靶向沉默膀胱癌T24細(xì)胞系VEGF對(duì)樹突狀細(xì)胞分化成熟及免疫功能的影響

黃一珂，刁思軍，梁 平，周婷婷，魏志濤，楊 偉，王 亮

(成都軍區(qū)總醫(yī)院 泌尿外科， 四川 成都 610083)

研究論文

靶向沉默膀胱癌T24細(xì)胞系VEGF對(duì)樹突狀細(xì)胞分化成熟及免疫功能的影響

黃一珂，刁思軍，梁 平，周婷婷，魏志濤，楊 偉，王 亮*

(成都軍區(qū)總醫(yī)院 泌尿外科， 四川 成都 610083)

目的觀察靶向沉默膀胱癌T24細(xì)胞系VEGF對(duì)樹突狀細(xì)胞(DC)分化成熟及免疫功能的影響。方法構(gòu)建VEGF慢病毒干擾載體LV-VEGFA-RNAi(實(shí)驗(yàn)組)及慢病毒陰性對(duì)照載體LV-CON(陰性對(duì)照組)，分別感染各組T24細(xì)胞，空白對(duì)照組不采取任何干預(yù)措施。分別用RT-PCR和ELISA檢測(cè)各組T24細(xì)胞VEGFmRNA和蛋白的表達(dá)。各組T24上清分別與PBMC來(lái)源的未成熟DC共培養(yǎng)，用流式細(xì)胞儀檢測(cè)DC分化成熟指標(biāo)CD1a、CD83及免疫功能指標(biāo)CD86。結(jié)果與空白對(duì)照組和陰性對(duì)照組相比，實(shí)驗(yàn)組T24細(xì)胞VEGFmRNA及蛋白的表達(dá)均受到顯著抑制(P<0.05)。實(shí)驗(yàn)組DC表型CD1a、CD83和CD86較空白對(duì)照組明顯增高(P<0.05)，較陰性對(duì)照組亦明顯增高(P<0.05)。結(jié)論靶向沉默膀胱癌T24細(xì)胞VEGF的表達(dá)，有利于微環(huán)境中DC的成熟及其免疫功能的發(fā)揮，可能通過(guò)修復(fù)DC被損害的免疫監(jiān)視功能，增加DC的抗腫瘤能力。

膀胱癌；微環(huán)境；血管內(nèi)皮生長(zhǎng)因子；樹突狀細(xì)胞

膀胱癌是泌尿系統(tǒng)最常見的惡性腫瘤，全世界每年大約有35～38萬(wàn)例發(fā)病報(bào)道[1]，免疫逃逸是導(dǎo)致膀胱癌復(fù)發(fā)和進(jìn)展的重要機(jī)制。樹突狀細(xì)胞(dendritic cells, DC)是目前發(fā)現(xiàn)的腫瘤微環(huán)境中最主要的，功能最強(qiáng)的抗原遞呈細(xì)胞(antigen-presenting cell, APC)，在機(jī)體的抗腫瘤免疫反應(yīng)中發(fā)揮著極為重要的作用。血管內(nèi)皮生成因子(vascular endothelial growth factor, VEGF)是目前活性最強(qiáng)的血管生成因子，同時(shí)也是腫瘤微環(huán)境中極為重要的免疫抑制因子。研究顯示[2]：腫瘤微環(huán)境中的免疫抑制因子會(huì)導(dǎo)致腫瘤患者外周血或腫瘤組織中DC前體細(xì)胞成熟度降低，成熟DC的數(shù)量減少，促使腫瘤發(fā)生免疫逃逸。因此推測(cè)，如果能夠沉默腫瘤細(xì)胞免疫抑制因子VEGF的分泌，將可能增加DC細(xì)胞的成熟度，修復(fù)DC細(xì)胞的抗原呈遞功能，甚至提高DC疫苗的療效。本研究利用RNAi技術(shù)，構(gòu)建VEGF慢病毒干擾載體LV-VEGFA-RNAi，靶向沉默膀胱癌T24細(xì)胞的VEGF表達(dá)，觀察T24細(xì)胞培養(yǎng)上清對(duì)PBMC來(lái)源的DC的分化成熟及免疫功能的影響。

