MCF-7荷瘤小鼠不同树突状细胞亚群凋亡的研究
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摘要
第一章MCF-7荷瘤小鼠的建立、树突状细胞的诱导分化及其表面分子标记的检测
研究背景和目的
乳腺癌是妇女最常见的恶性肿瘤,发病率无论在发达国家或发展中国家仍在逐年上升；随着手术、化放疗、内分泌治疗及靶向治疗等综合治疗的合理使用,乳腺癌的死亡率在发达国家已呈下降趋势,治疗效果亦进入了一个相对平台期。然而,晚期和转移性乳腺癌仍不可治愈,成为威胁女性尤其是发展中国家女性生命的重要原因；另一方面,随着医学科学发展,抗肿瘤免疫治疗作为乳腺癌治疗一个很有前景的治疗模式受到越来越多的关注,树突状细胞(dendritic cells,DCs)作为机体功能最强的抗原呈递细胞,能摄取、加工和递呈抗原从而使T细胞致敏、激活和扩增,有潜在的克服肿瘤耐受的能力和负载肿瘤抗原后诱导产生抗肿瘤免疫力的能力,在抗肿瘤免疫治疗中发挥着重要作用。多项实验证明人类和鼠的DCs主要有两种亚群：髓系来源的树突状细胞(]myeloid dendritic cell, mDC)和淋巴来源的树突状细胞(plasmacytoid dendritic cell, pDC),前者高表达CD11c,低表达CD123；后者高表达CD123,低表达CD11c,不同的DCs亚群在介导机体免疫反应中发挥不同的作用。本研究拟通过构建MCF-7荷瘤小鼠,研究其骨髓起源的树突状细胞分化、成熟的情况及是否存在不同亚群表达,为设计新的免疫治疗方法和新的疫苗的提供基础信息。
方法
1.取对数生长期MCF-7细胞调整细胞浓度为1×107/ml,用于以下实验。
2.20只5周龄无特定病原体(SPF)级BALB/C雌性裸鼠,随机分为2组,用1ml注射器向荷瘤组小鼠右侧胸壁第二对乳腺脂肪垫注入0.2mL上述MCF-7细胞悬液,对照组注入0.2mL无血清培养基。待肿块直径达5mm后处死小鼠。
3.取小鼠骨髓单个核细胞,RPMI1640(10%FBS)培养基调整细胞浓度为107/1.5m1,每皿(60mm)加入1.5m1细胞悬液,再加入1.5mlRPMI1640隔3天更换1次培养液。第3天吸弃全部培养基及悬浮细胞,加入含100ng/mlrmGM-CSF,100ng/ml rmIL-4的上述培养基共孵育,第7天更换培养基时补加脂多糖100ng/mL共孵育,第8天吹打疏松贴壁的增殖性细胞聚集体,次日收集悬浮的细胞即为富集的DCs。相差显微镜下观察细胞形态特点。收集细胞做以下实验。
4.用PBS(含1%FBS)调整细胞浓度为106/100u1,每流式管加入100ul细胞悬液,再加入1ug/管相应荧光素偶联一抗,上机检测。
5.荷瘤小鼠乳房肿块病理免疫组化检测：supervision二步法检测ER、PR、 Cerb-B2、Ki-67。
6.统计学方法：计量资料统计描述用均数±标准差表示,两组均数比较用独立样本t检验。检验水准α=0.05。
结果
1.对数生长期MCF-7细胞倒置显微镜下观察,贴壁生长,形态良好、折光性强。
2.荷瘤组小鼠在注射MCF-7后第16天乳腺肿块达5mm或以上。荷瘤组和对照组小鼠体重在荷瘤前及荷瘤后均无统计学差异,具可比性。
3.从骨髓中获取的PBMC培养至第九天,可见单个的细胞表面有明显毛刺状突起的悬浮的DCs。
4.荷瘤组和对照组DCs均高表达CD86、CD83及MHC-Ⅱ,且荷瘤组CD83表达为74.61±1.26%,比对照组(77.30±1.41)低,P<0.05。CD34表达荷瘤组表达为1.30±0.17%,对照组为1.71±0.18%,P<0.05。荷瘤组CD11c+CD123-细胞52.01±1.43%,较对照组(56.36±1.76%)低,P<0.05。荷瘤组CD11c-CD123+细胞32.19±1.71%,较对照组(28.95±1.39%)高,P<0.05。
5.荷瘤小鼠乳腺肿块病理活检可见乳腺癌细胞,免疫组化示：ER(+++),PR(++), Cerb-B2(-),Ki-67(+++)
结论
1.动物模型构建成功,动物骨髓单个核细胞体外能诱导分化出高质量和高纯度的成熟的DCs。
2.MCF-7荷瘤小鼠骨髓起源的DCs存在成熟障碍,肿瘤抗原递呈能力降低,导致T细胞激活、增殖能力下降,机体免疫杀伤肿瘤细胞效应降低。
3.MCF-7乳腺癌小鼠mDC数量减少,pDC数量增多,意味着荷瘤机体mDCs把肿瘤抗原递呈至T细胞产生抗肿瘤免疫反应能力下降,而由mDC分泌并活化的IL-12也相应减少,对肿瘤细胞的免疫杀伤能力减低。而pDC一方面具有弱的免疫应答对抗肿瘤,另一方面存在较强抑制抗肿瘤免疫反应能力,故其数量的增多,并不能增强机体的抗肿瘤免疫功能。
第二章MCF-7荷瘤小鼠不同树突状细胞亚群凋亡率的检测
研究背景和目的
细胞程序性死亡是体内DCs维持动态平衡的重要方式,DCs程序性死亡失调可改变DCs的生存期而导致正常免疫反应的受损,最近的研究表明诱导DCs的凋亡是肿瘤逃避免疫识别的另一重要机制,在体外,肿瘤细胞和肿瘤起源的相关因子互相作用后能诱导DCs凋亡。
人类T细胞分为Th1和Th2两个亚群,正常状态下,Th1和Th2类细胞功能处于一种动态平衡状态,而机体抗肿瘤免疫以介导细胞免疫的Th1类细胞为主。成熟DCs不仅可诱导细胞毒T淋巴细胞(cytotoxic t lymphocyte,CTL)增殖,还诱导CTL分泌细胞因子(如interleukin-12,IL-12),IL-12有强有力的诱导细胞毒性T细胞、自然杀伤细胞、淋巴因子激活杀伤细胞的能力,产生大量TNF-γ、穿孔素和颗粒酶,增强对靶细胞的溶解作用,产生Th1型免疫应答而发挥抗肿瘤作用。不同的DCs亚群表达不同的细胞毒分子,一般认为pDC诱导TH2反应,而mDC诱导THl反应。
使用DCs作为抗肿瘤免疫治疗是一种通过增强患者自身的免疫反应来消灭肿瘤细胞的强有力的手段,利用成熟DCs负载肿瘤抗原的树突状细胞疫苗已在多种肿瘤患者中应用,然而这些疗法技术上虽已较成熟,而疗效均不甚理想,因此需要研究更合理和有效的DC疫苗。了解乳腺癌DCs不同亚群的凋亡,可进一步为树突状细胞在抗肿瘤免疫治疗方面的应用提供更准确、更科学的信息。
方法
1.同上获取DCs悬液,调整细胞浓度为106/100ul,每流式管加入100ul细胞悬液。
2.分别加入CD11c-PC5, CD123-PE各1ug,室温避光放置30分钟。
3用含1%FBS的PBS洗一次,细胞沉淀后加入100ul Annexin-V Binding Buffer,5ul Annexin-V,室温避光放置15分钟。
4.加入400ul Annexin-V Binding Buffer,上机同时检测FITC、PE、PE/CY5三个荧光通道。
5.统计学方法：计量资料统计描述用均数±标准差表示,两组均数比较用独立样本t检验。检验水准a=0.05。
结果
1.MCF-7荷瘤小鼠经上述方法诱导分化的mDC凋亡率为23.64±2.43%,对照组为20.05-2.49%,前者比后者高,P<0.05。
2,MCF-7乳腺癌小鼠骨髓来源pDC凋亡率为11.53±2.92%,,对照组为14.96±2.96%,荷瘤组比对照组低,P<0.05。
结论
1.MCF-7荷瘤小鼠mDC凋亡率比对照组明显增高,说明MCF-7乳腺癌小鼠mDC诱导Th1免疫反应能力减低,调节Thl细胞分泌相关细胞因子包括IL-12等能力下降,一方面,使T细胞增殖、活化受损,导致细胞毒T细胞、自然杀伤细胞、淋巴因子激活杀伤细胞产生干扰素-γ(interferon-γ,IFN-γ)、穿孔素和颗粒酶等能力相应下降,对肿瘤细胞的溶解作用减弱,从而降低机体对肿瘤细胞的免疫杀伤作用。另一方面,改变了机体Th1/Th2的平衡,诱导荷瘤机体产生免疫耐受,抑制抗肿瘤免疫反应。
2.荷瘤组小鼠骨髓来源pDC凋亡较对照组小鼠明显减少,提示MCF-7乳腺癌小鼠体内,Th0向Th2细胞分化增多,Th2细胞分泌因子增多,刺激产生CD4+/CD25+调节T细胞逃避抗肿瘤免疫,并使抗原递呈和T细胞增殖、活化功能降低,诱导体内免疫耐受的发生,并通过促血管生成参与和促进乳腺癌生长和转移。是机体抗肿瘤免疫反应能力下降的另一个重要因素。
3.pDC凋亡减少,还可抑制单核细胞分化成DCs,减少mDC的抗原递呈能力或甚至使mDC转变成介导免疫耐受,从而进一步削弱机体抗肿瘤免疫反应。
第三章MCF-7荷瘤小鼠血清及树突状细胞上清液细胞因子的测定
研究背景和目的
细胞因子是一类在体内有广泛生物学效应的多肽物质,干扰素-6(interleukin-6, IL-6)、TNF-α、IL-12等是细胞因子网络中的重要成员,不仅介导机体的抗感染免疫和自身免疫,还影响恶性肿瘤细胞的分化、转移、血管生成等过程,在监视和杀灭肿瘤细胞中发挥了重要作用。
目前,TNF-α在乳腺癌中的作用尚存争议,高剂量的TNF-α可引起肿瘤中血管退行性改变和出血性坏死,但是,有研究表明,在体内,肿瘤细胞中内源性TNF-α的分泌并不杀伤肿瘤,反而通过诱导恶性乳腺上皮细胞等肿瘤细胞增殖,并促进血管再生、肿瘤细胞侵袭和转移。IL-6参与对肿瘤的免疫反应,可促进乳腺癌干细胞的生长和恶化,并具有促进肿瘤血管生成和侵袭的作用,还在肿瘤演进和雌激素调节中发挥了重要作用,另外,IL-6可通过参与多种细胞因子的分泌,与其他细胞因子共同发挥作用,破坏机体的自然杀伤能力,促进乳腺癌细胞的浸润和转移。作为引发细胞免疫的关键因素, IL-12促进NK细胞介导的HER2(+)肿瘤细胞的杀伤；IL-12既能通过DCs和B细胞的一种直接的相互作用诱导体液免疫发生,也能通过DCs和T细胞的直接相互作用诱导Thl细胞。
多种细胞因子在抗肿瘤免疫治疗中起重要作用,深入研究这些细胞因子在荷瘤机体内的变化,可以我们通过合理途径,改变体内一些重要细胞因子的水平,结合其他免疫治疗,提高机体抗肿瘤免疫效应。
方法
1.摘除裸鼠眼球采静脉血约1ml,在室温下静置40分钟,室温下离心3,000rpm,15分钟,取上层血清4℃保存。
2.ELISA kit检测血清中TNF-α, IL-6, IL-12浓度。
3.上清液准备：DCs诱导分化第九天的上清液。
4.ELISA kit检测DCs上清液TNF-α, IL-6, IL-12浓度。
