特異性小干擾RNA沉默Itch基因增強(qiáng)小鼠T細(xì)胞對MFC胃癌細(xì)胞的免疫殺傷作用

湖北民族學(xué)院附屬民大醫(yī)院急診科，湖北 恩施 445000

特異性小干擾RNA沉默Itch基因增強(qiáng)小鼠T細(xì)胞對MFC胃癌細(xì)胞的免疫殺傷作用

粟英 蘭亞明 盧義瓊 田國紅 胡烈獻(xiàn)

湖北民族學(xué)院附屬民大醫(yī)院急診科，湖北 恩施 445000

背景與目的：Itch蛋白是一種具有調(diào)節(jié)T細(xì)胞免疫應(yīng)答起始的關(guān)鍵分子，屬于E3泛素轉(zhuǎn)移酶家族，廣泛參與細(xì)胞內(nèi)多種信號蛋白如ZAP70、P85、VAV、PLC-γ和PKC-θ等的泛素化修飾過程，在腫瘤誘導(dǎo)機(jī)體免疫耐受中起著重要作用。Itch通過調(diào)節(jié)T細(xì)胞表面受體活性及轉(zhuǎn)移生長因子-β信號通路來介導(dǎo)T細(xì)胞免疫無反應(yīng)性，誘導(dǎo)外周組織Treg細(xì)胞增殖。因此，通過改變Itch蛋白表達(dá)活性有望成為一種治療自體免疫性疾病和腫瘤的有效途徑。我們利用特異性小干擾RNA(small interfering RNA，siRNA)沉默T細(xì)胞Itch基因的表達(dá)，觀察轉(zhuǎn)染T細(xì)胞對小鼠MFC胃癌細(xì)胞的體外免疫殺傷作用。方法：分離615小鼠脾臟T細(xì)胞，篩選高效特異性沉默Itch基因的siRNA序列轉(zhuǎn)染T細(xì)胞，蛋白質(zhì)印跡法檢測各分組Itch蛋白的表達(dá)水平；轉(zhuǎn)染72 h后，利用酶聯(lián)免疫吸附法(enzyme-linked immunosorbent assay，ELISA)檢測細(xì)胞因子IL-2、INF-γ分泌情況，比較空白組、空轉(zhuǎn)組及轉(zhuǎn)染組T細(xì)胞與小鼠MFC胃癌細(xì)胞混合培養(yǎng)腫瘤殺傷率。結(jié)果：轉(zhuǎn)染48 h后，與對照組相比，轉(zhuǎn)染T細(xì)胞Itch蛋白表達(dá)率降低至16%；轉(zhuǎn)染72 h后，檢測轉(zhuǎn)染組、空轉(zhuǎn)組、空白組細(xì)胞因子IL-2分泌水平分別為(1 891.96±141.91)pg/mL，(1 241.69±91.67)pg/mL，(1 175.03±89.14)pg/mL(P＜0.001)，轉(zhuǎn)染組、空轉(zhuǎn)組、空白組細(xì)胞因子INF-γ分泌水平分別為(958.33±75.46)pg/mL，(683.33±66.67)pg/mL，(691.72±68.72)pg/mL(P＜0.05)。在體外實(shí)驗(yàn)中，與空白組及空轉(zhuǎn)組T細(xì)胞相比，轉(zhuǎn)染組T細(xì)胞能更高效地殺傷小鼠MFC胃癌細(xì)胞，最高殺瘤率達(dá)到(54.18±2.96)%。結(jié)論：利用特異性siRNA技術(shù)沉默Itch基因能夠促進(jìn)小鼠T細(xì)胞因子IL-2、INF-γ分泌，增強(qiáng)T細(xì)胞對小鼠MFC胃癌細(xì)胞的體外免疫殺傷作用。

