本文選題：樹(shù)突狀細(xì)胞 + 流式細(xì)胞儀�。� 參考：《山東大學(xué)》2008年博士論文

【摘要】： 【研究背景與目的】 喉癌是耳鼻喉科常見(jiàn)的惡性腫瘤之一,發(fā)病率處于耳鼻咽喉各部分惡性腫瘤的第三位。在喉癌的病理類(lèi)型中,以喉鱗癌最為常見(jiàn),一般占全部喉惡性腫瘤的95～99%。目前喉癌的治療以手術(shù)、放療及化療為主。近年來(lái),雖然手術(shù)、放化療技術(shù)發(fā)展迅速,但喉鱗癌患者特別是晚期患者的的長(zhǎng)期生存率在過(guò)去10年中未見(jiàn)明顯提高。因此,進(jìn)一步改善喉癌患者預(yù)后的研究勢(shì)在必行。 以往的研究證實(shí),抗腫瘤免疫在腫瘤發(fā)生發(fā)展的過(guò)程中有重要作用。機(jī)體的抗腫瘤免疫應(yīng)答是以T淋巴細(xì)胞、NK細(xì)胞和巨噬細(xì)胞介導(dǎo)的細(xì)胞免疫應(yīng)答為主。在此過(guò)程中,首先由抗原遞呈細(xì)胞(antigen-presenting cell,APC)捕獲、加工處理抗原并將抗原信息傳遞給T淋巴細(xì)胞,才能進(jìn)而引發(fā)特異性免疫應(yīng)答。樹(shù)突狀細(xì)胞(dendritic cell,DC)是已知體內(nèi)功能最強(qiáng)的APC,能在體內(nèi)外直接激活初始T細(xì)胞(na(?)ve T cell),促進(jìn)輔助性T細(xì)胞和細(xì)胞毒性T細(xì)胞的生成,同時(shí)也能夠促進(jìn)B淋巴細(xì)胞生成抗體,是啟動(dòng)、調(diào)控并維持免疫應(yīng)答的中心環(huán)節(jié)。 由于DC在體內(nèi)免疫應(yīng)答中重要而獨(dú)特的作用,近年來(lái),以DC為基礎(chǔ)的腫瘤免疫治療研究越來(lái)越受到關(guān)注。已有研究證實(shí),通過(guò)體外細(xì)胞因子聯(lián)合培養(yǎng)擴(kuò)增DC,模擬DC體內(nèi)成熟過(guò)程,以不同形式的抗原物質(zhì),如人工合成腫瘤多肽、腫瘤細(xì)胞裂解產(chǎn)物、腫瘤蛋白抗原、凋亡腫瘤細(xì)胞、DNA或RNA負(fù)載DC,再將致敏DC回輸體內(nèi)作為疫苗可誘導(dǎo)機(jī)體產(chǎn)生有效的抗腫瘤免疫應(yīng)答。樹(shù)突狀細(xì)胞應(yīng)用于抗喉鱗癌的研究尚處于初級(jí)階段。由于目前尚未發(fā)現(xiàn)喉癌腫瘤特異性抗原,因此腫瘤全抗原成為制備疫苗的主要手段。已有報(bào)道應(yīng)用喉癌細(xì)胞裂解產(chǎn)物負(fù)載樹(shù)突狀細(xì)胞或用電融合的方法制備疫苗誘導(dǎo)產(chǎn)生有效的抗腫瘤免疫反應(yīng)。最近研究發(fā)現(xiàn),以RNA負(fù)載DC能夠引發(fā)更為強(qiáng)大的抗腫瘤效應(yīng),而且已有實(shí)驗(yàn)證實(shí)了其優(yōu)越的安全性及廣闊的應(yīng)用前景。但以RNA負(fù)載DC的抗喉鱗癌研究國(guó)內(nèi)外尚未見(jiàn)報(bào)道。 另外,樹(shù)突狀細(xì)胞免疫學(xué)功能的強(qiáng)弱取決于DC的免疫狀態(tài)。