5.统计学方法：计量资料统计描述用均数±标准差表示,两组均数比较用独立样本t检验。检验水准α=0.05。
结果
1.MCF-7荷瘤小鼠血清TNF-α浓度为314.81±103.36pg/ml,比对照组小鼠浓度(371.87±115.30pg/ml)低,但差别无统计学意义。MCF-7荷瘤小鼠树突状细胞上清液TNF-α浓度为1.88±0.10ng/ml,比对照组小鼠的浓度高(1.72±0.07ng/ml),差别有统计学意义,P<0.05。
2.荷瘤组血清IL-6浓度为170.72±54.51pg/ml,与对照组(122.35±20.65pg/m1)比较,前者显著增高,P<0.05。荷瘤组DCs上清液IL-6浓度为57.18±5.06ng/ml,与对照组(44.53±4.86ng/ml)相比,有统计意义的增高,P<0.05。
3.IL-12血清浓度荷瘤组为119.80±33.53pg/ml,对照组为186.22±51.91pg/ml,前者明显降低,差别有统计学意义,P<0.05。IL-12上清液浓度荷瘤组为89.76±8.89pg/ml,对照组为157.98±48.31pg/ml,前者明显降低,差别有统计学意义,P<0.05。
结论
1.荷瘤小鼠血清中的TNF-α浓度比对照组低,但无统计学意义,可能跟TNF-α多种产生形式有关,TNF-α可通过自分泌、旁分泌和内分泌的形式产生,故MCF-7乳腺癌体内多种细胞及其他细胞因子参与了TNF-α的分泌,从另一侧面反映TNF-α在这种乳腺癌的作用可能比较复杂,需要更多更全面的研究。MCF-7荷瘤小鼠DCs上清液TNF-α浓度比对照组高,DCs可能参与产生TNF-α,至于TNF-α在MCF-7乳腺癌中发挥抗肿瘤作用还是促进肿瘤侵袭和转移有待更深入的研究,且此作用可能与剂量、肿瘤类型等有关。
2.MCF-7乳腺癌小鼠血清IL-6水平比对照组显著升高,说明IL-6可能通过上述机制,参与这种乳腺癌肿瘤细胞的生长,促进乳腺癌细胞的浸润和转移。MCF-7荷瘤小鼠DCs上清液IL-6水平亦高于对照组,说明DCs促进IL-6的分泌,使其参与抑制抗肿瘤免疫反应。
3.MCF-7荷瘤小鼠血清IL-12浓度显著低于对照组,低水平的IL-12,使T细胞及NK细胞对肿瘤细胞的杀伤作用减弱；MCF-7荷瘤小鼠DCs上清液IL-12水平亦明显低于对照组,MCF-7乳腺癌DCs影响IL-12的分泌,从而使机体抗肿瘤免疫效应减低,提高机体的IL-12水平,尤其结合化疗及靶向治疗,应该可以大大提高这种乳腺癌的治疗效果。
第四章树突状细胞亚群凋亡与细胞因子的关系
研究背景和目的
DCs和细胞因子网络在机体抗肿瘤免疫中互相影响和互相作用,一方面,细胞因子在DCs分化成熟过程中发挥重要的调节作用,各种肿瘤起源的相关因子(VEGF, IL-6, IL-10, M-CSF,和GM-CSF)能在体内外抑制DCs从造血干细胞中分化；另一方面,成熟的DCs不同亚群在发挥免疫杀伤或免疫耐受过程亦分泌各种细胞因子来增强或减弱这些反应。
肿瘤产生一系列抑制因素而形成一个免疫抑制环境,其分泌的多种可溶性因子的变化,包括炎症因子分泌的减少、抗炎症因子分泌的增多、以及Treg调节T细胞分泌的增多,能够抑制DCs的产生和分化成熟,对肿瘤宿主DC的数量和功能缺陷起着重要的调控作用,从而抑制机体对肿瘤细胞的免疫杀伤作用。
各种受体、细胞因子、炎症趋化因子可在免疫反应的不同阶段影响DCs的生存期。尽管维持DCs动态平衡的确切机制尚未清楚,很多证据显示细胞因子可能是重要的调节。MCF-7乳腺瘤小鼠树突状细胞亚群的凋亡与重要的细胞因子互相影响和互相作用,了解这种复杂的相互关系,对于设计复合的抗肿瘤免疫治疗有重要意义。一方面,我们可以利用更有效的DCs亚群来递呈抗原,使可以成功诱导自体T淋巴细胞产生特异性抗肿瘤免疫反应,增强CTL介导的细胞免疫；另一方面,在此过程,结合细胞因子的应用,能进一步放大肿瘤细胞的免疫杀伤作用。
方法
1.上述方法分别计算荷瘤组和对照组mDC、pDC凋亡率
2.ELISA法检测小鼠血清及DCs上清液TNF-α、IL-6、IL-12浓度测定。
3.统计学方法：计量资料的统计描述用均数±标准差表示,两组均数比较用独立样本t检验；两计量指标的相关性统计用Pearson相关分析。检验水准α=0.05。
结果
1血清TNF-α浓度与mDC凋亡率呈负相关,r=-0.70,P=0.00；IL-6浓度与pDC凋亡率呈负相关,r=-0.83,P=0.00；IL-12与mDC凋亡率呈负相关,r=-0.83,P=0.00。
2.DCs上清液TNF-α浓度与pDC凋亡率呈负相关,r=-0.91,P=0.00；IL-6浓度与pDC凋亡率呈负相关,r=-0.85,P=0.00；IL-12与mDC凋亡率呈负相关,r=-0.70,P=0.00。
结论
1.血清TNF-α浓度随着mDC凋亡率增加而减低,由于血清中TNF-α的由多种细胞分泌,多种因素参与,故其浓度变化与mDC的相关性并无实际意义,而在骨髓起源的DCs上清液,TNF-α浓度随着pDC凋亡率降低而增加,而由于TNF-α在抗肿瘤免疫中的双向性,故pDC凋亡对其在乳腺癌,尤其是不同分子亚型中的作用有待深入的探讨。
2.IL-6浓度无论血清或DCs上清液,均随着pDC凋亡率降低而增加,在MCF-7乳腺癌小鼠,由于pDC凋亡的减少,Th1/Th2失衡,向Th2偏移,作为Th2细胞分泌的主要细胞因子之一,IL-6水平明显增高,在乳腺癌的进展、侵袭中起重要作用。
3.IL-12浓度无论血清或DCs上清液,均随着mDC凋亡率增加而减低,在MCF-7乳腺癌小鼠,由于mDC凋亡的增多,其分泌的IL-12显著减少,导致不能有效刺激T淋巴细胞增殖,抑制mDC的成熟和活化,并改变Th1/Th2的平衡,诱导机体产生免疫耐受,最终肿瘤发生进展、转移。
Chapter1Establishment of MCF-7xenograft model in nude mice, induction and differentiation of dendritic cells and detection of its surface marker
Background and Objection:Breast cancer is the most common magnicnant in women. No matter in developed countries or developing countries, the incidence rate is increasing year by year. As operation, radiotherapy, endocrine therapy and targeted therapy are used reasonably, the mortality of breast cancer in developed countries has been declined. The clinical responds has come into a relative plateau, however, advanced and metastatic disease remains incurable and has become the main cause of threatened the life of female, especially in developing countries. On the other hand, as a promising treatment pattern, antitumor immunotherapy is received more and more attention along with the progress of medicine. Dendritic cells(DCs) is the most powerful antigen presenting cell in organism. It can uptake, processing and presentation of antigen to the T cell, and result in the T cell sensitization, activation and amplification. DCs play an important role in antitumor immunotherapy because of its potential abilities to overcome tumor tolerance and to induce antitumor immune capacity. Two main populations of DCs are recognized in mouse and human tissues:myeloid dendritic cell (mDC) and plasmacytoid dendritic cell(pDCs). The former are characterized by the high expression of CD11c and low expression of CD123. The latter express CD123at high level and CD11c at low level. There are various antitumor effect of immune responds in defferent DCs subsets. We purpose to establish MCF-7tumor-bearing mice, study the differentiation and maturation of bone marrow-derived DCs and the data of DCs subsets, in order to give the foundation information to design the new immune-therapeutic method and new vaccines in breast cancer.