Itch；基因沉默；T淋巴細(xì)胞；胃腫瘤；免疫療法

胃癌是全球最常見的惡性腫瘤之一，全球超過40%的胃癌患者發(fā)生在中國，死亡率居全部惡性腫瘤第2位［1］。對于大多數(shù)胃癌患者，外科手術(shù)是目前最主要的治療手段，但術(shù)后5年生存率不超過20%［2］。對于晚期轉(zhuǎn)移性胃癌，因喪失手術(shù)切除時(shí)機(jī)，對放化療敏感性不高，易產(chǎn)生化療耐藥現(xiàn)象，難以獲得持久緩解，預(yù)后較差。腫瘤免疫治療通過激發(fā)或調(diào)動機(jī)體的免疫系統(tǒng)，增強(qiáng)腫瘤微環(huán)境抗腫瘤免疫力，從而控制和殺傷腫瘤細(xì)胞，通過增強(qiáng)T細(xì)胞抗腫瘤免疫可望成為一種治療晚期胃癌的有效途徑。Itch蛋白是一種與T細(xì)胞活化和耐受直接相關(guān)的E3泛素鏈接酶，其通過泛素化T細(xì)胞信號轉(zhuǎn)導(dǎo)通路中PLC-1、PKC-θ、JunB、notch等底物蛋白以負(fù)性調(diào)節(jié)受體信號和誘導(dǎo)免疫耐受［3］。有實(shí)驗(yàn)研究表明Itch-/-T淋巴細(xì)胞明顯活化，能直接通過MHC-TCR/CD3相互作用激活T細(xì)胞，誘導(dǎo)TH2細(xì)胞增殖分化，促進(jìn)細(xì)胞因子IL-4、IL-5分泌，提高血清IgE水平，增強(qiáng)機(jī)體抗腫瘤免疫［4］。在本實(shí)驗(yàn)中，我們體外觀察Itch基因沉默的轉(zhuǎn)染T細(xì)胞對于615小鼠MFC胃癌細(xì)胞的免疫殺傷作用，為以Itch基因?yàn)榘邢蛎庖咧委熚赴┳龀鲆欢ㄌ剿鳌?/p>

1 材料和方法

1.1 實(shí)驗(yàn)動物和細(xì)胞株

615小鼠購于上海中國科學(xué)院，SPF級，6周齡，體質(zhì)量約15～20 g。利用美天尼免疫磁珠分離615小鼠脾臟T細(xì)胞，T細(xì)胞純度達(dá)94.2%，培養(yǎng)基配置：90%1640培養(yǎng)基、10%胎牛血清、0.197 g NaHC03、0.000 5 g ConA(5 μg/mL)。小鼠MFC胃癌細(xì)胞源自615小鼠前胃鱗癌移植瘤FC瘤組織，小塊法培養(yǎng)得到，購自上海中國科學(xué)院細(xì)胞庫，培養(yǎng)基配置：90%1640培養(yǎng)基、10%胎牛血清、0.197 g NaHC03、青霉素(100 IU/mL)、鏈霉素(l00 μg/mL)。

1.2 Itch siRNA設(shè)計(jì)合成

根據(jù)pubmed GENE信息查詢615小鼠ItchmRNA序列，打開Ambion公司在線設(shè)計(jì)軟件確定針對小鼠Itch基因的siRNA靶序列，根據(jù)siRNA設(shè)計(jì)原則進(jìn)行篩選得出4條符合條件的siRNA。序列如下：siRNA-1：正義鏈5’-CCACAACACUCGGAUUACUTT-3’，反義鏈 5’-AGUAAUCCGAGUGUUGUGGTT-3’；siRNA-2：正義鏈 5’-GCAAGCAGAUCAUGUGG UUTT-3’，反義鏈5’-GCAAGCAGAUCAU GUGGUUTT-3’；siRNA-3：正義鏈 5’-CACCUUAGUUACAUUUCAUTT-3’，反義鏈 5’-AUGAAAUGUAACUAAGGUGTT-3’；siRNA-4：正義鏈5’-GUAAUCCAACACCUUAGU UTT-3’，反義鏈5’-AACUAAGGUGUUGGAUUA CTT-3’。所有siRNA序列均由上海吉凱基因技術(shù)有限公司合成。