眾所周知,腫瘤患者的DC存在缺陷,但由于T細(xì)胞活化的MHC限制性,臨床上應(yīng)用DC誘導(dǎo)抗腫瘤免疫應(yīng)答必須使用腫瘤患者自身的DC,因而研究惡性腫瘤患者DC的數(shù)目及功能顯得尤為重要。另一方面,雖然DC為基礎(chǔ)的免疫治療有其獨(dú)特的優(yōu)勢(shì),但近期內(nèi)仍無(wú)法作為主要的臨床治療手段。對(duì)于喉鱗癌患者,手術(shù)及放療仍然不失為目前最有效的治療方法。因此研究喉鱗癌患者DC免疫狀態(tài),除了考慮疾病本身對(duì)DC的影響,尚需考慮目前常規(guī)治療方法對(duì)DC的影響。 本研究檢測(cè)手術(shù)及術(shù)后輔助放療對(duì)喉癌患者外周血循環(huán)DC亞型及外周血單核細(xì)胞來(lái)源DC(monocyte-derived DCs,MoDes)功能的影響,以探討喉癌免疫治療的時(shí)機(jī);同時(shí),以EGFP標(biāo)記的喉癌Hep-2株總RNA轉(zhuǎn)染MoDCs,研究其誘導(dǎo)Hep-2細(xì)胞特異性的細(xì)胞毒性T淋巴細(xì)胞(cytotoxic T lymphocyte,CTL)的能力及對(duì)喉鱗癌的特異性殺傷效應(yīng),為臨床以DC為基礎(chǔ)的抗喉癌治療提供實(shí)驗(yàn)基礎(chǔ)和理論依據(jù)。 第一部分健康人外周血樹(shù)突狀細(xì)胞亞型檢測(cè)及培養(yǎng)、鑒定 【目的】建立簡(jiǎn)便可靠的外周血樹(shù)突狀細(xì)胞亞型檢測(cè)方法,培養(yǎng)鑒定外周血單核細(xì)胞來(lái)源樹(shù)突狀細(xì)胞。 【方法】采集15例健康人外周血,采用三色流式細(xì)胞計(jì)量術(shù)檢測(cè)外周血HLA-DR~+lineage~-樹(shù)突狀細(xì)胞及其亞型myeloid DC(mDC)、plasmacytoid DC(pDC)在外周血WBC所占百分比并計(jì)算其絕對(duì)數(shù)。采用淋巴細(xì)胞分離液自外周血分離獲得單核細(xì)胞,體外經(jīng)人重組細(xì)胞因子GM-CSF、IL-4和TNF-α誘導(dǎo)培養(yǎng),獲得成熟外周血單核細(xì)胞來(lái)源DC。倒置顯微鏡下觀察DC形態(tài),透射電鏡觀察DC超微結(jié)構(gòu),流式細(xì)胞儀檢測(cè)其表型,混合淋巴細(xì)胞反應(yīng)(MLR)檢測(cè)刺激自體T淋巴細(xì)胞增殖的能力。 【結(jié)果】健康人LIN~-DR~+細(xì)胞在WBC中所占百分比為0.41±0.13%,絕對(duì)數(shù)為22.75±5.22/μl,mDC在WBC中所占百分比為0.28±0.13%,絕對(duì)數(shù)為15.12±4.66/μl,pDC在WBC中所占百分比為0.068±0.032%,絕對(duì)數(shù)為3.78±1.40/μl;培養(yǎng)獲得具有典型形態(tài)特征的外周血單核細(xì)胞來(lái)源DC;健康人LIN陰性細(xì)胞中,共刺激分子CD80(B7-1)、CD86(B7-2)陽(yáng)性細(xì)胞所占百分比分別為46.5±7.1%和51.1±9.6%,同時(shí)多數(shù)DC表達(dá)DC成熟標(biāo)志CD83和HLA-DR,陽(yáng)性細(xì)胞百分比分別為62.6±10.3%和73.4±8.1%,表明單核細(xì)胞經(jīng)細(xì)胞因子誘導(dǎo)后大部分分化為成熟DC;證實(shí)少量DC即可刺激自體淋巴細(xì)胞產(chǎn)生較強(qiáng)的增殖效應(yīng)。 