Methods and materials5wk-old female BALB/c mice (ten mice/group) were injected subcutaneously (s.c.) with o.2ml(1×107/ml) MCF-7cells into the flank, while mice of the control group received o.2ml serum-free medium by s.c injection. Mice were sacrificed when tumor size reached5mm in diameter. All of the experiments were conducted in randomized fashion. The isolated cells from bone marrow were plated in nonadhesive Petri dishes at a density of1×107cells/1.5ml in medium (RPMI,10%FCS). The medium was replenished every3d. After3days in culture, all medium and nonadherent cells were discarded, fresh medium supplemented with murine recombinant GM-CSF and murine recombinant IL-4was added. For DCs maturation, the cells were treated overnight with100ng/ml LPS on day7. Nonadherent DCs were harvested on day9, and culture supernatants were collected, and used for following studies. DCs were identified by flow cytometry. DC subsets were verified by analyzing anti-CD11c PE or anti-CD123PE expression using flow cytometry, respectively. Immunohistochemistry was used to detect ER, PR, Cerb-B2and Ki-67of the tumor biopsy.
Comparisons of samples to establish statistical significance were determined by the two tailed Students't-test. Results were considered to be statistically significant when the p-value was<0.05.
Results:MCF-7cells were observed attached and in good shape under inverted microscope. The tumor size of tumor-bearing mice reached5mm in diameter after16days. There were no significant difference in the weight between both two groups before and after injected MCF-7cell.
We can observe the single nonadherent DCs with obvious burrs protuberance on the cellular surface when the bone marrow-derived PBMC has been cultured for9days.
High levels expression of CD86,CD83and MHC-Ⅱ were similarly observed in two groups, and the expression of CD83in tumor-bearing mice was significantly decreased than that in control mice.(74.61±1,26%, vs77.30±1.41%, P<0.05). Ther was a more significant descent on the expression of CD34(1.30±0.17%, vs1.71±0.18%, P<0.05). The cells of CD11c+CD123-in tumor-bearing mice was lower than that of control mice(52.01±1.43%, vs56.36±1.76%, P<0.05)。The cells of CD11c-CD123+in tumor-bearing mice was higher than that of control mice(32.19±1.71%, vs28.95±1.39%, P<0.05)。
The tumor biopsy can observe breast cancer cell, and immunohistochemistry shows that ER(+++), PR(++), Cerb-B2(-) and Ki-67(+++) in tumor cells.
Conclusion:The MCF-7Tumor Xenograft Model was established successfully. In vitro, bone marrow-derived PBMC can be induced high quality and high purity mature DCs.
The immature status of bone marrow-derived DCs in MCF-7tumor-bearing mice resulted in decreased the ability of tumor antigen presentation and subsequent lack the ability to induce T-cell activation and proliferation. The efficiency of immune killing tumor cells declined.
The number of mDC decreasing in MCF-7tumor-bearing mice showed the antitumor immune responses was diminished, which generated by presentation tumor antigen to T cell, in turn, the level of Il-12secreted and actived by mDC reduced, the effect of immune killing tumor cells lessened. While pDC has a weak immune responses against tumor, on the other hand, it has a strong ability to inhibit antitumor immune responses, so the high quantity of pDC can not enhance the antitumor immune function.
Chapter2The detection of apoptosis of dendritic cell subsets in MCF-7tumor-bearing mice
Background and Objection:Programmed cell death is important for maintaining the homeostasis of DCs in vivo. Dysregulated programmed cell death in DCs may change the lifespan of DCs and lead to the imparement of immune responses. Recent research suggests that induction apoptosis of DCs is another important mechanism of tumor escape immune recognition. In vitro, the interactions between tumor cells and tumor-derived cytokines can induce apoptosis of DCs.
There are two subsets T cell in human tissues:Thl cells and Th2cells. The function of Thl cells and Th2cells remains dynamic balance in normal organism. The antitumor immune responses is mainly depending on cellular immunity mediated by Thl cells. Matuer DCs not only can induce cytotoxic T lymphocyte(CTL) proliferation, also induce CTL secreting cytokines (such as IL-12).11-12has a strong capability of stimulating cytotoxic T cells, natural killer cells and lymphokine-activated killer cells, result in generating abundant of IFN-y, perforin and granzyme, the effect of lysing the target cell enhances. A Thl response is induced to antitumor. Different DCs subsets express various cellutoxic molecule. It was believed that pDC induce a Th2response, whereas mDC induce a Thl response.
Using DCs as antitumor immunotherapy is a powerful approach to eradicate tumor cells by enhancing the immune responses of the organism. The vaccines of tumor antigen with mature DCs have been applied in several cancers, however, although the technique of the therapy is ripe, the clinical effect is not ideal. More reasonable and more effectively DCs vaccines exploration would be required. We purpose to study the apoptosis of DCs subsets, in order to provide more accurate and more scientific iomformation on antitumor immune therapy.
Methods and materials:DCs suspensions were prepared as above. Cells were treated with CD11c-PC5and CD123-PE, respectively. And then were put at room temperature for30min. Annexin V Binding Buffer and FITC Annexin V were added after being washed with PBS, Three colour analysis was performed using flow cytometry for apoptosis of DC subsets.
Comparisons of samples to establish statistical significance were determined by the two tailed Students't-test. Results were considered to be statistically significant when the p-value was<0.05.
Results:The proportion of apoptotic mDCs in tumor-bearing mice was significantly increased (23.64±2.43%, vs20.05±2.49%, P<0.05). In contrast, in tumor-bearing mice, the percentage of apoptotic pDC was lower than that in control mice(11.53±2.92%, vs14.96±2.96%, P<0.05).
Conclusion The rate of apoptotic mDCs in tumor-bearing mice is higher significantly than that in control mice. It suggested that mDC in MCF-7breast cancer mice induced a weak Thl response, and the level of cytokines secreted by Thl cells including IL-12decreased. The consequence on one hand impaired the T-cell activation and proliferation, result in decreasing the capability of stimulating cytotoxic T cells, natural killer cells and lymphokine-activated killer cells to generate IFN-y, perforin and granzyme, in turn, the effect of lysing the target cell declined, on the other hand, changed the balance of Thl/Th2in the organism, induces immune tolerance and inhibited antitumor immune responses.
The rate of apoptotic pDCs in tumor-bearing mice is lower significantly than that in control mice. It suggested that pDC promoted ThO cell differentiation into Th2cells. Cytokines secreted by Th2response increased, induced producing CD4+/CD25+regulatory T cells to escape antitumor immune response. The high percentage of apoptotic pDC can also induce immune tolerance by declining the ability of antigen presentation, T-cell activation and proliferation, participate and promote breast cancer growth and metastasis by angiogenesis. There was another important factor on the descent of antitumor immune ability.
