【作者】 雷明盛；

【导师】 胡成平；

【作者基本信息】 中南大学 ， 内科学， 2009， 博士

【摘要】 目的:肺癌已成为全球发病率、致死率最高的恶性肿瘤。近几年来,世界各国肺癌的发病率和死亡率都有升高,每年新发肺癌超过百万,我国肺癌的发病率已高达61.4/10万,到2015年,我国将成为世界第一肺癌大国。尽管肺癌的化学治疗、放射治疗、分子靶向治疗等技术不断发展,肺癌治疗的总体疗效仍然很差。生物反应调节剂（biology response modulation BRM）作为肿瘤防治的第四程式受到医学界广泛的重视。BRM的作用机制,主要是通过激活机体抗肿瘤的免疫,增强或恢复宿主对肿瘤的免疫反应。在机体肿瘤免疫监护功能及肿瘤特异性免疫应答产生的过程中,树突状细胞（dendriticcells,DCs）是功能强大的抗原提呈细胞,在肿瘤免疫抑制中起重要作用,具有激活静息型T细胞及诱导初级免疫应答的能力。然而肿瘤细胞能够逃避宿主的免疫监视系统,使肿瘤得以生长及转移;肿瘤对DC还有抑制作用,可造成免疫耐受,使T细胞的特异性杀伤性失去靶向。因此,DC特有的生物学特性使其成为了当今肿瘤生物治疗领域备受关注的焦点之一。由于DCs的重要功能使人们越来越多地关注DCs的临床应用,特别是其抗肿瘤作用,目前应用DC治疗肿瘤已取得了初步成果,一系列动物实验研究证明DC可以诱导特异性抗肿瘤保护效应,使肿瘤消退或稳定。但是仍有许多问题有待进一步研究解决,怎样恢复肿瘤内环境受抑的DC,如何提高DC介导的T细胞的杀伤活性?因此,寻找简单、经济、有效的刺激物作为免疫佐剂,更有效地激发机体抗肿瘤的免疫反应,是今后肺癌防治的一个方向。茶氨酸是绿茶的提取物,可通过多种机制防治肿瘤,在生物化学及分子水平对肿瘤发生的多个环节进行干预,调节免疫器官功能,防止免疫抑制,延缓荷瘤动物免疫衰退,增强肿瘤特异性和非特异性免疫,对机体抗癌“主力军”细胞毒T细胞杀伤肿瘤细胞功能有明显增强作用。而茶氨酸作为生物反应调节剂,未发现任何毒性反应。预实验采用模拟肿瘤细胞分泌细胞因子抑制DC环境,用蛙皮素与DC细胞共培养（蛙皮素是肿瘤细胞分泌主要抑制DC的细胞因子之一）,证实蛙皮素与DC前体细胞共培养时可显著减少DC的数量,DC表达HLA-DR、CD86、CD83、CD80、CD40显著减低,DC刺激淋巴细胞反应的能力下降,而茶氨酸刺激蛙皮素抑制DC,HLA-DR、CD80、CD86、CD83、CD40表面分子表达明显增强,接近正常对照组。因此,推测茶氨酸可拮抗肿瘤内环境肺癌细胞分泌蛙皮素对DC的抑制,提高DC刺激T淋巴细胞增殖的能力,增强对肺癌细胞的杀伤力。故行模拟肺癌内环境下蛙皮素抑制DC,以及SCID小鼠（A549）移植瘤实验,采用茶氨酸作为受试因素,观察对蛙皮素抑制DC以及对SCID小鼠移植瘤的影响。方法:健康人外周血来源单个核细胞,严格按照DC的分离培养、扩增方法,行形态学,DC特征性表面分子检查,进行DC鉴定。与蛙皮素共培养,行DC电镜、特征性表面分子检查、凋亡检测,建立蛙皮素抑制DC的模型。茶氨酸拮抗蛙皮素对DC的抑制实验:模拟肺癌内环境肺癌细胞分泌蛙皮素对DC抑制作用,建立蛙皮素抑制DC模型,用茶氨酸进行干预,通过对DC形态学,功能成熟的影响及COX-2表达等证明茶氨酸对肿瘤环境下肿瘤细胞分泌蛙皮素抑制DC的保护和活化效应。分茶氨酸+蛙皮素DC组;蛙皮素DC组;PBSDC组。通过蛙皮素抑制DC,用茶氨酸干预后与正常组DC对比,观察不同茶氨酸浓度对蛙皮素抑制DC表面分子（CD40、CD80 CD83、CD86、HLA-DR）和白介素-12（IL-12）的影响（剂量-效应关系）,对抑制DC凋亡的分析,DC的形态学检查,以及对COX-2表达的影响（剂量.效应关系）。体内实验:通过SCID小鼠体内注射人外周血淋巴细胞,重建免疫后,以茶氨酸受试因素刺激DC细胞,用A549细胞株接种于SCID小鼠皮下,通过对SCID小鼠移植瘤模型作用观察,证实茶氨酸对DC免疫保护和免疫治疗作用。培养A549细胞,按原方法分离培养、培养DC。分茶氨酸刺激DC组、DC组、茶氨酸组、PBS组,DC以同质培养液调细胞浓度为6.25×10⁵/ml,加入24孔培养板中,每孔2ml,37℃、5%CO₂中培养7d备用,而茶氨酸组取上述培养2d的DC悬液,添加抽滤的茶氨酸,使其终浓度为200μmol/L（第一章最佳刺激浓度）,按上述培养方法培养。DC、A549、PBS、茶氨酸分别腹腔注射,行SCID小鼠致瘤性实验,观察各自致瘤性,在光镜下行组织病理学检查。SCID小鼠免疫重建实验,ELISA法检测SCID小鼠IgG水平。诱导抗肿瘤的免疫保护作用实验,观察肿瘤生长的潜伏期、肿瘤的大小、重量、存活率、生存时间,组织病理学检查。抗肿瘤治疗作用实验,观察肿瘤的大小、重量,并行组织病理学,免疫组织化学检查,观察各组瘤体组织中VEGF的表达情况。结果:显微镜下观察、电镜检测,呈典型的DC形态学特点;CD40、CD80、CD83、CD86、HLA-DR的表达增强,CD83是DC特征性表面分子,从形态学和特征性的表面分子鉴定是DC。纯度、活细胞数检查,符合DC的后续实验要求。加入蛙皮素后共培养,电镜检查,包膜突起少,凋亡实验,其凋亡率是16%,特征性表面分子表达降低,与肺癌细胞和DC共培养的一致,故蛙皮素抑制DC的模型成功建立。模拟肺癌细胞分泌蛙皮素抑制DC,加入茶氨酸培养,7天后流式细胞仪检测DC表面分子的表达情况,茶氨酸能够上调CD40、CD80、CD83、CD86、HLA-DR的表达,用不同浓度的茶氨酸（25μmol/L、50μmol/L、100μmol/L、200μmol/L、400μmol/L）处理DC,结果其效应呈浓度依赖性,并且最佳浓度为200μmol/l,因为浓度大于200μmol/l时（如400μmol/l）表面分子的表达降低。在DC培养的第7天,观察茶氨酸对DC凋亡的影响,与蛙皮素组相比,茶氨酸处理组能够抑制蛙皮素促使DC凋亡的作用。其凋亡率分别是:蛙皮素组16.0%;茶氨酸处理DC组8.4%;PBS组4.0%。茶氨酸能够促进DC分泌IL-12,ELISA结果显示DC在未做任何处理时（即蛙皮素组）IL-12分泌的量很少（63.5±3.6pg/ml）,茶氨酸和正常组的DC能够分泌高水平的IL-12,200μmol/l浓度时为:90.4±4.9pg/ml,此试验结果与对表面分子表达的影响、抗凋亡实验的结果综合起来说明茶氨酸能够拮抗蛙皮素对DC的抑制作用,促进DC功能的成熟,并且呈浓度依赖性。茶氨酸处理组的DC扫描电镜照片展示出比较成熟的形态学特征,DC培养至第7天时收集,茶氨酸处理组和正常组的DC表面可见很长的突起,这是DC成熟的特征;而未做处理的DC（即蛙皮素组）的突起很短甚至没有突起。茶氨酸对树突状细胞COX-2 mRNA表达的影响,不同浓度茶氨酸处理DC 7天,RT-PCR检测COX-2 mRNA的表达,随着药物浓度的增加,表达降低,呈浓度依赖性。对树突状细胞COX-2蛋白表达,不同浓度茶氨酸处理DC 7天Western-blot检测COX-2蛋白表达。结果显示,茶氨酸作用后,可见COX-2蛋白表达的抑制,随着药物浓度增加,抑制效应增强。