【結(jié)論】流式細(xì)胞儀檢測(cè)外周血DC亞群簡(jiǎn)便、可靠;聯(lián)合應(yīng)用細(xì)胞因子GM-CSF、IL-4和TNF-α能夠自外周血中成功分離、誘導(dǎo)培養(yǎng)具有典型形態(tài)特征及功能活性的單核細(xì)胞來(lái)源DC。 第二部分手術(shù)及術(shù)后放療對(duì)喉鱗癌患者外周血樹(shù)突狀細(xì)胞亞型及功能的影響 【目的】研究手術(shù)及術(shù)后輔助放療對(duì)喉鱗癌患者外周血樹(shù)突狀細(xì)胞亞型及外周血單核細(xì)胞來(lái)源樹(shù)突狀細(xì)胞功能的影響。 【方法】采集46例經(jīng)病理證實(shí)的喉鱗癌患者及15例健康志愿者外周血。46名喉鱗癌患者中男性44例,女性2例,年齡分布為32～76歲,平均年齡為57.57±10.09歲。根據(jù)2002年國(guó)際抗癌聯(lián)盟(UICC)制定的TNM分期標(biāo)準(zhǔn),Ⅰ期14例,Ⅱ期8例,Ⅲ期11例,Ⅳ期13例。46例患者入院后均接受手術(shù)治療,28例患者手術(shù)后接受頸部直線加速器放射治療。接受單純手術(shù)治療的患者(n=18)及接受手術(shù)+放療的患者(n=28)均于治療前、治療中及治療后分三次采集外周血標(biāo)本。采用三色流式細(xì)胞計(jì)量術(shù)檢測(cè)外周血HLA-DR~+lineage~-樹(shù)突狀細(xì)胞及其亞型mDC、pDC在外周血WBC所占百分比并計(jì)算其絕對(duì)數(shù)。采用淋巴細(xì)胞分離液自外周血分離獲得單核細(xì)胞,體外經(jīng)人重組細(xì)胞因子GM-CSF、IL-4和TNF-α誘導(dǎo)培養(yǎng),獲得外周血單核細(xì)胞來(lái)源DC,流式細(xì)胞儀檢測(cè)其表型,混合淋巴細(xì)胞反應(yīng)(MLR)檢測(cè)刺激自體T淋巴細(xì)胞增殖的能力。 【結(jié)果】治療前喉鱗癌患者外周血mDC計(jì)數(shù)、MoDC表面分子表達(dá)及刺激自體T淋巴細(xì)胞增殖能力均低于健康人;經(jīng)手術(shù)治療后,接受輔助放療和未接受輔助放療的患者其mDC計(jì)數(shù)、MoDC表面分子CD80,CD83,HLA-DR的表達(dá)均明顯高于治療前,但僅在術(shù)后未接受輔助放療的患者中觀察到CD86表達(dá)及刺激自體T淋巴細(xì)胞增殖能力的恢復(fù)。 【結(jié)論】喉鱗癌患者DC存在缺陷;手術(shù)治療可提高喉鱗癌患者外周血mDC計(jì)數(shù)及MoDC的功能;術(shù)后輔助放療不影響外周血mDC計(jì)數(shù)的正�；�,但對(duì)MoDC功能具有抑制作用。 第三部分以EGFP標(biāo)記的喉癌Hep-2細(xì)胞株總RNA轉(zhuǎn)染樹(shù)突狀細(xì)胞的特異性抗喉癌實(shí)驗(yàn)研究 【目的】探討以EGFP為標(biāo)記觀察喉鱗癌Hep-2細(xì)胞株總RNA轉(zhuǎn)染樹(shù)突狀細(xì)胞的可行性;探討轉(zhuǎn)染后DC誘導(dǎo)特異性識(shí)別Hep-2細(xì)胞的細(xì)胞毒性T淋巴細(xì)胞的可能性及特異性殺傷效應(yīng)。 