1 材料與方法

1.1 材料

人膀胱癌T24細(xì)胞系(中國(guó)科學(xué)院上海生命科學(xué)研究院細(xì)胞資源中心)；人外周血單個(gè)核細(xì)胞(PMBC)(成都軍區(qū)總醫(yī)院干細(xì)胞實(shí)驗(yàn)室)；慢病毒干擾載體LV-VEGFA-RNAi和陰性對(duì)照載體LV-CON和(上海吉?jiǎng)P基因化學(xué)技術(shù)有限公司)；RPMI- 1640培養(yǎng)基和1%PBS緩沖液(Hyclone公司)；胎牛血清(Gibco公司)；胰蛋白酶和青鏈霉素(Millipore公司)；重組人IL- 4、重組人GM-CSF和重組人TNF-α(Peprotech公司)；RNA提取試劑盒(Sigma公司)；PrimeScriptTMRT reagent Kit with gDNA Eraser(Perfect Real Time)試劑盒和SYBR? Premix Ex TaqTMⅡ(Tli RNaseH Plus)(TaKaRa公司)；PCR引物(成都擎科梓熙生物技術(shù)有限公司)；人VEGF酶聯(lián)免疫吸附測(cè)定試劑盒(福因德科技有限公司)；FITC Mouse Anti-Human CD1a、APC Mouse Anti-Human CD83及PE Mouse Anti-Human CD86(Becton,Dickinson公司)。

1.2 方法

1.2.1 慢病毒干擾載體的分組：由上海吉?jiǎng)P基因化學(xué)技術(shù)有限公司合成的含有針對(duì)VEGF的有效干擾序列的慢病毒載體命名為L(zhǎng)V-VEGFA-RNAi(實(shí)驗(yàn)組)，不含有效干擾VEGF的陰性對(duì)照序列(無(wú)關(guān)序列)的慢病毒載體命名為L(zhǎng)V-CON(陰性對(duì)照組)。兩種載體所含序列見表1。

表1 兩種慢病毒載體所含序列Table 1 Sequences of two lentiviral vectors

1.2.2 膀胱癌T24細(xì)胞的感染：感染前用孔稀釋法對(duì)各組病毒進(jìn)行滴度測(cè)定，選擇所測(cè)滴度最高的病毒進(jìn)行后續(xù)實(shí)驗(yàn)。根據(jù)上海吉?jiǎng)P提供的慢病毒感染手冊(cè)規(guī)范，在正式感染前進(jìn)行預(yù)實(shí)驗(yàn)，測(cè)試出最佳感染參數(shù)。正式感染需根據(jù)比例，計(jì)算病毒用量。人膀胱癌T24細(xì)胞按實(shí)驗(yàn)組、陰性對(duì)照組和空白對(duì)照組的設(shè)置接種于12孔板，每孔0.5×105個(gè)細(xì)胞，每組設(shè)3個(gè)復(fù)孔，采用含體積分?jǐn)?shù)為10%胎牛血清和1%雙抗的RPMI-1640培養(yǎng)基，置于37 ℃、5% CO2的孵箱中培養(yǎng)，當(dāng)細(xì)胞培養(yǎng)至第3天對(duì)數(shù)增殖期時(shí)，按預(yù)實(shí)驗(yàn)得出的最佳感染參數(shù)(表2)分別對(duì)各組T24細(xì)胞進(jìn)行慢病毒的感染，12 h后更換培養(yǎng)基，熒光顯微鏡下觀察慢病毒感染效率，待各組細(xì)胞增殖至感染后第4天提取總RNA，使用GAPDH作內(nèi)參，具體引物信息如表3。按說(shuō)明書操作，反轉(zhuǎn)錄及定量PCR檢測(cè)各組細(xì)胞VEGF mRNA的表達(dá)，ELISA檢測(cè)各組細(xì)胞上清液中的VEGF的量，計(jì)算慢病毒干擾載體對(duì)目的基因VEGF的沉默效率。

1.2.3 T24細(xì)胞上清與DC共培養(yǎng)：使用含rh GM-CSF和rh IL- 4各100 ng/mL， 1%雙抗10% FBS的1640培養(yǎng)基將PBMC(含未成熟DCs)等量接種于底面積為25 cm2帶透氣閥的培養(yǎng)瓶，分別對(duì)應(yīng)前面的T24細(xì)胞分組，置于37 ℃、5%的CO2孵箱中培養(yǎng)至第4天和第6天，分別加入?yún)R合度達(dá)90%的各組T24細(xì)胞培養(yǎng)上清(100 μL/mL)，第9天時(shí)加入rh TNF-α(20 ng/mL)，培養(yǎng)至第14天時(shí)收集成熟DC。用鼠抗人CD1a、CD83、CD86單克隆抗體分別對(duì)各組DC標(biāo)記染色，進(jìn)行流式細(xì)胞分析。

表2 各組感染參數(shù)Table 2 Infection parameters of each group

*CM(complete medium).