1.3 T淋巴細(xì)胞轉(zhuǎn)染

T細(xì)胞在6孔板中培養(yǎng)至細(xì)胞80%～90%融合時(shí)，用PBS緩沖液洗滌細(xì)胞3次，用Opti-MEM培養(yǎng)基稀釋吹打成單細(xì)胞懸液，按濃度1×105個(gè)/mL接種96孔板，每孔100 μL。按轉(zhuǎn)染內(nèi)容和目的不同，設(shè)轉(zhuǎn)染組、空轉(zhuǎn)組和空白組共3組，轉(zhuǎn)染組將LipofectamineTM2000脂質(zhì)體和4條siRNA、陰性對照分別溶于5 μL opti-MEM培養(yǎng)基中，LipofectamineTM2000脂質(zhì)體和siRNA含量各為0.5、1.5 μL(siRNA終濃度為200 nmol/L)，室溫放置5 min。輕輕混勻上述2管液體，室溫溫育20 min。將轉(zhuǎn)染混合物加入96孔板中，混勻后，加入小鼠CD3功能性抗體15 μL。37 ℃溫育6 h，加入20 μL胎牛血清?？辙D(zhuǎn)組中每孔加入等量陰性對照siRNA序列和LipofectamineTM2000脂質(zhì)體，空白組則僅加入等量opti-MEM培養(yǎng)基，其余處理同轉(zhuǎn)染組。另外利用熒光標(biāo)記的siRNA(FAM-siRNA)檢測轉(zhuǎn)染效率，方法同上，轉(zhuǎn)染6 h后熒光顯微鏡下觀察轉(zhuǎn)染效果，流式細(xì)胞儀檢測轉(zhuǎn)染效率。

1.4 Western blot檢測蛋白表達(dá)

T細(xì)胞轉(zhuǎn)染后繼續(xù)培養(yǎng)48 h，1 000 r/min離心5 min，PBS緩沖液洗1遍，全蛋白提取試劑盒提取T淋巴細(xì)胞蛋白，Lowry法測定蛋白濃度，取蛋白樣品對目的蛋白Itch的表達(dá)水平進(jìn)行檢測，轉(zhuǎn)PVDF膜80 V 2 h后，TBST稀釋的BSA(濃度1%)封閉l h，剪膜，加入一抗(1∶1 000稀釋) 4 ℃過夜溫育，加入HRP標(biāo)記二抗(l∶1 500稀釋)室溫溫育45 min，ECL顯色系統(tǒng)定影顯色，觀察雜交條帶。

1.5 ELISA檢測細(xì)胞因子IL-2，INF-γ分泌變化

參照凱基ELISA試劑盒說明要求，轉(zhuǎn)染48 h后，取細(xì)胞培養(yǎng)上清液，把標(biāo)準(zhǔn)品、稀釋液和待測樣品分別加入酶標(biāo)板中，每孔0.1 mL。37 ℃反應(yīng)120 min后，加入0.1 mL生物素抗小鼠細(xì)胞因子IL-2、INF-γ抗體工作液，37 ℃反應(yīng)60 min。加入親和素過氧化物酶復(fù)合物的工作液0.1 mL，37 ℃反應(yīng)30 min。每孔依次加入TMB顯色液，37 ℃避光反應(yīng)30 min，加入0.1 mL終止液，酶聯(lián)免疫監(jiān)測儀450 nm測定A值。

1.6 腫瘤殺傷率測定

轉(zhuǎn)染T細(xì)胞24 h后，將轉(zhuǎn)染T細(xì)胞、陰性對照T細(xì)胞及單純T細(xì)胞分別與MFC胃癌細(xì)胞按照效靶比40∶1、20∶1、10∶1接種于96孔板，每組3個(gè)復(fù)孔，體積各為100 μL。同時(shí)設(shè)單純T細(xì)胞和單純腫瘤細(xì)胞作為效應(yīng)細(xì)胞組和靶細(xì)胞組，每孔100 μL。共同培養(yǎng)72 h后，CCK-8試劑盒測定各組T細(xì)胞對小鼠MFC胃癌細(xì)胞的腫瘤殺傷作用。腫瘤殺傷率計(jì)算公式如下［5］：