第一部分得出最佳刺激浓度为200μmol/L,使用该浓度刺激DC,行肺癌A549细胞株SCID小鼠移植瘤实验,腹腔注射人外周血淋巴细胞免疫重建,行SCID小鼠致瘤性实验,A549组SCID小鼠肺组织表面转移瘤结节数量较多,结节较大,部分结节相互融合,结节中央可见坏死。在光镜下行组织病理学检查,均可见肿瘤组织呈实体样结构或腺腔样结构,肿瘤细胞长梭形,核大,核仁明显,可见核分裂象,胞浆微呈透明稍嗜碱,细胞大小不等,癌细胞被纤维组织分隔成大小不等的癌巢,肺转移瘤模型成功建立。DC组未见肿瘤的生成。因此,A549具致瘤性,符合腺癌的特征。诱导抗肿瘤的免疫保护作用实验,茶氨酸刺激DC组肿瘤生长的潜伏期延长、生存时间较对照组延长,肿瘤的体积较对照组小,重量较对照组轻,组织病理学检查可见不同程度的坏死。抗肿瘤治疗作用实验,肿瘤体积和重量两两比较有差异性,并行组织病理学检查可见不同程度的坏死。免疫组织化学检测,观察各组瘤体组织中VEGF的表达情况比较。结果显示茶氨酸刺激DC能明显抑制SCID小鼠移植瘤细胞表达VEGF。免疫组织化学检测VEGF在肿瘤中的表达,分析显示棕黄色颗粒主要定位于胞浆,VEGF在PBS、茶氨酸组肿瘤组织中呈高表达,而在茶氨酸刺激组和/或DC组肿瘤组织中表达减弱,VEGF的表达总记分情况:茶氨酸刺激DC组＜C组＜茶氨酸组＜PBS组,两两比较,茶氨酸、PBS组没差别,余有明显差别。结论:1.本实验成功分离、扩增、分化树突状细胞。以健康人外周血单核细胞为树突状细胞前体细胞诱导成熟树突状细胞,是体外大量生成树突状细胞的一条重要途径。GM-CSF是生成和维持树突状细胞发育和分化的最根本的细胞因子一,IL-4抑制粒细胞和巨噬细胞产生,促进单核细胞向树突状细胞的转化,并维持树突状细胞成熟。2.成功建立模拟肿瘤内环境蛙皮素抑制树突状细胞的模型。3.茶氨酸可能是通过抑制COX-2的表达,减少内源性的IL-10合成,促使树突状细胞的成熟,从而促使IL-12的分泌,调节IL-10/IL-12的平衡机制来实现。4.SCID小鼠实验中的细胞和体液免疫缺陷,行腹腔注射T细胞,成功实现免疫重建。腹腔注射A549细胞,成功建立肺腺癌模型。5.茶氨酸刺激树突状细胞介导T细胞抑制SCID小鼠肺腺癌移植瘤作用,提示茶氨酸刺激树突状细胞调节T细胞,可能是通过免疫监视及清除畸变细胞作用,以及介导VEGF的表达等机制来实现。更多还原

【Abstract】 Objective:Lung cancer has become the malignant tumor of highest incidence and death rate in the world.In recent years,morbidity of lung cancer and mortality have increased,every year over one million new patients appeard in the world.Lung cancer incidence in China has been an upward trend,in the past 30 years,has been the incidence of lung cancer up to 61.4/10.By 2015,our country will become the first lung cancer in the world.Although chemotherapy,radiation therapy,harrow to the treatment of molecular technologies have developed continuously, efficacy of lung cancer treatment is still poor in overall.Biological response modifier（BRM） has opened up a new field of biological treatment in Cancer,the current BRM is a wide range of great importance to the prevention and treatment program of Cancer,which is commonly thought high of by medical science realm.it is the regulation and recoverying the immune function of the body.The effect mechanism of BRM is regulating the immune system by activating the special regulatory immune function of the body,stimulating the body’s natural immune function,activating host immune defense system,and enhancing or restoring the host immune response to tumors.Cancer cells can evade the host’s immune surveillance system,so that the growth and metastasis can be conduced.In the course of the generation of the body immune custody function and tumor-specific immune response,DC is the important joint of immunosuppressive to cancer,cancer show inhibition effect on DC,it can be conduce immune tolerance to tumor cells,so that T lymphocyte is leaded to destruction and loss of targeting specificity.DCs are antigen presentation cells which have strong function.,they possessed the ability of activating resting-type T cell and inducing ability.So,DC has become one of the attention focus of nowadays tumour biotherapy sphere because of its biology speciality,people have been paying more attention to DCs’ clinical application because of its impotant function on