表3 目的基因及內(nèi)參引物Table 3 Primers of VEGF and GAPDH

1.3 統(tǒng)計(jì)學(xué)分析

2 結(jié)果

2.1 慢病毒感染膀胱癌T24細(xì)胞

實(shí)驗(yàn)組和陰性對(duì)照組熒光強(qiáng)度相當(dāng)，而空白對(duì)照組因未進(jìn)行帶熒光的病毒感染，故熒光視野下無(wú)表達(dá)綠色熒光的T24細(xì)胞(圖1)

與陰性對(duì)照組和空白對(duì)照組相比，實(shí)驗(yàn)組T24細(xì)胞VEGFmRNA表達(dá)受到明顯抑制(72.39%，P<0.05)(表4)。

圖1 各組T24細(xì)胞的熒光表達(dá)情況Fig 1 Fluorescence expression of each T24 cell group(×40)

groupexpression(2-△△Ct)Ctnegativecontrolgroup 1.141±0.28920.45±0.04blankcontrolgroup 1.144±0.21320.90±0.20experimentalgroup 0.316±0.066*22.11±0.20

*P<0.05 compared with negative control group and blank control group.

與陰性對(duì)照組和空白對(duì)照組相比，實(shí)驗(yàn)組T24細(xì)胞上VEGF 蛋白的表達(dá)亦受到顯著抑制(75.26%,P<0.05)(表5)。

表5 各組T24細(xì)胞VEGF 蛋白表達(dá)情況

*P<0.05 compared with negative control group and blank control group.

2.2 T24細(xì)胞上清與DC共培養(yǎng)

T24細(xì)胞上清與PBMC來(lái)源的未成熟DC共培養(yǎng)至第14天，光鏡下觀察結(jié)果(圖2，箭頭所指為成熟DC表面的樹突)?？梢钥闯觯瑢?shí)驗(yàn)組DC變大變圓，出現(xiàn)了明顯的毛刺狀，呈懸浮增殖，為成熟DC特征；而陰性對(duì)照組和空白對(duì)照組DC則更多仍處于半貼壁狀態(tài)，毛刺不明顯，成熟程度較實(shí)驗(yàn)組低。

各組DC 3種標(biāo)志物表達(dá)情況如表6。從表中可以看出，實(shí)驗(yàn)組DC成熟標(biāo)志物CD1a、CD83和CD86的表達(dá)量明顯高于陰性對(duì)照組和空白對(duì)照組(P<0.05)(圖3)。

圖2 第14天各組DC在倒置顯微鏡下的生長(zhǎng)情況Fig 2 Growth of each group’s DC by inverted microscope on the 14th day

groupCD1aCD83 CD86 negativecontrol24.18±4.8724.12±5.0449.54±4.57blankcontrol21.62±3.7120.39±4.0545.42±5.03experimental49.47±5.16*45.81±5.12*83.92±5.67*

*P<0.05 compared with negative control group and blank control group.

圖3 各組DC經(jīng)流式細(xì)胞計(jì)分析結(jié)果Fig 3 Flow cytometry result of each group’s DC