腫瘤細(xì)胞殺傷率(%)=［(AE+AT)- AE+T］/AT×100%

AE+T=效應(yīng)細(xì)胞+靶細(xì)胞孔A值

AE=相應(yīng)濃度單獨(dú)效應(yīng)細(xì)胞的A值

AT=相應(yīng)濃度單獨(dú)靶細(xì)胞的A值

1.7 統(tǒng)計(jì)學(xué)處理

2 結(jié) 果

2.1 T淋巴細(xì)胞轉(zhuǎn)染率測定及Itch蛋白表達(dá)

LipofectamineTM2000脂質(zhì)體轉(zhuǎn)染T淋巴細(xì)胞6 h后，流式細(xì)胞儀檢測T淋巴細(xì)胞轉(zhuǎn)染率達(dá)81.39%(圖1)，熒光鏡下觀察視野中綠色熒光亮點(diǎn)(圖2)。Western blot檢測各組蛋白表達(dá)，可見4條siRNA抑制率分別是52%、24%、18%和84%，陰性對照組與空白組差異不大，siRNA-1抑制率最高，達(dá)84%，表明siRNA-1序列能高效特異性抑制615小鼠脾臟T細(xì)胞Itch蛋白表達(dá)(圖3)。

2.2 沉默Itch基因能增加T細(xì)胞因子IL-2，INF-γ分泌水平

轉(zhuǎn)染48 h后，ELISA檢測各分組T細(xì)胞上清液中細(xì)胞因子IL-2、INF-γ分泌水平變化，測定結(jié)果表明轉(zhuǎn)染組、空轉(zhuǎn)組、空白組細(xì)胞因子IL-2分泌水平分別為(1 891.96±141.91) pg/mL、(1 241.69±91.67)pg/mL和(1 175.03± 89.14)pg/mL，轉(zhuǎn)染組IL-2水平較空轉(zhuǎn)組和空白組均明顯升高(P＜0.001)；轉(zhuǎn)染組、空轉(zhuǎn)組、空白組細(xì)胞因子INF-γ分泌水平分別為(958.33±75.46)pg/mL、(683.33±66.67)pg/mL和(691.72±68.72)pg/mL，轉(zhuǎn)染組INF-γ水平較空轉(zhuǎn)組和空白組有所增加(P＜0.05，圖4)。

圖1 FAM-siRNA轉(zhuǎn)染T淋巴細(xì)胞效率Fig. 1 Transfection ef fi ciency of FAM-siRNA for T lymphocyte

圖2 T淋巴細(xì)胞熒光轉(zhuǎn)染圖Fig. 2 T lymphocyte fl uorescence transfection picture(×100)

圖3 Itch蛋白表達(dá)Fig. 3 The expression of The Itch protein

圖4 轉(zhuǎn)染48 h后細(xì)胞因子IL-2、INF-γ分泌水平Fig. 4 The secretion level of IL-2, INF-γ after 48 hours of transfection

2.3 沉默Itch基因能增強(qiáng)小鼠T細(xì)胞對MFC胃癌細(xì)胞免疫殺傷

轉(zhuǎn)染T細(xì)胞與MFC胃癌細(xì)胞共同培養(yǎng)72 h后，在各個(gè)不同效靶比水平轉(zhuǎn)染組T淋巴細(xì)胞均較空轉(zhuǎn)組及空白組T細(xì)胞殺瘤活性增強(qiáng)(P＜0.05)，隨著靶效比升高，T細(xì)胞對于MFC胃癌細(xì)胞殺傷率亦逐漸增加，當(dāng)效靶比為40∶1時(shí)，轉(zhuǎn)染組T細(xì)胞殺傷腫瘤細(xì)胞活性最高，達(dá)到(54.18±2.96)%(圖5)。

圖5 共同培養(yǎng)72 h后后腫瘤殺傷率測定Fig. 5 The killing activity measurement after co-cultured 48 h