body of immunity especially on its anti-tumor effect.At present,initial achievement had showed about the application of DC’s tumourtherapy,a series of animal experiment showed that DC could induce special effection of anti-tumor protection,and then tumour could be resolved and stabilized.But many problem must be researched and solved,such as:the technique of non-serum cultivation is still immature,many stimulating factor must been added to promote DC generating and ripening,but these factors must be identified;it also isn’t identified that which method allergizing DC could provocate more effectively the immunoreaction of body anti-tumor;the best method of DC retro-transfusion and the quantity,path,frequency and interval time definition etal of inoculating DC clinical application are immature.So,looking for simple,economic and effective stimulator as immunoadjuvant is one direction of lung cancer therpy in future.With the investigative deepenness and the technic flawless,DCs have extensive clinical application prospect on the tumor precaution and therapy,they would be generally utilized clinically.Theanine can prevent and treat tumour through a variety of mechanisms,it could interfere in many joints of tumor occurrence on biochemistry and molecular level,including tumor initiation,promotion and development,regulating the immune organ function and preventing immune suppression,delaying tumor-bearing animals immune recession, enhancing immune function and enhancing tumor-specific and non-specific immunity,obviously enhancing the body anti-cancer"main force"of cytotoxic T lymphocyte killing tumor cells.Theanine is a type of safe and atoxic food.Above of that,Theanine has been confirmed safe material by Japan and USA,which isn’t limited dosage regulation in use. Preliminary experiment use mimicry tumor cell excreting cell factor environment and use bombesin（lung cancer cell excrete） co-culture with DC,and it confirm bombesin could obviously decrease DC quantity when its’ co-culture with DC pre-cell,and HLA-DR、CD86、CD83、CD80、CD40 express obviously lower in DC,the capability of DC stimulating leukomonocyte reaction descends,HLA-DR、CD80、CD86、CD83、CD40 expression obviously increase after Theanine stimulating DC,Theanine could strengthen the ability of stimulating leukomonocyte reaction,and they got close to normal control group.So,Theanine could inhibit DC apoptosis and increase DC function with bombesin,it can increase the ability of DC stimulating T cell proliferation,we presumpt Theanine might prevent the inhibition to DC in the tumor environment,and then study,if Theanine could prevent and cure cancer as food additive.Methods:Simulate the inhibition of DC caused by bombesin that secreted by tumor cells in tumor environment,set up a bombesin suppress DC model,with intervention of theanine,prove the protection and activation influence on secreting bombesin to inhibit DC in tumor environment caused by theanine through the influence on DC morphology and functional maturation,and the expression