【方法】采用淋巴細(xì)胞分離液自健康人外周血分離獲得單核細(xì)胞,體外經(jīng)人重組細(xì)胞因子GM-CSF、IL-4誘導(dǎo)培養(yǎng),獲得未成熟DC,觀察細(xì)胞形態(tài),流式細(xì)胞技術(shù)鑒定其表型;培養(yǎng)喉癌Hep-2細(xì)胞株,采用脂質(zhì)體轉(zhuǎn)染技術(shù)將攜帶綠色熒光蛋白質(zhì)粒pEGFP-N1轉(zhuǎn)染至Hep-2細(xì)胞株,通過(guò)G418篩選穩(wěn)定表達(dá)EGFP的細(xì)胞株;Trizol一步法提取穩(wěn)定轉(zhuǎn)染的Hep-2細(xì)胞株總RNA,檢測(cè)其完整性及純度;以共孵育及電穿孔法將RNA導(dǎo)入未成熟DC,并以TNF-α促成熟;應(yīng)用熒光顯微鏡觀察并計(jì)算轉(zhuǎn)染效率;通過(guò)流式細(xì)胞技術(shù)、混合淋巴細(xì)胞反應(yīng)檢測(cè)RNA轉(zhuǎn)染后對(duì)DC表型、刺激T細(xì)胞增殖能力的影響;將轉(zhuǎn)染后DC與同種異體T淋巴細(xì)胞共培養(yǎng)7天,收集效應(yīng)細(xì)胞作為特異性CTL,通過(guò)~(51)Cr釋放法檢測(cè)其對(duì)Hep-2細(xì)胞株的特異性殺傷效應(yīng)。 【結(jié)果】pEGFP-N1質(zhì)粒轉(zhuǎn)染喉癌Hep-2細(xì)胞株,經(jīng)G418篩選可獲得穩(wěn)定表達(dá)EGFP的細(xì)胞株,熒光顯微鏡下可見(jiàn)綠色熒光,經(jīng)20次傳代后熒光仍無(wú)消失,Hep-2細(xì)胞形態(tài)及生長(zhǎng)特性未見(jiàn)明顯改變;經(jīng)Trizol一步法提取穩(wěn)定轉(zhuǎn)染的Hep-2細(xì)胞株總RNA 80～100μg/10~7細(xì)胞,分光光度法測(cè)定OD260/OD280=1.83,RNA電泳顯示18S、28S條帶,證實(shí)RNA的完整性;共孵育及電穿孔轉(zhuǎn)染法轉(zhuǎn)染效率分別為7～9%和21～23%;RNA導(dǎo)入未成熟DC后,細(xì)胞表達(dá)綠色熒光持續(xù)20～24小時(shí);Hep-2-EGFP-RNA轉(zhuǎn)染組DC表面分子CD83、HLA-DR表達(dá)較轉(zhuǎn)染前及對(duì)照轉(zhuǎn)染組顯著升高,陽(yáng)性細(xì)胞百分比達(dá)89.5%、95.4%,刺激同種異體T淋巴細(xì)胞增殖的能力顯著提高;Hep-2-EGFP-RNA轉(zhuǎn)染組DC體外誘導(dǎo)的CTLs可特異性殺傷喉癌Hep-2細(xì)胞,但對(duì)結(jié)腸癌HT-29細(xì)胞及卵巢癌OVCAR3細(xì)胞無(wú)殺傷活性。 【結(jié)論】綠色熒光蛋白可作為喉癌Hep-2細(xì)胞RNA轉(zhuǎn)染DC的標(biāo)記;轉(zhuǎn)染喉癌Hep-2細(xì)胞總RNA促進(jìn)DC成熟,提高DC刺激同種異體T淋巴細(xì)胞增殖的能力,并能夠產(chǎn)生喉癌Hep-2細(xì)胞特異性殺傷效應(yīng)。
[Abstract]:Background and purpose of research