3 討 論

目前認(rèn)為誘導(dǎo)抗腫瘤免疫應(yīng)答較其他腫瘤治療方法具有明顯優(yōu)勢，表現(xiàn)在一方面刺激活化的特異性T淋巴細(xì)胞不斷地增殖活化并在體內(nèi)游走，能清除體內(nèi)一切表達(dá)有特異性腫瘤免疫原性的腫瘤細(xì)胞；另一方面，免疫系統(tǒng)會產(chǎn)生對腫瘤細(xì)胞的記憶和監(jiān)視能力從而有效防止腫瘤復(fù)發(fā)［6］。胃癌發(fā)生、發(fā)展與機(jī)體免疫密切相關(guān)，有學(xué)者證實(shí)胃癌患者血液中能夠分離出針對癌細(xì)胞的腫瘤特異性T淋巴細(xì)胞，而腫瘤特異性T細(xì)胞在腫瘤組織中的大量增殖浸潤可明顯改善患者預(yù)后［7-8］。而胃癌患者腫瘤組織中髓源性抑制細(xì)胞和Treg細(xì)胞增殖可明顯抑制抗腫瘤免疫應(yīng)答，減少細(xì)胞毒性T細(xì)胞的產(chǎn)生，此類患者通常預(yù)后較差［9］。目前應(yīng)用于胃癌免疫療法研究主要包括非特異性免疫刺激劑、腫瘤疫苗和單克隆抗體及過繼免疫治療等，并取得一定進(jìn)展。已有Ⅲ期臨床實(shí)驗(yàn)結(jié)果表明胃癌患者接受CTLA-4單克隆抗體治療，其總體生存期和無進(jìn)展生存期均優(yōu)于安慰劑組，且副作用小，機(jī)體耐受良好［10］。而Bazas等［11］研究結(jié)果也表明，在應(yīng)用了自體腫瘤細(xì)胞疫苗的86例胃癌患者中，超過30%的Ⅲ期胃癌患者和14%的Ⅳ期胃癌患者3年生存率有一定程度提高。T細(xì)胞通過介導(dǎo)細(xì)胞免疫直接殺傷腫瘤細(xì)胞，在機(jī)體抗腫瘤免疫過程中扮演關(guān)鍵作用，而Itch蛋白在T淋巴細(xì)胞活化增殖及分化誘導(dǎo)過程中起著重要的負(fù)性調(diào)節(jié)作用。T淋巴細(xì)胞活化是一系列復(fù)雜的級聯(lián)反應(yīng)，需要多種活性蛋白酶的參與和調(diào)節(jié)［12］，而腫瘤從啟動到效應(yīng)整個(gè)階段中均伴隨著CD4+CD25+FOX3+調(diào)節(jié)性T細(xì)胞增殖，后者有利于腫瘤免疫耐受微環(huán)境形成［13］。E3泛素連接酶Itch蛋白通過泛素化T淋巴細(xì)胞活化信號通路的酪氨酸激酶及其下游重要信號蛋白分子來負(fù)性調(diào)節(jié)受體信號和誘導(dǎo)免疫無反應(yīng)性，以達(dá)到精細(xì)調(diào)節(jié)抗原受體信號和免疫應(yīng)答［14］。研究表明Itch 基因缺失的人［15］或小鼠［16］均發(fā)生嚴(yán)重的自身免疫和炎性反應(yīng)疾病，表現(xiàn)為肝脾腫大，廣泛的多器官(肺、肝臟、腸等)自體免疫性疾病，病理活檢示：肝、脾、肺等臟器淋巴細(xì)胞和巨噬細(xì)胞大量增殖與廣泛浸潤。因此，Itch蛋白的負(fù)性免疫調(diào)控作用為以Itch基因?yàn)榘邢蚣せ頣細(xì)胞免疫治療胃癌提供了可能。