of COX-2 mRNA and protein.There are 3 groups:the bombesin+Theanine DC group; bombesin+DC group;PBS+DCgroup.Use bombesin to suppress DC,compare to the DC in normal group after the intervention with theanine,then observe the influence of different theanine’s concentration on the inhibition of DC surface molecules and IL-12 caused by bombesin（dose-effect of growth curve）,analysis of the inhibition of DC apoptosis,DC morphological inspection,as well as the influence on expression COX-2 mRNA and protein.In vivo experiment,inject human peripheral blood lymphocytes into SCID mice to rebuild the immune,then use theanine subjects factors to stimulate DC cells,use A549 line to inoculate under the SCID mice’s skin,approve the immune protection and immune therapy influence of theanine on DC through observing the transplanted tumor SCID mice model.Cultivate A549 cells,isolate and cultivate DC according to the original method.There are theanine stimulating DC group,DC group,theanine group and PBS group,adjust DC to the same cell concentration of 6.25×10⁵/ml with homogeneity cultivate solution,add it to 24-hole culture plate with 2ml for each hole, cultivate in 37℃and 5%CO₂ for 7days to be reserve,while for theanine group taking the DC suspension cultivated by the above-mentioned method for 2 days,add the leaching of theanine to make the final concentration to be 200μmol/L（optimal stimulus concentration）,cultivate according to the method mentioned above.In the SCID mouse tumorigenicity experiments,by intraperitoneal injection with DC, A549,DC,PBS and theanine separately,and then doing the histopathological examination by using Optical microscope to observe the tumorigenicity.Immune reconstitution in SCID mice experiments.To test the IgG levels in SCID mice by ELASE.The experiment to test inducing anti-tumor immune protective effects,observe the latency of tumor,size,weight,survival time and histopathological examination.The anti-tumor therapy experiments,observe the incubation period of tumor,tumor size,weight,survival time,parallel histopathological examination.Histopathological immunohistochemical detection,observe the expression of VEGF in each tumor tissues. Results:The healthy human peripheral blood source of DC,analog tumor cells secrete cytokines（add bombesin） inhibited DC,add Theanine cultivate,the CD40,CD80,CD83,CD86 and HLA-DR expression of DC surface,detected by flow cytometry after 7 days,Theanine can increase the CD40,CD80,CD83,CD86 and HLA-DR expression with different concentrations of theanine（25μmol/ L,50μmol/L,100μmol/L,200μmol/L,400μmol/L） deal with DC,the results of a dose-dependent manner.Theanine,and the best concentration is 200μmol/L,when the concentration is greater than 200μmol/L（such as 400μmol/L） then the CD40,CD80,CD83,CD86 and HLA-DR expression reduced.DC cultured in the first 7 days to observe the post-theanine inhibited bombesin on DC apoptosis with bombesin group,in treatment group,Theanine can inhibite to DC apoptosis.The apoptosis rate are:bombesin group is 