The rate of apoptotic mDC, which is mainly important in killing tumor cell by immune responses, in MCF-7breast cancer mice increasing can impair directly antitumor immune responses. However, decreasing of apoptotic pDC can inhibit monocyte cell differentiation into DCs, and decline the ability of antigen presentation of mDC even make mDC mediate immune tolerance, further impair antitumor immune responses in vivo.
Chapter3The detection of the levels of cytokines in MCF-7murine serum and DCs-derived supernatans
Background and Objection:Cytokines is a kinds of polypeptide substances with varied biological effects. IL-6, TNF-a and11-12are the important members of the cytokines net. They not only mediate the anti-infection immunity and autoimmunity, but also contribute to differentiation, metastasis and angiogenesis of tumor cells. They play a important role in surveillance and killin g tumor cells.
Now the role of TNF-a in breast cancer remains controversy. High dose of TNF-a can cause the vascular degeneration and hemorrhagic necrosis in tumor. However, other studies showed endogenousTNF-a in tumor cell can not kill tumor cell, but induce malignant mammary epithelial cell proliferation and promote angiogenesis, invasion and metastasis of tumor cells. IL-6involves in the antitumor immune responses. It can promote breast cancer stem cell growth and progression, and promote angiogenesis, and invasion of tumor cells. It plays an important role in tumor progression and estrogen regulation. As a key role of trigger cellular immunity, IL-12promotes NK-cell mediated killing of HER2-positive tumor cells. IL-12appears to contribute to humoral immunity in humans through a direct path in DC-B interaction, and an indirect path in DC-T cell interaction and induction of Th cells.
Varied cytokines play important roles in antitumor therapy. We purpose to study the change of these cytokines in tumor-bearing organism, and then modify the level of cytokines in vivo in reasonable ways and combine other immune therapy to enhance antitumor immune responses.
Methods and materials:For measurements of TNF-a, IL-6and IL-12secretion, murine serum and DCs-derived supernatans were collected, and assayed using commercially available ELISA kits. Samples were analyzed in duplicate according to manufacturer's instruction.
Comparisons of samples to establish statistical significance were determined by the two tailed Students't-test. Results were considered to be statistically significant when the p-value was<0.05.
Results:No major difference in the levels of TNF-a in serum was observed between control mice and tumor-bearing mice(314.81±103.36pg/ml, vs371.87±115.30pg/ml, P>0.05). however, TNF-a concentration in DCs-derived supernatans of tumor-bearing mice was higher than that of control mice (1.88±0.1Ong/ml, vs1.72±0.07ng/ml, P<0.05).
The IL-6level was significantly increased in serum of tumor-bearing mice(170.72±54.51pg/ml, vs122.35±20.65pg/ml, P<0.05). A similar increase in the IL-6level in DCs-derived supernatans was also observed in tumor-bearing mice(57.18±5.06pg/ml, vs44.53±4.86pg/ml, P<0.05).
On the other hand, IL-12level was significantly decreased either in serum or in DCs-derived supernatans in tumor-bearing mice(119.80±33.53pg/ml, vs186.22±51.91pg/ml, P<0.05)(89.76±8.89pg/ml, vs157.98±48.31pg/ml, P<0.05).
Conclusion:There was not significantly differences in TNF-α concentration in serum between tumor-bearing mice and control mice. It probably correlate with a variety production ways. TNF-α can product by the way of autocine, paracrine and endocrine. So a variety of cells and other cytokines involved in producting the TNF-α in MCF-7breast cancer. That is to say the role of TNF-α in breast cancer is more complex and need more comprehensive study. The level of TNF-α in DCs-derived supernatans of tumor-bearing mice was higher than that of control mice. DCs maybe participate to produce TNF-α. Further investigations in the role of TNF-α in antitumor immune in breast cancer should be needed, and the role maybe correlate with the dose of TNF-α and the type of tumor.
The level of IL-6showed a significant increase in serum in tumor bearing mice when compared with control mice. IL-6plays an important role in participating the breast cancer cell growth and promoting breast cancer cells infiltrating and metastasis as described above previous mechanism. IL-6concentration in DCs-derived supernatans of tumor-bearing mice was higher than that of control mice. Our data revealed that that IL-6may also display its suppressive ability in antitumor immune by decreasing the level of IL-6secreted from DCs.
IL-12concentration in serum of tumor-bearing mice was lower significantly than that of control mice. The ability of killing tumor cell by T cells and NK cells decreased because of low-level IL-12. In our study, the level of IL-12in DCs-derived supernatans had s significant reduction in tumor-bearing mice when compared with control mice. The result demonstrate that decreasing of IL-121evel may suppress exerting potent antitumor activity in breast cancer. Enhancement the level of IL-12in vivo, especially combining chemotherapy and target therapy, can powerfully improve the clinical effectiveness of the breast cancer.
Chapter4The relationships between apoptosis of DC subsets and cytokines level
Background and Objection:
The interplays between DCs and cytokines network can influence the antitumor immune response in organism. On one hand, cytokines play an important regulating role in the differentiation and maturation of DCs, arious tumor-derived factors (VEGF, IL-6, IL-10, M-CSF, and GM-CSF) can inhibit DC differentiation from hematopoietic progenitor cells (HPCs) in vitro and in vivo. On the other hand, defferent mature DC subsets can secret various cytokines to enhance or decrease the ability of immune killing response or immune tolerance responses.
Tumor can generate a series of immunosuppressive factors to form a immunosuppression environment. The changes of various soluble cytokines, including decreasing of inflammatory factors, increasing anti-inflammatory factors and increasing of Treg regulated T cell, can inhibit differentiation and maturation of DCs. With detrimental effects on DC quantity and function can significantly prevent the establishment of effective antitumor immune responses
The effects of various receptors, cytokines and chemokines may affect the lifespan of DCs at various stages of immune responses. Although the precise mechanism for the maintenance of DC homeostasis is not clear, emerging evidence suggests that cytokines may be important regulators. The investigation of interaction between DCs subsets and key cytokines in MCF-7breast cancer can bring important significance in designing composite antitumor immune therapy. On one hand, we can use the more effective DCs subsets to present antigen, induce T cell to generate specific antitumor immune responses, enhance cellular immunity mediated by CTL. On the other hand, combining cytokines can further enhance the immune killing response.
Methods and materials:Detecting the rate of mDC and oDC in tumor-bearing mice and control mice as described above, assayed the levels of TNF-a, IL-6and IL-12in serum and DCs-derived supernatans using commercially available ELISA kits.
Comparisons of samples to establish statistical significance were determined by the two tailed Students't-test. Pearson correlation analysis was used to assess associations between apoptosis of DC subsets and cytokines levels in DCs-derived supernatans. Results were considered to be statistically significant when the p-value was<0.05.
Results:The negative correlation betweenTNF-alevel in serum and proportion of apoptotic mDC was shown. r=-0.70, P=0.00. There was the negative correlation between IL-6level in serum and proportion of apoptotic pDC. r=-0.83,P=0.00. The IL-12level in serum and proportion of apoptoticmDC also showed a negative correlation. r=-0.83, P=0.00.
There was the negative correlation between TNF-a level in DCs-derived supernatans and proportion of apoptotic pDC. r=-0.91, P=0.00. The negative negative correlation betweenIL-6in in DCs-derived supernatans and proportion of apoptotic pDC was shown. r=-0.85, P=0.00. IL-12level and the percentage of apoptotic mDC in DCs-derived supernatans have also been shown negative correlation. r=-0.70, P=0.00.
Conclusion:TNF-a level and the percentage of apoptotic mDC in serum showed negative correlation, and the correlation has no significance because of various cells and factors participating the secretion of TNF-a. There was the negative correlation between TNF-a level in DCs-derived supernatans and proportion of apoptotic pDC. It showed that the level of TNF-a were significantly increased as proportion of apoptotic pDC was reduction, however, because of the bidirectional action on antitumor immunity in breast cancer, more investigations on the role of TNF-a in defferent molecule isoforms of breast cancer would be need.
Thl/Th2was imbalance due to reduction of apoptosis of pDC in MCF-7breast cancer and deflected to Th2status. As the main cytokines secreted by Th2cell, the level of IL-6increased significantly. IL-6can promote breast cancer stem cell growth and progression, and promote angiogenesis. It plays an important role in breast cancer progression and invasive. Moreover, IL-6can produce a marked effect with other cytokines, impair natural killer ability and promote breast cancer cell invasion and metastasis.
The level of IL-12decreased significantly due to the proportion of apoptotic mDC increased in MCF-7breast cancer mice. The status can stimulate ineffectively T cell proliferation and inhibit the maturation and activation of mDC, in turn, the balance of Thl/Th2changed, and immune tolerance induced, Finally lead to tumor progression and metastasis.
