以GFP為標(biāo)記的肝癌細(xì)胞RNA轉(zhuǎn)染樹(shù)突狀細(xì)胞的體外效應(yīng)

1、    以GFP為標(biāo)記的肝癌細(xì)胞RNA轉(zhuǎn)染樹(shù)突狀細(xì)胞的體外效應(yīng)            作者：時(shí)間：2007-11-22 11:33:00                          &

2、#160;     作者：張利旺，張紅梅，劉文超，潘伯榮，斯曉明，任軍 【關(guān)鍵詞】  樹(shù)突狀細(xì)胞；綠色熒光蛋白；RNA；轉(zhuǎn)染Effect of dendritic cells transfected with total RNA of HepG2 cell line using GFP marker in vitro【Abstract】 AIM: To investigate the feasibility of GFP as a marker to observe the dendritic cells (DCs) transfected w

3、ith total RNA of tumor cells and the feasibility of the transfected DCs serving as a vaccine for potential immunotherapy. METHODS: Plasmid pGFPC3 was transfected into HepG2 stably. Total RNA was extracted from the HepG2GFP using Trizol; DCs were induced by liver cancer patients peripheral blood mono

4、nuclear cells (PBMCs), and transfected with the total RNA. The effect of transfection was observed by a fluorescence microscope, the changes of phenotype were detected by flow cytometry, and the change of IL12 secretion in the supernatant of DCs was detected by ELISA assay. The cytotoxic effect of C

5、TLs was assessed by MTT assay. RESULTS: GFP was expressed stably in the HepG2GFP cells that presented green fluorescence under a fluorescence microscope, so did DCs transfected with total HepG2GFP cell RNA. After transfection, the expression of membrane molecules such as CD80 (13.2% to 86.7%), HLADR

6、 (38.9% to 97.9%), CD83 (0.9% to 97.1%), CD86 (31.2% to 92.5%) was increased dramatically, IL12 secretion in the supernatant was elevated significantly (61.3±8.1) ng/L to (287.4±29.3) ng/L, P0.05. The CTLs activated by DCs transfected with total HepG2GFP RNA showed a potent specific lysis

7、to HepG2 cells. CONCLUSION: GFP could be used as a marker to observe the effect of transfection of DCs with total tumor cell RNA. DCs transfected with total tumor cell RNA may serve as a promising vaccine of immunotherapy.【Keywords】 dendritic cell；green fluorescent protein；RNA；transfection【摘要】 目的：探討

8、GFP作為標(biāo)記觀察肝癌細(xì)胞RNA轉(zhuǎn)染DCs效果的可行性及腫瘤細(xì)胞RNA轉(zhuǎn)染DCs制備疫苗的可行性. 方法：綠色熒光蛋白質(zhì)粒載體 pGFPC3穩(wěn)定轉(zhuǎn)染肝癌細(xì)胞HepG2，Trizol法提取篩選后細(xì)胞HepG2GFP總RNA；分離肝癌患者外周血單核細(xì)胞體外誘導(dǎo)DCs細(xì)胞，總RNA轉(zhuǎn)染DCs，熒光顯微鏡下觀察轉(zhuǎn)染效果，流式細(xì)胞儀檢測(cè)轉(zhuǎn)染前后DCs表型變化；ELISA法檢測(cè)轉(zhuǎn)染前后上清中IL12變化情況；MTT法檢測(cè)效應(yīng)細(xì)胞對(duì)靶細(xì)胞的殺傷率. 結(jié)果：pGFPC3穩(wěn)定轉(zhuǎn)染肝癌細(xì)胞HepG2后可得到穩(wěn)定表達(dá)GFP的細(xì)胞HepG2GFP，熒光顯微鏡下呈綠色熒光；總RNA轉(zhuǎn)染的DCs熒光顯微鏡下呈綠色熒光，C

9、D80(13.2%上升至86.7%)，HLADR(38.9%上升至97.9%)，CD83(0.9%上升至97.1%)，CD86(31.2%上升至92.5%)表達(dá)明顯升高，上清IL12分泌量ng/L顯著增高(61.3±8.1287.4±29.3，P0.05)；誘導(dǎo)的CTL能夠?qū)Ω伟┘?xì)胞株HepG2起特異性殺傷作用. 結(jié)論：GFP可以作為腫瘤細(xì)胞RNA轉(zhuǎn)染樹(shù)突狀細(xì)胞的觀察標(biāo)記，腫瘤細(xì)胞RNA轉(zhuǎn)染DCs可作為一種有效的腫瘤疫苗.【關(guān)鍵詞】 樹(shù)突狀細(xì)胞；綠色熒光蛋白；RNA；轉(zhuǎn)染【中圖號(hào)】 R739.410引言肝細(xì)胞癌(hepatocellular carcinoma, HCC)是