16.0%;Theanine DC treatment group is 8.4%;PBS group is 4.0%. Theanine stimulate bombesin-DC that is able to promote secretion of IL-12,ELISA results showed that without making any deal with DC（ie bombesin group） IL-12 secretion is very little volume（63.5±3.6pg/ml）,Theanine and normal group DC can secrete high levels of IL-12,200μmol/L,when the concentration of 90.4±4.9pg/ml,the test results with the anti-apoptosis results together prove that theanine can be bombesin antagonist on the DC that inhibit the function of the promotion of DC maturation,and there is a concentration-dependent manner.Theanine treatment of DC scanning electron microscope photographs showing the morphological characteristics of more mature: DC cultivate to 7 days to collect,Theanine,and the normal group of DC surface can be seen a very long process,which is characteristic of mature DC;and done deal with the DC（ie bombesin group） did not even have a very short neurite processes.Theanine on dendritic cells COX-2 expression of the mRNA.and protein Theanine treatment of different concentrations of DC 7 days,RT-PCR detection of COX-2 expression,as the drug concentration increased,the expression decreased dose-dependent manner.Dendritic cells to expression of COX-2 protein, then different concentrations of Theanine bombesin-DC 7 days deal with Western-blot detection of COX-2 protein expression,results showed that expression of COX-2 protein was inhibited and with the increase in drug concentration,inhibitory effect significantly.HLA-DR,CD86,CD83, CD80,CD40 expression of the DC were detected by flow cytometry after 7 days.PBS as control,Theanine can increase HLA-DR,CD86,CD83, CD80,CD40 expression,namely,Theanine can promote the phenotype maturation of DC and Theanine dose-dependent manner,and the best stimulation concentration of Theanine is 200μmol/L,DC regulator T cells through the suppression SCID mice experiments,intraperitoneal injection of peripheral blood lymphocytes of all immune reconstitution,the SCID mice tumorigenicity experiment,in A549 group SCID mice surface metastases in lung tissue of nodules were found lots of nodules,some of the nodular was mutually integrated,nodular central necrosis can be seen.But the DC group can’t be seen.In the light microscope line histopathological examination,all visible tumor tissue was entity-like structure or glandular cavity-like structure,tumor cells long spindle,nuclear,and nucleolar clearly shows mitotic cytoplasm micro alkalophilic slightly transparent,cell size,cells were separated by fibrous tissue into cancer nests of varying sizes,which can be seen glandular whose cavity like structure.Induced anti-tumor effect of immune protection experiments,Theanine DC group to stimulate the growth of the incubation period of the extension of tumor,survival time is longer than the control group,tumor size,weight is smaller than the control group.The role of anti-tumor therapy experiments,observation of the survival time of tumor growth is longer than the control group,tumor