研究背景和目的
乳腺癌是妇女最常见的恶性肿瘤,发病率无论在发达国家或发展中国家仍在逐年上升；随着手术、化放疗、内分泌治疗及靶向治疗等综合治疗的合理使用,乳腺癌的死亡率在发达国家已呈下降趋势,治疗效果亦进入了一个相对平台期。然而,晚期和转移性乳腺癌仍不可治愈,成为威胁女性尤其是发展中国家女性生命的重要原因；另一方面,随着医学科学发展,抗肿瘤免疫治疗作为乳腺癌治疗一个很有前景的治疗模式受到越来越多的关注,树突状细胞(dendritic cells,DCs)作为机体功能最强的抗原呈递细胞,能摄取、加工和递呈抗原从而使T细胞致敏、激活和扩增,有潜在的克服肿瘤耐受的能力和负载肿瘤抗原后诱导产生抗肿瘤免疫力的能力,在抗肿瘤免疫治疗中发挥着重要作用。多项实验证明人类和鼠的DCs主要有两种亚群：髓系来源的树突状细胞(]myeloid dendritic cell, mDC)和淋巴来源的树突状细胞(plasmacytoid dendritic cell, pDC),前者高表达CD11c,低表达CD123；后者高表达CD123,低表达CD11c,不同的DCs亚群在介导机体免疫反应中发挥不同的作用。本研究拟通过构建MCF-7荷瘤小鼠,研究其骨髓起源的树突状细胞分化、成熟的情况及是否存在不同亚群表达,为设计新的免疫治疗方法和新的疫苗的提供基础信息。
方法
1.取对数生长期MCF-7细胞调整细胞浓度为1×107/ml,用于以下实验。
2.20只5周龄无特定病原体(SPF)级BALB/C雌性裸鼠,随机分为2组,用1ml注射器向荷瘤组小鼠右侧胸壁第二对乳腺脂肪垫注入0.2mL上述MCF-7细胞悬液,对照组注入0.2mL无血清培养基。待肿块直径达5mm后处死小鼠。
3.取小鼠骨髓单个核细胞,RPMI1640(10%FBS)培养基调整细胞浓度为107/1.5m1,每皿(60mm)加入1.5m1细胞悬液,再加入1.5mlRPMI1640隔3天更换1次培养液。第3天吸弃全部培养基及悬浮细胞,加入含100ng/mlrmGM-CSF,100ng/ml rmIL-4的上述培养基共孵育,第7天更换培养基时补加脂多糖100ng/mL共孵育,第8天吹打疏松贴壁的增殖性细胞聚集体,次日收集悬浮的细胞即为富集的DCs。相差显微镜下观察细胞形态特点。收集细胞做以下实验。
4.用PBS(含1%FBS)调整细胞浓度为106/100u1,每流式管加入100ul细胞悬液,再加入1ug/管相应荧光素偶联一抗,上机检测。
5.荷瘤小鼠乳房肿块病理免疫组化检测：supervision二步法检测ER、PR、 Cerb-B2、Ki-67。
6.统计学方法：计量资料统计描述用均数±标准差表示,两组均数比较用独立样本t检验。检验水准α=0.05。
结果
1.对数生长期MCF-7细胞倒置显微镜下观察,贴壁生长,形态良好、折光性强。
2.荷瘤组小鼠在注射MCF-7后第16天乳腺肿块达5mm或以上。荷瘤组和对照组小鼠体重在荷瘤前及荷瘤后均无统计学差异,具可比性。
3.从骨髓中获取的PBMC培养至第九天,可见单个的细胞表面有明显毛刺状突起的悬浮的DCs。
4.荷瘤组和对照组DCs均高表达CD86、CD83及MHC-Ⅱ,且荷瘤组CD83表达为74.61±1.26%,比对照组(77.30±1.41)低,P<0.05。CD34表达荷瘤组表达为1.30±0.17%,对照组为1.71±0.18%,P<0.05。荷瘤组CD11c+CD123-细胞52.01±1.43%,较对照组(56.36±1.76%)低,P<0.05。荷瘤组CD11c-CD123+细胞32.19±1.71%,较对照组(28.95±1.39%)高,P<0.05。
5.荷瘤小鼠乳腺肿块病理活检可见乳腺癌细胞,免疫组化示：ER(+++),PR(++), Cerb-B2(-),Ki-67(+++)
结论
1.动物模型构建成功,动物骨髓单个核细胞体外能诱导分化出高质量和高纯度的成熟的DCs。
2.MCF-7荷瘤小鼠骨髓起源的DCs存在成熟障碍,肿瘤抗原递呈能力降低,导致T细胞激活、增殖能力下降,机体免疫杀伤肿瘤细胞效应降低。
3.MCF-7乳腺癌小鼠mDC数量减少,pDC数量增多,意味着荷瘤机体mDCs把肿瘤抗原递呈至T细胞产生抗肿瘤免疫反应能力下降,而由mDC分泌并活化的IL-12也相应减少,对肿瘤细胞的免疫杀伤能力减低。而pDC一方面具有弱的免疫应答对抗肿瘤,另一方面存在较强抑制抗肿瘤免疫反应能力,故其数量的增多,并不能增强机体的抗肿瘤免疫功能。
第二章MCF-7荷瘤小鼠不同树突状细胞亚群凋亡率的检测
研究背景和目的
细胞程序性死亡是体内DCs维持动态平衡的重要方式,DCs程序性死亡失调可改变DCs的生存期而导致正常免疫反应的受损,最近的研究表明诱导DCs的凋亡是肿瘤逃避免疫识别的另一重要机制,在体外,肿瘤细胞和肿瘤起源的相关因子互相作用后能诱导DCs凋亡。
人类T细胞分为Th1和Th2两个亚群,正常状态下,Th1和Th2类细胞功能处于一种动态平衡状态,而机体抗肿瘤免疫以介导细胞免疫的Th1类细胞为主。成熟DCs不仅可诱导细胞毒T淋巴细胞(cytotoxic t lymphocyte,CTL)增殖,还诱导CTL分泌细胞因子(如interleukin-12,IL-12),IL-12有强有力的诱导细胞毒性T细胞、自然杀伤细胞、淋巴因子激活杀伤细胞的能力,产生大量TNF-γ、穿孔素和颗粒酶,增强对靶细胞的溶解作用,产生Th1型免疫应答而发挥抗肿瘤作用。不同的DCs亚群表达不同的细胞毒分子,一般认为pDC诱导TH2反应,而mDC诱导THl反应。
使用DCs作为抗肿瘤免疫治疗是一种通过增强患者自身的免疫反应来消灭肿瘤细胞的强有力的手段,利用成熟DCs负载肿瘤抗原的树突状细胞疫苗已在多种肿瘤患者中应用,然而这些疗法技术上虽已较成熟,而疗效均不甚理想,因此需要研究更合理和有效的DC疫苗。了解乳腺癌DCs不同亚群的凋亡,可进一步为树突状细胞在抗肿瘤免疫治疗方面的应用提供更准确、更科学的信息。
方法
1.同上获取DCs悬液,调整细胞浓度为106/100ul,每流式管加入100ul细胞悬液。
2.分别加入CD11c-PC5, CD123-PE各1ug,室温避光放置30分钟。
3用含1%FBS的PBS洗一次,细胞沉淀后加入100ul Annexin-V Binding Buffer,5ul Annexin-V,室温避光放置15分钟。
4.加入400ul Annexin-V Binding Buffer,上机同时检测FITC、PE、PE/CY5三个荧光通道。
5.统计学方法：计量资料统计描述用均数±标准差表示,两组均数比较用独立样本t检验。检验水准a=0.05。
结果
1.MCF-7荷瘤小鼠经上述方法诱导分化的mDC凋亡率为23.64±2.43%,对照组为20.05-2.49%,前者比后者高,P<0.05。
2,MCF-7乳腺癌小鼠骨髓来源pDC凋亡率为11.53±2.92%,,对照组为14.96±2.96%,荷瘤组比对照组低,P<0.05。
结论
1.MCF-7荷瘤小鼠mDC凋亡率比对照组明显增高,说明MCF-7乳腺癌小鼠mDC诱导Th1免疫反应能力减低,调节Thl细胞分泌相关细胞因子包括IL-12等能力下降,一方面,使T细胞增殖、活化受损,导致细胞毒T细胞、自然杀伤细胞、淋巴因子激活杀伤细胞产生干扰素-γ(interferon-γ,IFN-γ)、穿孔素和颗粒酶等能力相应下降,对肿瘤细胞的溶解作用减弱,从而降低机体对肿瘤细胞的免疫杀伤作用。另一方面,改变了机体Th1/Th2的平衡,诱导荷瘤机体产生免疫耐受,抑制抗肿瘤免疫反应。
2.荷瘤组小鼠骨髓来源pDC凋亡较对照组小鼠明显减少,提示MCF-7乳腺癌小鼠体内,Th0向Th2细胞分化增多,Th2细胞分泌因子增多,刺激产生CD4+/CD25+调节T细胞逃避抗肿瘤免疫,并使抗原递呈和T细胞增殖、活化功能降低,诱导体内免疫耐受的发生,并通过促血管生成参与和促进乳腺癌生长和转移。是机体抗肿瘤免疫反应能力下降的另一个重要因素。
3.pDC凋亡减少,还可抑制单核细胞分化成DCs,减少mDC的抗原递呈能力或甚至使mDC转变成介导免疫耐受,从而进一步削弱机体抗肿瘤免疫反应。
第三章MCF-7荷瘤小鼠血清及树突状细胞上清液细胞因子的测定
研究背景和目的
细胞因子是一类在体内有广泛生物学效应的多肽物质,干扰素-6(interleukin-6, IL-6)、TNF-α、IL-12等是细胞因子网络中的重要成员,不仅介导机体的抗感染免疫和自身免疫,还影响恶性肿瘤细胞的分化、转移、血管生成等过程,在监视和杀灭肿瘤细胞中发挥了重要作用。
目前,TNF-α在乳腺癌中的作用尚存争议,高剂量的TNF-α可引起肿瘤中血管退行性改变和出血性坏死,但是,有研究表明,在体内,肿瘤细胞中内源性TNF-α的分泌并不杀伤肿瘤,反而通过诱导恶性乳腺上皮细胞等肿瘤细胞增殖,并促进血管再生、肿瘤细胞侵袭和转移。IL-6参与对肿瘤的免疫反应,可促进乳腺癌干细胞的生长和恶化,并具有促进肿瘤血管生成和侵袭的作用,还在肿瘤演进和雌激素调节中发挥了重要作用,另外,IL-6可通过参与多种细胞因子的分泌,与其他细胞因子共同发挥作用,破坏机体的自然杀伤能力,促进乳腺癌细胞的浸润和转移。作为引发细胞免疫的关键因素, IL-12促进NK细胞介导的HER2(+)肿瘤细胞的杀伤；IL-12既能通过DCs和B细胞的一种直接的相互作用诱导体液免疫发生,也能通过DCs和T细胞的直接相互作用诱导Thl细胞。
多种细胞因子在抗肿瘤免疫治疗中起重要作用,深入研究这些细胞因子在荷瘤机体内的变化,可以我们通过合理途径,改变体内一些重要细胞因子的水平,结合其他免疫治疗,提高机体抗肿瘤免疫效应。
方法
1.摘除裸鼠眼球采静脉血约1ml,在室温下静置40分钟,室温下离心3,000rpm,15分钟,取上层血清4℃保存。
2.ELISA kit检测血清中TNF-α, IL-6, IL-12浓度。
3.上清液准备：DCs诱导分化第九天的上清液。
4.ELISA kit检测DCs上清液TNF-α, IL-6, IL-12浓度。
5.统计学方法：计量资料统计描述用均数±标准差表示,两组均数比较用独立样本t检验。检验水准α=0.05。