本實(shí)驗(yàn)首先選取驗(yàn)證有效的特異性Itch siRNA序列，通過脂質(zhì)體轉(zhuǎn)染615小鼠脾臟T細(xì)胞以高效抑制Itch蛋白表達(dá)。細(xì)胞因子通過影響免疫細(xì)胞增殖分化，廣泛參與機(jī)體免疫應(yīng)答，具有抗腫瘤、免疫調(diào)節(jié)等多種生物活性。我們的實(shí)驗(yàn)結(jié)果表明，轉(zhuǎn)染48 h后，轉(zhuǎn)染組細(xì)胞因子IL-2、INF-γ分泌水平明顯升高，表明沉默Itch基因能誘導(dǎo)T淋巴細(xì)胞活化。我們進(jìn)一步體外觀察siRNA沉默Itch基因能否提高T淋巴細(xì)胞對于MFC胃癌細(xì)胞的體外免疫殺傷作用。T細(xì)胞通過識別腫瘤細(xì)胞表面的腫瘤抗原肽-MHCⅠ類分子復(fù)合物，進(jìn)而增殖分化，并通過FasL/Fas、顆粒酶等多種途徑殺傷腫瘤細(xì)胞。T細(xì)胞與靶細(xì)胞混合培養(yǎng)72 h，在不同靶效比情況下，轉(zhuǎn)染組T細(xì)胞均較空轉(zhuǎn)組及空白組具有更強(qiáng)的殺瘤活性。當(dāng)效靶比為40∶1時(shí)，腫瘤殺傷力最強(qiáng)，達(dá)到(54.18±2.96)%，這提示通過siRNA技術(shù)沉默T細(xì)胞Itch基因以增強(qiáng)其對于MFC胃癌細(xì)胞的細(xì)胞毒作用是可能的。當(dāng)然，這種腫瘤抑制作用還需要進(jìn)一步驗(yàn)證，必要時(shí)需要進(jìn)行相關(guān)動物實(shí)驗(yàn)研究?？傊?，我們的體外實(shí)驗(yàn)證實(shí)利用特異性小干擾RNA技術(shù)沉默Itch基因能夠一定程度地增強(qiáng)T淋巴細(xì)胞對MFC胃癌細(xì)胞的免疫殺傷作用。

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Silencing itch by small speci fi c interfering RNA enhance immune activity of mouse T lymphocyte to kill MFC stomach neoplasms cells in vitro

SU Ying, LAN Ya-ming, LU Yi-qiong, TIAN Guo-hong, HU

Lie-xian
(Emergency Department, the Affiliated MinDa Hospital of Hubei University for Nationalities, EnShi Hubei 445000, China)

SU-Ying E-mail: fl y100_inthesky@163.com

Background and purpose: Itch protein is an established regulator of T cell immune response thresholds, belong to a class of E3 ubiquitin-transferring enzymes, widely involve in the ubiquitination of several key signaling molecules, such as ZAP70, P85, VAV, PLC-γ, PKC-θ, etc, plays a critical role in tumor induced immunosuppression. Itch ligase activity regulate T-cell anergy and development of regulatory T cells in the periphery by modulating key components of T-cell receptor and transforming growth factor-β signaling. Therefore, manipulation of Itch activities may provide the opportunities to develop future therapies for immune disorders such as autoimmunity and cancer. speci fi c small interfering RNA(siRNA) was utilized to silence the expression of Itch gene of T-lymphocytes and investigate the cytotoxicity activity of transfected T lymphocytes against MFC stomach neoplasms cells in vitro. Methods: T lymphocytes were isolated from the spleen of 615 mice and transfected by speci fi c siRNA to silence the expression of Itch gene, The expression of Itch protein were examined by Western bolt in each group; 72 hours aftertransfection, The secretion level of IL-2, INF-γ were measured by enzyme-linked immunosorbent assay (ELISA). At the end, the cytotoxicity activity changes against MFC stomach neoplasms cells was compared between transfected T lymphocytes, negative control and blank control in vitro. Results: Compared with control group, the expression rate of Itch protein of transfected T-lymphocytes was decreased to 16% after transfection 48 hours; 72 hours after transfection, the secretion level of IL-2 in transfection group, negative control and blank control respectively were (1 891.96±141.91)pg/ mL, (1 241.69±91.67)pg/mL and (1 175.03±89.14)pg/mL (P＜0.001), the secretion level of INF-γ in transfection group, negative control and blank control respectively were (958.33±75.46)pg/mL, (683.33±66.67)pg/mL and (691.72±68.72) pg/mL (P＜0.05). Transfected T lymphocyte also showed more ef fi cient killing ability against MFC stomach neoplasms cells than negative control and blank control in vitro, the highest killing rate has reached (54.18±2.96)%. Conclusion: Silencing Itch gene can signi fi cantly promoted the secretion level of IL-2, INF-γ of mice T lymphocyte, enhanced the cytotoxicity activity of T lymphocyte against MFC stomach neoplasms cells in vitro.

Itch; Gene silencing; T-lymphocytes; Stomach neoplasms; Adoptive immunotherapy

10.3969/j.issn.1007-3969.2014.10.011

R73-36;R735.2

A

1007-3639(2014)10-0777-06

2014-07-11

2014-08-25）

粟英 E-mail: fl y100_inthesky@163.com