10、一種常見(jiàn)的惡性腫瘤，適合手術(shù)治療的患者只占一少部分，具有較高的復(fù)發(fā)率，預(yù)后很差，嚴(yán)重威脅人類(lèi)健康1. DCs(dendritic cells, DCs)是體內(nèi)功能強(qiáng)大的專(zhuān)職性抗原提呈細(xì)胞，它能夠高效的攝取、處理抗原，并將處理后的多肽呈遞給靜息型T細(xì)胞，引起針對(duì)該抗原的特異性免疫反應(yīng)2. 國(guó)內(nèi)外類(lèi)似的研究中，都沒(méi)有明確的可以觀察腫瘤RNA轉(zhuǎn)染DCs效果的指標(biāo)，我們應(yīng)用綠色熒光蛋白質(zhì)粒載體 pGFPC3穩(wěn)定轉(zhuǎn)染肝癌細(xì)胞HepG2，提取篩選后細(xì)胞HepG2GFP總RNA轉(zhuǎn)染DCs，觀察轉(zhuǎn)染后DCs綠色熒光蛋白表達(dá)及特異性表面標(biāo)志及功能相關(guān)分子表達(dá)，檢測(cè)其上清細(xì)胞因子分泌，探討其作為腫瘤疫苗的可行性.

11、1材料和方法1.1材料肝癌患者外周血單個(gè)核細(xì)胞來(lái)源于我科造血干細(xì)胞移植患者；HepG2肝癌細(xì)胞株由我科常規(guī)傳代培養(yǎng). 流式細(xì)胞儀（BD），DG3022A型酶聯(lián)免疫檢測(cè)儀（華東電子管廠(chǎng)），熒光顯微鏡Optiphot XIF（Nicon）等均為我院常備. FITCCD80，F(xiàn)ITCHLADR，PECD83，PECD86抗體購(gòu)自BD公司；rhGMCSF，rhIL4購(gòu)自Peprorotec公司；TNF購(gòu)自我校生物技術(shù)中心；綠色熒光蛋白載體pGFPC3由陜西省腫瘤醫(yī)院賈軍博士惠贈(zèng)；脂質(zhì)體Transfection2000，Trizol Reagent購(gòu)自Invitrogen；RPMI 1640培養(yǎng)基、胎牛

12、血清、G418購(gòu)自Gibco公司，Xvivo無(wú)血清培養(yǎng)基購(gòu)自BioWhittaker；IL12 ELISA檢測(cè)試劑盒購(gòu)自Bioscience公司；MTT購(gòu)自Sigma公司.1.2方法參照Transfection2000說(shuō)明書(shū)，pGFPC3轉(zhuǎn)染HepG2，G418篩選2 mon. 篩選細(xì)胞命名HepG2GFP，熒光顯微鏡下觀察. Trizol試劑提取HepG2GFP總RNA，紫外燈下觀察，-80凍存?zhèn)溆? 同樣方法提取HepG2總RNA. 取肝癌患者造血干細(xì)胞分離液，貼壁法分離單核細(xì)胞，貼壁細(xì)胞在細(xì)胞因子rhGMCSF，rhIL4作用下，誘導(dǎo)DCs. 倒置顯微鏡、電子顯微鏡觀察細(xì)胞形態(tài)，流式細(xì)胞

13、儀檢測(cè)細(xì)胞表型.2結(jié)果2.1HepG2GFP總RNA轉(zhuǎn)染DCs穩(wěn)定轉(zhuǎn)染及篩選后得到的HepG2GFP細(xì)胞，能夠穩(wěn)定的表達(dá)GFP，熒光顯微鏡下所有細(xì)胞都呈現(xiàn)綠色熒光（圖1），傳代20次以上仍無(wú)消失. 與對(duì)照細(xì)胞相比，GFP基因修飾細(xì)胞在形態(tài)、生長(zhǎng)等特性上均無(wú)顯著改變，也未見(jiàn)GFP表達(dá)對(duì)靶細(xì)胞的毒性. 使用Trizol提取HepG2GFP細(xì)胞總RNA后，取2 L總RNA進(jìn)行凝膠電泳，可見(jiàn)5 s，18 s和28 s條帶（圖2）. 單個(gè)核細(xì)胞貼壁后，貼壁細(xì)胞在細(xì)胞因子GMCSF，IL4的作用下，培養(yǎng)第2日出現(xiàn)細(xì)胞集落，但細(xì)胞集落較小. 5 d后，集落明顯增加、變大，出現(xiàn)具有毛刺狀突起的細(xì)胞. 至7 d

14、，集落開(kāi)始減少，產(chǎn)生大量具有樹(shù)枝狀突起的細(xì)胞（圖3A，B）. 熒光顯微鏡下，HepG2GFP總RNA轉(zhuǎn)染DCs組細(xì)胞呈綠色熒光，而HepG2總RNA轉(zhuǎn)染組及空白對(duì)照組則無(wú)明顯熒光表達(dá)（圖4A，B，C）.圖1熒光顯微鏡下pGFPC3穩(wěn)定轉(zhuǎn)染后HepG2胞漿熒光陽(yáng)性 ×250（略）圖2HepG2GFP細(xì)胞的總RNA電泳（略）圖3樹(shù)突狀細(xì)胞形態(tài)(第7日)（略）2.2流式細(xì)胞儀檢測(cè)細(xì)胞表型誘導(dǎo)d 7，DCs表達(dá)CD80 13.2%，HLADR 38.9%，CD86 31.2%，CD83低表達(dá)，僅0.9%；轉(zhuǎn)染后上述分子表達(dá)明顯升高，分別為CD80 86.7%，HLADR 97.9%，CD86