结果
1.MCF-7荷瘤小鼠血清TNF-α浓度为314.81±103.36pg/ml,比对照组小鼠浓度(371.87±115.30pg/ml)低,但差别无统计学意义。MCF-7荷瘤小鼠树突状细胞上清液TNF-α浓度为1.88±0.10ng/ml,比对照组小鼠的浓度高(1.72±0.07ng/ml),差别有统计学意义,P<0.05。
2.荷瘤组血清IL-6浓度为170.72±54.51pg/ml,与对照组(122.35±20.65pg/m1)比较,前者显著增高,P<0.05。荷瘤组DCs上清液IL-6浓度为57.18±5.06ng/ml,与对照组(44.53±4.86ng/ml)相比,有统计意义的增高,P<0.05。
3.IL-12血清浓度荷瘤组为119.80±33.53pg/ml,对照组为186.22±51.91pg/ml,前者明显降低,差别有统计学意义,P<0.05。IL-12上清液浓度荷瘤组为89.76±8.89pg/ml,对照组为157.98±48.31pg/ml,前者明显降低,差别有统计学意义,P<0.05。
结论
1.荷瘤小鼠血清中的TNF-α浓度比对照组低,但无统计学意义,可能跟TNF-α多种产生形式有关,TNF-α可通过自分泌、旁分泌和内分泌的形式产生,故MCF-7乳腺癌体内多种细胞及其他细胞因子参与了TNF-α的分泌,从另一侧面反映TNF-α在这种乳腺癌的作用可能比较复杂,需要更多更全面的研究。MCF-7荷瘤小鼠DCs上清液TNF-α浓度比对照组高,DCs可能参与产生TNF-α,至于TNF-α在MCF-7乳腺癌中发挥抗肿瘤作用还是促进肿瘤侵袭和转移有待更深入的研究,且此作用可能与剂量、肿瘤类型等有关。
2.MCF-7乳腺癌小鼠血清IL-6水平比对照组显著升高,说明IL-6可能通过上述机制,参与这种乳腺癌肿瘤细胞的生长,促进乳腺癌细胞的浸润和转移。MCF-7荷瘤小鼠DCs上清液IL-6水平亦高于对照组,说明DCs促进IL-6的分泌,使其参与抑制抗肿瘤免疫反应。
3.MCF-7荷瘤小鼠血清IL-12浓度显著低于对照组,低水平的IL-12,使T细胞及NK细胞对肿瘤细胞的杀伤作用减弱；MCF-7荷瘤小鼠DCs上清液IL-12水平亦明显低于对照组,MCF-7乳腺癌DCs影响IL-12的分泌,从而使机体抗肿瘤免疫效应减低,提高机体的IL-12水平,尤其结合化疗及靶向治疗,应该可以大大提高这种乳腺癌的治疗效果。
第四章树突状细胞亚群凋亡与细胞因子的关系
研究背景和目的
DCs和细胞因子网络在机体抗肿瘤免疫中互相影响和互相作用,一方面,细胞因子在DCs分化成熟过程中发挥重要的调节作用,各种肿瘤起源的相关因子(VEGF, IL-6, IL-10, M-CSF,和GM-CSF)能在体内外抑制DCs从造血干细胞中分化；另一方面,成熟的DCs不同亚群在发挥免疫杀伤或免疫耐受过程亦分泌各种细胞因子来增强或减弱这些反应。
肿瘤产生一系列抑制因素而形成一个免疫抑制环境,其分泌的多种可溶性因子的变化,包括炎症因子分泌的减少、抗炎症因子分泌的增多、以及Treg调节T细胞分泌的增多,能够抑制DCs的产生和分化成熟,对肿瘤宿主DC的数量和功能缺陷起着重要的调控作用,从而抑制机体对肿瘤细胞的免疫杀伤作用。
各种受体、细胞因子、炎症趋化因子可在免疫反应的不同阶段影响DCs的生存期。尽管维持DCs动态平衡的确切机制尚未清楚,很多证据显示细胞因子可能是重要的调节。MCF-7乳腺瘤小鼠树突状细胞亚群的凋亡与重要的细胞因子互相影响和互相作用,了解这种复杂的相互关系,对于设计复合的抗肿瘤免疫治疗有重要意义。一方面,我们可以利用更有效的DCs亚群来递呈抗原,使可以成功诱导自体T淋巴细胞产生特异性抗肿瘤免疫反应,增强CTL介导的细胞免疫；另一方面,在此过程,结合细胞因子的应用,能进一步放大肿瘤细胞的免疫杀伤作用。
方法
1.上述方法分别计算荷瘤组和对照组mDC、pDC凋亡率
2.ELISA法检测小鼠血清及DCs上清液TNF-α、IL-6、IL-12浓度测定。
3.统计学方法：计量资料的统计描述用均数±标准差表示,两组均数比较用独立样本t检验；两计量指标的相关性统计用Pearson相关分析。检验水准α=0.05。
结果
1血清TNF-α浓度与mDC凋亡率呈负相关,r=-0.70,P=0.00；IL-6浓度与pDC凋亡率呈负相关,r=-0.83,P=0.00；IL-12与mDC凋亡率呈负相关,r=-0.83,P=0.00。
2.DCs上清液TNF-α浓度与pDC凋亡率呈负相关,r=-0.91,P=0.00；IL-6浓度与pDC凋亡率呈负相关,r=-0.85,P=0.00；IL-12与mDC凋亡率呈负相关,r=-0.70,P=0.00。
结论
1.血清TNF-α浓度随着mDC凋亡率增加而减低,由于血清中TNF-α的由多种细胞分泌,多种因素参与,故其浓度变化与mDC的相关性并无实际意义,而在骨髓起源的DCs上清液,TNF-α浓度随着pDC凋亡率降低而增加,而由于TNF-α在抗肿瘤免疫中的双向性,故pDC凋亡对其在乳腺癌,尤其是不同分子亚型中的作用有待深入的探讨。
2.IL-6浓度无论血清或DCs上清液,均随着pDC凋亡率降低而增加,在MCF-7乳腺癌小鼠,由于pDC凋亡的减少,Th1/Th2失衡,向Th2偏移,作为Th2细胞分泌的主要细胞因子之一,IL-6水平明显增高,在乳腺癌的进展、侵袭中起重要作用。
3.IL-12浓度无论血清或DCs上清液,均随着mDC凋亡率增加而减低,在MCF-7乳腺癌小鼠,由于mDC凋亡的增多,其分泌的IL-12显著减少,导致不能有效刺激T淋巴细胞增殖,抑制mDC的成熟和活化,并改变Th1/Th2的平衡,诱导机体产生免疫耐受,最终肿瘤发生进展、转移。
Chapter1Establishment of MCF-7xenograft model in nude mice, induction and differentiation of dendritic cells and detection of its surface marker
Background and Objection:Breast cancer is the most common magnicnant in women. No matter in developed countries or developing countries, the incidence rate is increasing year by year. As operation, radiotherapy, endocrine therapy and targeted therapy are used reasonably, the mortality of breast cancer in developed countries has been declined. The clinical responds has come into a relative plateau, however, advanced and metastatic disease remains incurable and has become the main cause of threatened the life of female, especially in developing countries. On the other hand, as a promising treatment pattern, antitumor immunotherapy is received more and more attention along with the progress of medicine. Dendritic cells(DCs) is the most powerful antigen presenting cell in organism. It can uptake, processing and presentation of antigen to the T cell, and result in the T cell sensitization, activation and amplification. DCs play an important role in antitumor immunotherapy because of its potential abilities to overcome tumor tolerance and to induce antitumor immune capacity. Two main populations of DCs are recognized in mouse and human tissues:myeloid dendritic cell (mDC) and plasmacytoid dendritic cell(pDCs). The former are characterized by the high expression of CD11c and low expression of CD123. The latter express CD123at high level and CD11c at low level. There are various antitumor effect of immune responds in defferent DCs subsets. We purpose to establish MCF-7tumor-bearing mice, study the differentiation and maturation of bone marrow-derived DCs and the data of DCs subsets, in order to give the foundation information to design the new immune-therapeutic method and new vaccines in breast cancer.
