育24 h，miR-7表達(dá)水平顯著下調(diào)(圖3C，P<0.05)。

2.4CD4+T細(xì)胞相關(guān)膜分子CD62L、CD69分子表達(dá)變化我們進(jìn)一步用FACS檢測(cè)CD4+T細(xì)胞CD62L、CD69分子表達(dá)水平。如圖4所示，與對(duì)照組相比，PD98059處理組細(xì)胞膜CD62L的表達(dá)水平較對(duì)照組顯著增加(圖4A，P<0.01)，而CD69分子的表達(dá)水平， PD98059組顯著降低(圖4B ，P<0.05)。

表1基因名稱及引物序列

Tab.1Gene names and prime sequences

MousegeneForwardprimerReverseprimerGAPDH5'-GAGCCAAACGGGTCATCATCT-3'5'-GAGGGGCCATCCACAGTCTT-3'IL-45'-AAGAGGACACTGGAGCAA-3'5'-AGACATCCCGAAGGTTCC-3'IL-65'-CCCAATTTCCAATGCTCTCCTA-3'5'-AGGAATGTCCACAAACTGATATGCT-3'IFN-γ5'-CACTCGACCTTAATCTA-3'5'-CTCTTGATTCAATGCTTCA-3'

圖1　CD4+CD62L+T細(xì)胞經(jīng)抗CD3/CD28抗體刺激后miR-7和CD69的表達(dá)變化Fig.1　Change on expression of miR-7 and CD69 in CD4+CD62L+T cells after anti-CD3/CD28 antibody stimulationNote: A,B.Real-time PCR assay;C.FACS assay.*.P<0.05,**.P<0.01，compared with control group.

圖2　CD4+T細(xì)胞活化的不同時(shí)間點(diǎn) miR-7表達(dá)情況Fig.2　Expression of miR-7 in activated CD4+T cells at different time pointsNote: A.Microscope(Arrow indicates cell clone)； B.Real-time PCR assay；*.P<0.05,compared with control group.

圖3　抑制劑PD98059作用后CD4+T細(xì)胞增殖和miR-7表達(dá)改變Fig.3　Effect of inhibitor PD98059 on proliferation of CD4+T cells and change of expression of miR-7Note: A.Microscope;B.CCK-8 assay;C.Real-time PCR assay.*.P<0.05，compared with control group.

圖4　抑制劑 PD98059作用后CD4+T細(xì)胞膜 CD62L和CD69分子的表達(dá)變化Fig.4　Effect of inhibitor PD98059 on expression of CD62L and CD69 in CD4+ T cellsNote: *.P<0.05,compared with control group.

圖5　抑制劑 PD98059作用后CD4+T細(xì)胞相關(guān)細(xì)胞因子的表達(dá)變化Fig.5　Effect of inhibitor PD98059 on expression of cytokines in CD4+T cellsNote: *.P<0.05;**.P<0.01.

2.5CD4+T細(xì)胞相關(guān)細(xì)胞因子的表達(dá)變化Real-time PCR結(jié)果顯示，與對(duì)照組相比，PD98059處理組CD4+T細(xì)胞的IL-4，IL-6和IFN-γ表達(dá)水平均顯著降低(如圖5A，P<0.05)。

3　討論

近年來(lái)，大量文獻(xiàn)報(bào)道m(xù)iRNAs與T細(xì)胞增殖、活化以及凋亡密切相關(guān)[13,14]。如miR-17-92在CD4+T細(xì)胞和CD8+T細(xì)胞上高表達(dá)，可促進(jìn)T淋巴細(xì)胞的增殖，導(dǎo)致血清抗體增加，致使自身免疫性疾病的發(fā)生[15]；類似的，miR-146a缺陷型的T細(xì)胞對(duì)T細(xì)胞受體(T cell receptor，TCR)信號(hào)有高度反應(yīng)性，促進(jìn)炎癥的發(fā)生[16]。對(duì)于miR-7而言，我們前期發(fā)現(xiàn)miR-7敲減后腸系膜淋巴結(jié)中αβCD4+T細(xì)胞的CD62L的表達(dá)水平顯著下調(diào)，而CD69的表達(dá)水平卻明顯上調(diào)，提示miR-7可能作為一個(gè)負(fù)性調(diào)控分子，參與CD4+T的活化等生物學(xué)功能調(diào)控[11]；而在本研究中，我們發(fā)現(xiàn)CD4+CD62L+T細(xì)胞較CD4+CD62L-T細(xì)胞低表達(dá)miR-7，進(jìn)一步發(fā)現(xiàn)CD4+T活化后，miR-7表達(dá)水平明顯增加，提示miR-7與CD4+T細(xì)胞的活化密切相關(guān)。有意義的是，進(jìn)一步發(fā)現(xiàn)在活化不同時(shí)間點(diǎn)上，miR-7在CD4+T細(xì)胞中表達(dá)水平呈現(xiàn)先下降后升高的趨勢(shì)，該趨勢(shì)與我們前期發(fā)現(xiàn)miR-7可能是CD4+T細(xì)胞重要的負(fù)調(diào)控分子是一致的。上述研究表明，特定miRNAs分子在CD4+T細(xì)胞的發(fā)育和功能中發(fā)揮了重要的調(diào)控作用。然而，miR-7對(duì)CD4+T細(xì)胞功能的確切調(diào)控效應(yīng)及機(jī)制有待后續(xù)研究闡明。