In recent years , the long - term survival rate of laryngeal squamous cell carcinoma , especially advanced patients , has not been significantly improved in the past 10 years . Therefore , it is imperative to further improve the prognosis of laryngeal cancer patients .


Dendritic cells ( DCs ) are the most powerful APC in vivo , which can promote the production of T - cells and cytotoxic T - cells , but also promote the production of antibody against B - lymphocytes , which is the central link of starting , regulating and maintaining immune response .


Dendritic cells ( DCs ) have been used in the study of anti - laryngeal squamous cell carcinoma ( SCC ) .


It is well known that DC - induced antitumor immune responses in patients with laryngeal squamous cell carcinoma have a unique advantage . However , although DC - based immunotherapy has its unique advantages , the DC - induced immune response in patients with laryngeal squamous cell carcinoma is not currently the most effective treatment . In addition to the effect of the disease itself on DC , the effect of conventional treatment methods on DC is still needed .


The effects of surgical and postoperative adjuvant radiotherapy on peripheral circulating DC ( DC ) and peripheral blood mononuclear cells ( DCs ) in peripheral blood mononuclear cells ( DCs ) of patients with laryngeal cancer were investigated . The cytotoxic T lymphocyte ( CTL ) and specific killing effects on laryngeal squamous cell carcinoma ( SCC ) were investigated by using EGFP - labeled total RNA of laryngeal cancer .


Detection , Culture and Identification of Dendritic Cell Subtypes in Human Peripheral Blood in the First Part


Objective To establish a simple and reliable method for detecting peripheral blood mononuclear cells ( DCs ) from peripheral blood mononuclear cells ( PBMC ) .


Peripheral blood mononuclear cells ( HLA - DR ~ + ) ~ - dendritic cells and their subtypes , DC ( mDC ) and plasmacytoid DC ( pDC ) were measured by three - color flow cytometry .


The percentage of DCs in WBC was 0 . 28 鹵 0 . 13 % , the absolute number was 22.75 鹵 5.22 / 渭l , the percentage of mDC in WBC was 0 . 068 鹵 0 . 32 % , the absolute number was 3.78 鹵 1.40 / 渭l , the percentage of positive cells was 62.6 鹵 10.3 % and 73.4 鹵 8.1 % , respectively .


Conclusion Flow cytometry is simple and reliable in detecting peripheral blood DC subgroup , and the combination of cytokine GM - CSF , IL - 4 and TNF - 偽 can be successfully isolated from peripheral blood , which can induce DC of monocytes with typical morphological characteristics and functional activity .


Effects of second partial surgery and postoperative radiotherapy on the subtypes and functions of peripheral blood dendritic cells in patients with laryngeal squamous cell carcinoma


Objective To investigate the effects of surgical and postoperative adjuvant radiotherapy on the function of dendritic cells in peripheral blood dendritic cells and peripheral blood mononuclear cells in patients with laryngeal squamous cell carcinoma .


Forty - six patients with laryngeal squamous cell carcinoma and 15 healthy volunteers were collected from peripheral blood of 46 patients with laryngeal squamous cell carcinoma .


The expression of CD80 , CD83 and HLA - DR in peripheral blood of patients with laryngeal squamous cell carcinoma was significantly higher than that before treatment . The expression of CD80 , CD83 and HLA - DR on the surface of MoDC was significantly higher than that before treatment , but only in patients who did not receive adjuvant radiotherapy after surgery .


Conclusion There is a deficiency in DC in patients with laryngeal squamous cell carcinoma . Surgical treatment can improve the peripheral blood mDC count and MoDC function of patients with laryngeal squamous cell carcinoma . The postoperative adjuvant radiotherapy does not affect the normalization of mDC count in peripheral blood , but has an inhibitory effect on MoDC function .


Experimental study on the specific anti - laryngocarcinoma of dendritic cells transfected with EGFP - labeled total RNA of laryngeal cancer cells


Objective : To investigate the feasibility of using EGFP as a marker to observe the expression of dendritic cells transfected with total RNA from laryngeal squamous cell carcinoma cells .


Cells were isolated from the peripheral blood of healthy individuals using lymphocyte separation liquid . The cells were cultured in vitro by human recombinant cytokines GM - CSF and IL - 4 . The cell lines were transfected with green fluorescent protein particles by G418 selection , the integrity and purity of the cells were observed . The transfection efficiency was observed by G418 selection . After transfection , the cells were cultured for 7 days . The effect cells were collected as specific CTL .


Results The expression of CD83 and HLA - DR was significantly higher than that in control transfected group . The expression of CD83 , HLA - DR was 89.5 % and 95.4 % , respectively , and the expression of CD83 and HLA - DR was 89.5 % and 95.4 % respectively .


Conclusion The green fluorescent protein can be used as a marker for the transfection of DC by RNA transfection of laryngeal cancer 2 - cell RNA . The transfection of total RNA of laryngeal cancer 2 - 2 cells promotes DC maturation , enhances the ability of DC to stimulate the proliferation of allogeneic T lymphocytes , and can produce the specific killing effect of laryngeal cancer Hep2 cells .
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R392;R739.65

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 林嘉盈;李曉艷;;治療頭頸部腫瘤的樹(shù)突狀細(xì)胞疫苗的研究進(jìn)展[J];醫(yī)學(xué)綜述;2010年22期

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