Methods and materials5wk-old female BALB/c mice (ten mice/group) were injected subcutaneously (s.c.) with o.2ml(1×107/ml) MCF-7cells into the flank, while mice of the control group received o.2ml serum-free medium by s.c injection. Mice were sacrificed when tumor size reached5mm in diameter. All of the experiments were conducted in randomized fashion. The isolated cells from bone marrow were plated in nonadhesive Petri dishes at a density of1×107cells/1.5ml in medium (RPMI,10%FCS). The medium was replenished every3d. After3days in culture, all medium and nonadherent cells were discarded, fresh medium supplemented with murine recombinant GM-CSF and murine recombinant IL-4was added. For DCs maturation, the cells were treated overnight with100ng/ml LPS on day7. Nonadherent DCs were harvested on day9, and culture supernatants were collected, and used for following studies. DCs were identified by flow cytometry. DC subsets were verified by analyzing anti-CD11c PE or anti-CD123PE expression using flow cytometry, respectively. Immunohistochemistry was used to detect ER, PR, Cerb-B2and Ki-67of the tumor biopsy.
Comparisons of samples to establish statistical significance were determined by the two tailed Students't-test. Results were considered to be statistically significant when the p-value was<0.05.
Results:MCF-7cells were observed attached and in good shape under inverted microscope. The tumor size of tumor-bearing mice reached5mm in diameter after16days. There were no significant difference in the weight between both two groups before and after injected MCF-7cell.
We can observe the single nonadherent DCs with obvious burrs protuberance on the cellular surface when the bone marrow-derived PBMC has been cultured for9days.
High levels expression of CD86,CD83and MHC-Ⅱ were similarly observed in two groups, and the expression of CD83in tumor-bearing mice was significantly decreased than that in control mice.(74.61±1,26%, vs77.30±1.41%, P<0.05). Ther was a more significant descent on the expression of CD34(1.30±0.17%, vs1.71±0.18%, P<0.05). The cells of CD11c+CD123-in tumor-bearing mice was lower than that of control mice(52.01±1.43%, vs56.36±1.76%, P<0.05)。The cells of CD11c-CD123+in tumor-bearing mice was higher than that of control mice(32.19±1.71%, vs28.95±1.39%, P<0.05)。
The tumor biopsy can observe breast cancer cell, and immunohistochemistry shows that ER(+++), PR(++), Cerb-B2(-) and Ki-67(+++) in tumor cells.
Conclusion:The MCF-7Tumor Xenograft Model was established successfully. In vitro, bone marrow-derived PBMC can be induced high quality and high purity mature DCs.
The immature status of bone marrow-derived DCs in MCF-7tumor-bearing mice resulted in decreased the ability of tumor antigen presentation and subsequent lack the ability to induce T-cell activation and proliferation. The efficiency of immune killing tumor cells declined.
The number of mDC decreasing in MCF-7tumor-bearing mice showed the antitumor immune responses was diminished, which generated by presentation tumor antigen to T cell, in turn, the level of Il-12secreted and actived by mDC reduced, the effect of immune killing tumor cells lessened. While pDC has a weak immune responses against tumor, on the other hand, it has a strong ability to inhibit antitumor immune responses, so the high quantity of pDC can not enhance the antitumor immune function.
Chapter2The detection of apoptosis of dendritic cell subsets in MCF-7tumor-bearing mice
Background and Objection:Programmed cell death is important for maintaining the homeostasis of DCs in vivo. Dysregulated programmed cell death in DCs may change the lifespan of DCs and lead to the imparement of immune responses. Recent research suggests that induction apoptosis of DCs is another important mechanism of tumor escape immune recognition. In vitro, the interactions between tumor cells and tumor-derived cytokines can induce apoptosis of DCs.
There are two subsets T cell in human tissues:Thl cells and Th2cells. The function of Thl cells and Th2cells remains dynamic balance in normal organism. The antitumor immune responses is mainly depending on cellular immunity mediated by Thl cells. Matuer DCs not only can induce cytotoxic T lymphocyte(CTL) proliferation, also induce CTL secreting cytokines (such as IL-12).11-12has a strong capability of stimulating cytotoxic T cells, natural killer cells and lymphokine-activated killer cells, result in generating abundant of IFN-y, perforin and granzyme, the effect of lysing the target cell enhances. A Thl response is induced to antitumor. Different DCs subsets express various cellutoxic molecule. It was believed that pDC induce a Th2response, whereas mDC induce a Thl response.
Using DCs as antitumor immunotherapy is a powerful approach to eradicate tumor cells by enhancing the immune responses of the organism. The vaccines of tumor antigen with mature DCs have been applied in several cancers, however, although the technique of the therapy is ripe, the clinical effect is not ideal. More reasonable and more effectively DCs vaccines exploration would be required. We purpose to study the apoptosis of DCs subsets, in order to provide more accurate and more scientific iomformation on antitumor immune therapy.
Methods and materials:DCs suspensions were prepared as above. Cells were treated with CD11c-PC5and CD123-PE, respectively. And then were put at room temperature for30min. Annexin V Binding Buffer and FITC Annexin V were added after being washed with PBS, Three colour analysis was performed using flow cytometry for apoptosis of DC subsets.
Comparisons of samples to establish statistical significance were determined by the two tailed Students't-test. Results were considered to be statistically significant when the p-value was<0.05.
Results:The proportion of apoptotic mDCs in tumor-bearing mice was significantly increased (23.64±2.43%, vs20.05±2.49%, P<0.05). In contrast, in tumor-bearing mice, the percentage of apoptotic pDC was lower than that in control mice(11.53±2.92%, vs14.96±2.96%, P<0.05).
Conclusion The rate of apoptotic mDCs in tumor-bearing mice is higher significantly than that in control mice. It suggested that mDC in MCF-7breast cancer mice induced a weak Thl response, and the level of cytokines secreted by Thl cells including IL-12decreased. The consequence on one hand impaired the T-cell activation and proliferation, result in decreasing the capability of stimulating cytotoxic T cells, natural killer cells and lymphokine-activated killer cells to generate IFN-y, perforin and granzyme, in turn, the effect of lysing the target cell declined, on the other hand, changed the balance of Thl/Th2in the organism, induces immune tolerance and inhibited antitumor immune responses.
The rate of apoptotic pDCs in tumor-bearing mice is lower significantly than that in control mice. It suggested that pDC promoted ThO cell differentiation into Th2cells. Cytokines secreted by Th2response increased, induced producing CD4+/CD25+regulatory T cells to escape antitumor immune response. The high percentage of apoptotic pDC can also induce immune tolerance by declining the ability of antigen presentation, T-cell activation and proliferation, participate and promote breast cancer growth and metastasis by angiogenesis. There was another important factor on the descent of antitumor immune ability.