15、 92.5%，CD83 97.1%，提示轉(zhuǎn)染后細(xì)胞具有成熟DCs特征.圖4總RNA轉(zhuǎn)染DCs×250（略）2.3ELISA檢測(cè)IL12分泌HepG2GFP總RNA    轉(zhuǎn)染DCs組上清中IL12分泌明顯增加，顯著高于轉(zhuǎn)染前（287.4±29.3) ng/L vs (61.3±8.1) ng/L； n=6，P0.05.2.4CTL介導(dǎo)細(xì)胞毒效應(yīng)HepG2GFP總RNA轉(zhuǎn)染DCs刺激的CTL對(duì)HepG2細(xì)胞殺傷率為（62.6±2.9）%，未轉(zhuǎn)染的DCs刺激的T細(xì)胞對(duì)HepG2的殺傷作用為(20.8±1.5)

16、%，無(wú)DCs刺激T細(xì)胞對(duì)HepG2的殺傷作用為(20.8±1.5)%，總RNA轉(zhuǎn)染DCs刺激的CTL對(duì)HepG2細(xì)胞殺傷率明顯高于兩個(gè)對(duì)照組（n=6，P0.05）. HepG2GFP總RNA轉(zhuǎn)染DCs刺激的CTL對(duì)SMMC7721和K562細(xì)胞殺傷率分別為(37.7±1.8)%和(26.8±1.1)%，對(duì)HepG2細(xì)胞殺傷率明顯高于SMMC7721和K562細(xì)胞（n=6，P0.05）.3討論DCs是體內(nèi)功能最強(qiáng)大的專(zhuān)職性抗原提呈細(xì)胞，它在T細(xì)胞免疫中發(fā)揮著極其重要的作用2. 應(yīng)用各種腫瘤抗原致敏DCs制備疫苗是近年來(lái)研究的熱點(diǎn) 3-8. 腫瘤細(xì)胞總RNA轉(zhuǎn)染DCs

17、制備疫苗與其他方法相比，具有如下優(yōu)越性：首先，它不受已知腫瘤抗原的限制，可誘導(dǎo)出多克隆的CTL；其次，轉(zhuǎn)染所需的RNA可以通過(guò)擴(kuò)增方法得到，很少量的腫瘤組織樣本即可擴(kuò)增得到大量的RNA8. 但目前國(guó)內(nèi)外的研究中，尚未有可以明確觀察總RNA轉(zhuǎn)染DCs效率的方法. 我們應(yīng)用穩(wěn)定表達(dá)GFP的HepG2GFP總RNA轉(zhuǎn)染DCs，以GFP為標(biāo)記觀察總RNA轉(zhuǎn)染DCs的效率，探討其作為腫瘤疫苗的可行性，為肝癌臨床治療提供實(shí)驗(yàn)基礎(chǔ).本試驗(yàn)中，HepG2GFP總RNA轉(zhuǎn)染后的DCs在熒光顯微鏡下觀察，胞質(zhì)呈現(xiàn)綠色熒光，證實(shí)GFP mRNA可以在DCs胞質(zhì)內(nèi)正常表達(dá)，同時(shí)說(shuō)明腫瘤細(xì)胞總RNA同樣也可以在DCs胞

18、漿內(nèi)進(jìn)行翻譯表達(dá). 轉(zhuǎn)染后CD83，CD80，CD86，HLADR表達(dá)明顯升高，IL12分泌明顯增加，說(shuō)明RNA轉(zhuǎn)染，促進(jìn)了DCs的體外成熟. 成熟DCs主要通過(guò)MHCI類(lèi)途徑，將腫瘤細(xì)胞總RNA在DCs胞質(zhì)內(nèi)進(jìn)行翻譯表達(dá)的腫瘤抗原呈遞給T淋巴細(xì)胞. 此途徑誘導(dǎo)的T淋巴細(xì)胞主要為CD8+細(xì)胞毒性T淋巴細(xì)胞（CTL）. 本試驗(yàn)中，誘導(dǎo)的CTL對(duì)HepG2的細(xì)胞毒效應(yīng)明顯高于對(duì)照組T細(xì)胞，證實(shí)誘導(dǎo)的T淋巴細(xì)胞為腫瘤特異性的.  因此，在當(dāng)前仍未發(fā)現(xiàn)肝癌特異性抗原情況下，應(yīng)用肝癌細(xì)胞總RNA轉(zhuǎn)染DCs制備瘤苗，誘導(dǎo)針對(duì)肝癌細(xì)胞的多克隆CTL，可以作為肝癌DCs疫苗的一個(gè)發(fā)展方向.【參考文獻(xiàn)

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