胞外調(diào)節(jié)蛋白激酶(Extracellular regulated protein kinases，ERK)信號(hào)轉(zhuǎn)導(dǎo)通路存在于大多數(shù)細(xì)胞內(nèi)，在將細(xì)胞外刺激信號(hào)轉(zhuǎn)導(dǎo)至細(xì)胞及其核內(nèi)，并引起細(xì)胞生物學(xué)反應(yīng)(如細(xì)胞增殖、分化、轉(zhuǎn)化及凋亡等)的過(guò)程中具有重要作用[17]。新近研究報(bào)道，ERK信號(hào)通路與miRNAs分子表達(dá)密切相關(guān)，如Wu等[18]發(fā)現(xiàn)在C2C12成肌細(xì)胞中，肌生成抑制素 (Myostatin，MSTN)可通過(guò)ERK信號(hào)通路調(diào)節(jié)miR-431的表達(dá)水平；Wang等[19]也報(bào)道在活化的CD4+T細(xì)胞中，抑制ERK信號(hào)后，miR-21表達(dá)水平明顯降低，同時(shí)，miR-21作為T(mén)細(xì)胞活化的一個(gè)反饋環(huán)節(jié)的重要調(diào)控因子，又可明顯促進(jìn)ERK磷酸化信號(hào)的上調(diào)。類似地，在本研究中，我們發(fā)現(xiàn)ERK信號(hào)通路的抑制劑PD98059可顯著抑制CD4+T細(xì)胞中miR-7的表達(dá)。與此同時(shí)，CD4+T細(xì)胞的增殖能力及活化相關(guān)分子CD69和CD62L的表達(dá)明顯改變。此外，我們還發(fā)現(xiàn)CD4+T細(xì)胞中細(xì)胞因子IL-4、IL-6和IFN-γ的表達(dá)均明顯下降。這些結(jié)果提示T細(xì)胞受體信號(hào)活化后，miR-7水平的改變可能與ERK信號(hào)通路密切相關(guān)。然而CD4+T細(xì)胞的增殖、活化等生物學(xué)功能的改變是否是由于miR-7水平改變所致，以及ERK信號(hào)通路改變miR-7表達(dá)水平的具體用機(jī)制仍不清楚。此外，CD4+T細(xì)胞活化過(guò)程中miR-7表達(dá)與其他信號(hào)途徑，如：信號(hào)轉(zhuǎn)導(dǎo)子和轉(zhuǎn)錄激活子(Signal transducer and activator of transcr-iption，Stat)、磷脂酰肌醇3 激酶/蛋白激酶B(Phosphatidylinositol 3 kinase /protein kinase B，PI3K/AKT)等是否存在關(guān)聯(lián)等問(wèn)題仍需進(jìn)一步深入研究。

總之，本研究中我們首次發(fā)現(xiàn)miR-7的表達(dá)水平在CD4+T細(xì)胞活化前后明顯改變，且該變化與ERK信號(hào)途徑密切相關(guān)，這為后續(xù)深入研究miR-7分子在CD4+T細(xì)胞活化及功能中的作用提供了重要前期實(shí)驗(yàn)基礎(chǔ)。

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[收稿2016-01-04修回2016-02-23]

(編輯許四平)

Significance and expression of miR-7 in activated CD4+T cells

XU Hua-Lin,ZHAO Juan-Juan,CUI Pan-Pan,GUO Meng-Meng,TAO Yi-Jing,CHEN Chao,XU Lin.

Department of Immunology,Zunyi Medical University,Talent Base of Biological Therapy of Guizhou Province,Zunyi 563099,China

Objective:To investigate the change of expression of miR-7 in activated CD4+T cells in vitro,and preliminary explore its possible significance.Methods: CD4+CD62L+T cells was purified from splenocytes of FVB mice by magnetic cell sorting system (MACS).After stimulation with anti-CD3/CD28 antibody,the relative expression of miR-7 was examined by Real-time PCR,and the expression level of CD69 molecular was analyzed by FACS.Furthermore,the relative expression of miR-7 in CD4+T cells was detected at different time points during stimulation.With the treatment of ERK inhibitor PD98059,change of miR-7 expression was determined by Real-time PCR.Meanwhile,the proliferation of CD4+T cells was examined by CCK-8 assay and the expression level of CD69 and CD62L molecular were analyzed by FACS.Finally,the expression of cytokines IL-6,IL-10,and IFN-γ were determined by Real-time PCR.Results: Compared with control group,the relative expression of miR-7 was increased significantly after stimulation with anti-CD3/CD28 antibody,as well as expression level of CD69 molecular was augmented(P<0.05).In contrasted with 0 h and 24 h,the expression of miR-7 was significantly increased after 48 h and 72 h during stimulation(P<0.05).Furthermore,the relative expression of miR-7 was significantly declined in CD4+T cells in ERK inhibitor PD98059 treatment group.Finally,the expression level of CD69 molecular,as well as cytokines IL-6,IL-10 and IFN-γ,were also decreased significantly(P<0.05).Conclusion: The relative expression of miR-7 was significantly increased in activated CD4+T cells,closely related to ERK pathway,which provided an important foundation for successive research work on exploring the functional role of miR-7 in the CD4+T cells.

miR-7;CD4+T cells;ERK pathway;Cytokines

10.3969/j.issn.1000-484X.2016.10.003

①本文受國(guó)家自然科學(xué)基金(No.31370918)、貴州省高層次創(chuàng)新人才計(jì)劃[黔科合人才(2016)4031號(hào)]和遵義醫(yī)學(xué)院優(yōu)秀青年人才計(jì)劃項(xiàng)目(15ZY-001)資助。

徐華林(1989年-)，男，在讀碩士，主要從事分子免疫學(xué)的研究。

及指導(dǎo)教師：徐林(1977年-)，男，博士，教授，主要從事腫瘤免疫學(xué)和分子免疫學(xué)研究，E-mail：xulinzhouya@163.com。

R392

A

1000-484X(2016)10-1419-05