The rate of apoptotic mDC, which is mainly important in killing tumor cell by immune responses, in MCF-7breast cancer mice increasing can impair directly antitumor immune responses. However, decreasing of apoptotic pDC can inhibit monocyte cell differentiation into DCs, and decline the ability of antigen presentation of mDC even make mDC mediate immune tolerance, further impair antitumor immune responses in vivo.
Chapter3The detection of the levels of cytokines in MCF-7murine serum and DCs-derived supernatans
Background and Objection:Cytokines is a kinds of polypeptide substances with varied biological effects. IL-6, TNF-a and11-12are the important members of the cytokines net. They not only mediate the anti-infection immunity and autoimmunity, but also contribute to differentiation, metastasis and angiogenesis of tumor cells. They play a important role in surveillance and killin g tumor cells.
Now the role of TNF-a in breast cancer remains controversy. High dose of TNF-a can cause the vascular degeneration and hemorrhagic necrosis in tumor. However, other studies showed endogenousTNF-a in tumor cell can not kill tumor cell, but induce malignant mammary epithelial cell proliferation and promote angiogenesis, invasion and metastasis of tumor cells. IL-6involves in the antitumor immune responses. It can promote breast cancer stem cell growth and progression, and promote angiogenesis, and invasion of tumor cells. It plays an important role in tumor progression and estrogen regulation. As a key role of trigger cellular immunity, IL-12promotes NK-cell mediated killing of HER2-positive tumor cells. IL-12appears to contribute to humoral immunity in humans through a direct path in DC-B interaction, and an indirect path in DC-T cell interaction and induction of Th cells.
Varied cytokines play important roles in antitumor therapy. We purpose to study the change of these cytokines in tumor-bearing organism, and then modify the level of cytokines in vivo in reasonable ways and combine other immune therapy to enhance antitumor immune responses.
Methods and materials:For measurements of TNF-a, IL-6and IL-12secretion, murine serum and DCs-derived supernatans were collected, and assayed using commercially available ELISA kits. Samples were analyzed in duplicate according to manufacturer's instruction.
Comparisons of samples to establish statistical significance were determined by the two tailed Students't-test. Results were considered to be statistically significant when the p-value was<0.05.
Results:No major difference in the levels of TNF-a in serum was observed between control mice and tumor-bearing mice(314.81±103.36pg/ml, vs371.87±115.30pg/ml, P>0.05). however, TNF-a concentration in DCs-derived supernatans of tumor-bearing mice was higher than that of control mice (1.88±0.1Ong/ml, vs1.72±0.07ng/ml, P<0.05).
The IL-6level was significantly increased in serum of tumor-bearing mice(170.72±54.51pg/ml, vs122.35±20.65pg/ml, P<0.05). A similar increase in the IL-6level in DCs-derived supernatans was also observed in tumor-bearing mice(57.18±5.06pg/ml, vs44.53±4.86pg/ml, P<0.05).
On the other hand, IL-12level was significantly decreased either in serum or in DCs-derived supernatans in tumor-bearing mice(119.80±33.53pg/ml, vs186.22±51.91pg/ml, P<0.05)(89.76±8.89pg/ml, vs157.98±48.31pg/ml, P<0.05).
Conclusion:There was not significantly differences in TNF-α concentration in serum between tumor-bearing mice and control mice. It probably correlate with a variety production ways. TNF-α can product by the way of autocine, paracrine and endocrine. So a variety of cells and other cytokines involved in producting the TNF-α in MCF-7breast cancer. That is to say the role of TNF-α in breast cancer is more complex and need more comprehensive study. The level of TNF-α in DCs-derived supernatans of tumor-bearing mice was higher than that of control mice. DCs maybe participate to produce TNF-α. Further investigations in the role of TNF-α in antitumor immune in breast cancer should be needed, and the role maybe correlate with the dose of TNF-α and the type of tumor.
The level of IL-6showed a significant increase in serum in tumor bearing mice when compared with control mice. IL-6plays an important role in participating the breast cancer cell growth and promoting breast cancer cells infiltrating and metastasis as described above previous mechanism. IL-6concentration in DCs-derived supernatans of tumor-bearing mice was higher than that of control mice. Our data revealed that that IL-6may also display its suppressive ability in antitumor immune by decreasing the level of IL-6secreted from DCs.
IL-12concentration in serum of tumor-bearing mice was lower significantly than that of control mice. The ability of killing tumor cell by T cells and NK cells decreased because of low-level IL-12. In our study, the level of IL-12in DCs-derived supernatans had s significant reduction in tumor-bearing mice when compared with control mice. The result demonstrate that decreasing of IL-121evel may suppress exerting potent antitumor activity in breast cancer. Enhancement the level of IL-12in vivo, especially combining chemotherapy and target therapy, can powerfully improve the clinical effectiveness of the breast cancer.
Chapter4The relationships between apoptosis of DC subsets and cytokines level
Background and Objection:
The interplays between DCs and cytokines network can influence the antitumor immune response in organism. On one hand, cytokines play an important regulating role in the differentiation and maturation of DCs, arious tumor-derived factors (VEGF, IL-6, IL-10, M-CSF, and GM-CSF) can inhibit DC differentiation from hematopoietic progenitor cells (HPCs) in vitro and in vivo. On the other hand, defferent mature DC subsets can secret various cytokines to enhance or decrease the ability of immune killing response or immune tolerance responses.
Tumor can generate a series of immunosuppressive factors to form a immunosuppression environment. The changes of various soluble cytokines, including decreasing of inflammatory factors, increasing anti-inflammatory factors and increasing of Treg regulated T cell, can inhibit differentiation and maturation of DCs. With detrimental effects on DC quantity and function can significantly prevent the establishment of effective antitumor immune responses
The effects of various receptors, cytokines and chemokines may affect the lifespan of DCs at various stages of immune responses. Although the precise mechanism for the maintenance of DC homeostasis is not clear, emerging evidence suggests that cytokines may be important regulators. The investigation of interaction between DCs subsets and key cytokines in MCF-7breast cancer can bring important significance in designing composite antitumor immune therapy. On one hand, we can use the more effective DCs subsets to present antigen, induce T cell to generate specific antitumor immune responses, enhance cellular immunity mediated by CTL. On the other hand, combining cytokines can further enhance the immune killing response.
Methods and materials:Detecting the rate of mDC and oDC in tumor-bearing mice and control mice as described above, assayed the levels of TNF-a, IL-6and IL-12in serum and DCs-derived supernatans using commercially available ELISA kits.
Comparisons of samples to establish statistical significance were determined by the two tailed Students't-test. Pearson correlation analysis was used to assess associations between apoptosis of DC subsets and cytokines levels in DCs-derived supernatans. Results were considered to be statistically significant when the p-value was<0.05.
Results:The negative correlation betweenTNF-alevel in serum and proportion of apoptotic mDC was shown. r=-0.70, P=0.00. There was the negative correlation between IL-6level in serum and proportion of apoptotic pDC. r=-0.83,P=0.00. The IL-12level in serum and proportion of apoptoticmDC also showed a negative correlation. r=-0.83, P=0.00.
There was the negative correlation between TNF-a level in DCs-derived supernatans and proportion of apoptotic pDC. r=-0.91, P=0.00. The negative negative correlation betweenIL-6in in DCs-derived supernatans and proportion of apoptotic pDC was shown. r=-0.85, P=0.00. IL-12level and the percentage of apoptotic mDC in DCs-derived supernatans have also been shown negative correlation. r=-0.70, P=0.00.
Conclusion:TNF-a level and the percentage of apoptotic mDC in serum showed negative correlation, and the correlation has no significance because of various cells and factors participating the secretion of TNF-a. There was the negative correlation between TNF-a level in DCs-derived supernatans and proportion of apoptotic pDC. It showed that the level of TNF-a were significantly increased as proportion of apoptotic pDC was reduction, however, because of the bidirectional action on antitumor immunity in breast cancer, more investigations on the role of TNF-a in defferent molecule isoforms of breast cancer would be need.
Thl/Th2was imbalance due to reduction of apoptosis of pDC in MCF-7breast cancer and deflected to Th2status. As the main cytokines secreted by Th2cell, the level of IL-6increased significantly. IL-6can promote breast cancer stem cell growth and progression, and promote angiogenesis. It plays an important role in breast cancer progression and invasive. Moreover, IL-6can produce a marked effect with other cytokines, impair natural killer ability and promote breast cancer cell invasion and metastasis.
The level of IL-12decreased significantly due to the proportion of apoptotic mDC increased in MCF-7breast cancer mice. The status can stimulate ineffectively T cell proliferation and inhibit the maturation and activation of mDC, in turn, the balance of Thl/Th2changed, and immune tolerance induced, Finally lead to tumor progression and metastasis.
引文
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