3 討論

DC是腫瘤微環(huán)境中功能最強(qiáng)大的專職APC，能激活初始型T淋巴細(xì)胞針對(duì)特異性抗原產(chǎn)生免疫應(yīng)答[3]。DC具有未成熟(immature DC, imDC)及成熟(mature DC, mDC)兩種狀態(tài)，二者在形態(tài)、表型和功能等方面都有較大差異[4]。形態(tài)方面：imDC較小較圓，在鏡下如葡萄串樣集結(jié)成簇，呈半貼壁增殖，細(xì)胞表面較光滑；而mDC則呈分散懸浮增殖，細(xì)胞表面有明顯不規(guī)則突起[5]。在本實(shí)驗(yàn)中，T24細(xì)胞上清與PBMC來(lái)源的未成熟DC共培養(yǎng)至第14天，光鏡下觀察結(jié)果與上述特征相符。表型方面，imDC少量表達(dá)主要組織相容性復(fù)合體(major histocompatibility complex，MHC)分子[6]、共刺激分子(如CD80和CD86)[7]等；而mDC則高表達(dá)上述分子及一些特征性標(biāo)記(如CDla、 CD11c和CD83)[8]等。因此，本實(shí)驗(yàn)選取CD1a和CD83，以及CD86，分別用來(lái)反映DC的成熟度和免疫功能。功能方面，imDC負(fù)責(zé)抗原的攝取、加工和處理，誘導(dǎo)機(jī)體產(chǎn)生免疫耐受，體外激發(fā)混合淋巴細(xì)胞反應(yīng)的能力較弱；隨著imDC的不斷成熟，其攝取、加工和處理抗原的能力逐漸喪失[9]，取而代之的是不斷增強(qiáng)的呈遞抗原的能力以及活化T細(xì)胞的能力[10]。由此可見，VEGF通過(guò)將DC抑制在未成熟狀態(tài)，使之只具備加工、處理抗原的能力，而不具備呈遞抗原及活化T細(xì)胞的能力，故而使DC的抗腫瘤效應(yīng)無(wú)法發(fā)揮。

研究證實(shí)絕大多數(shù)腫瘤都有VEGF分泌，并且在患者的腫瘤組織及血清中都可檢測(cè)到VEGF異常增高[11]，膀胱癌也不例外[12]。而腫瘤細(xì)胞分泌的VEGF能夠抑制DC的分化成熟，誘發(fā)DC凋亡，通過(guò)阻礙DC的抗原遞呈功能，促使腫瘤發(fā)生免疫逃避[13- 14]。本實(shí)驗(yàn)中，針對(duì)VEGF設(shè)計(jì)的干擾序列經(jīng)慢病毒干擾載體感染T24細(xì)胞后，PCR和ELISA驗(yàn)證T24細(xì)胞VEGF表達(dá)下降，感染后的T24細(xì)胞上清與未成熟DC共培養(yǎng)之后，經(jīng)流式細(xì)胞計(jì)鑒定，實(shí)驗(yàn)組DC的CD1a、CD83和CD86的表達(dá)量明顯高于陰性對(duì)照組和空白對(duì)照組。證明T24細(xì)胞分泌VEGF，抑制了處于同一微環(huán)境中DC分化與成熟及免疫功能指標(biāo)的表達(dá)。

綜上所述，本研究提示通過(guò)對(duì)VEGF作用于DC途徑的干擾，可增加腫瘤微環(huán)境中DC的成熟度，修復(fù)DC的免疫功能，同時(shí)還可能提高DC疫苗的療效。

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Influence of targeted geneVEGFsilencing of bladder cancer cell line T24 on differentiation, maturation and function of dendritic cells

HUANG Yi-ke, DIAO Si-jun, LIANG Ping, ZHOU Ting-ting, WEI Zhi-tao, YANG Wei, WANG Liang*

(Dept. of Urology, Chengdu Military General Hospital, Chengdu 610083, China)

ObjectiveTo observe the influence of targeted geneVEGFsilencing of bladder cancer cell line T24 on differentiation, maturation and function of dendritic cells.MethodsA lentiviral vector named LV-VEGFA-RNAi(experimental group) for gene silencing targetingVEGFand a lentiviral vector named LV-CON(negative control group) without any valid sequences were constructed. The blank control group accepted no intervention measures. The expression of VEGF’s mRNA and protein of T24 cells from each group were detected by RT-PCR and ELISA respectively. Then the immature DCs were co-cultured respectively with the supernatant of all the groups as mentioned above. CD1a, CD83 as the maturation marker and CD86 as the immunity marker of the DCs were detected by flow cytometry.ResultsThe expression of VEGF’s mRNA and protein of T24 cells in the experimental group were obviously inhibited (P<0.05) as compared with that in the negative control group and the blank control group. DCs of the experimental group had an obviously increased(P<0.05) expression of CD1a, CD83 and CD86 compared with the negative control group and the blank control group.ConclusionsTargeted geneVEGFsilencing by RNAi has advantages to the growth and immunity of DCs, which may strengthen the anti-tumor capacity of the DCs by repairing their damaged immune monitoring function.

bladder cancer; microenvironment; VEGF; DC

2016- 10- 31

2017- 05- 02

吳階平基金(320.6750.13249)

*通信作者(correspondingauthor)：wangliangcd@sina.com

1001-6325(2018)01-0057-06

R694；R737.14；R392.12

A