size,weight is too,Histopathological immunohistochemical detection, observation of tumor tissues in each group the expression of VEGF compared with the control group.The results showed that theaine stimulated DC significantly inhibited the transplanted tumor in SCID mice and inhibites expression of VEGF,Histopathological immunohistochemical detection,observation of tumor tissues in each group the expression of VEGF compared to the situation.The results showed that DC stimulated theanine significantly inhibited the SCID mice transplanted tumor cells expressed VEGF,immunohistochemical detection of VEGF expression in the tumor,analysis showed that brown granules mainly localized in the cytoplasm,VEGF in PBS,Theanine Group Cancer tissues with high expression,while stimulation at theanine and/or DC group reduced the expression of tumor tissue,the total points of expression of VEGF:to stimulate the DC group＜DC group＜Theanine group＜PBS group,Comparing theanine,PBS no difference between groups,there is significant difference others.Conclusion:1.In this experiment the successful separation,amplification and differentiation of dendritic cells from the health of human peripheral blood mononuclear cells,is induced to a large number of mature dendritic cells in vitro generation,which is an important way.GM-CSF is the most fundamental cytokine of the generation and maintenance of dendritic cells development,IL-4 inhibit neutrophil and macrophage cells, promoting monocyte to dendritic cell transformation and maintenance of dendritic mature dendritic cells2.Set up a bombesin suppressed DC model which analogs lung cancer cells cytokine secretion inhibitory effect on DC under the lung cancer environment.Wich are used to improve the model is successful.3.Theanine can stimulate the bombesin to inhibit the maturation of DC and inhibit the effect that is bombesin to DC apoptosis.3.Observed theanine on the suppression by bombesin in the form of dendritic cells,apoptosis,function,COX-2 expression,theanine may be by inhibiting the expression of COX-2 to reduce endogenous Synthesis of IL-10,to promote the mature dendritic cells,thereby promote the secretion of IL-12,regulate the balance of IL-10/IL-12 through this mechanisms.4.SCID mice is the sever cellular and humoral immune deficiency, the intraperitoneal injection of T cells,the successful implementation of immune reconstitution.Intraperitoneal injection of A549 cells, successfully established model of lung adenocarcinoma.5.Using A549 line inoculated into SCID mice and stimulating DC cells by theanine.Observation of the SCID mice transplanted tumor,the immune protection and the immune therapeutic effects of metastatic tumor model is to prove the protection and activation effects of theanine on inhibited DC which is under tumor environment,and further confirms the mechanism that theanine regulats T lymphocytes through dendritic cells to inhibit lung adenocarcinoma（A549）.theanine stimulate DC can inhibit the expression of VEGF,thereby inhibite the growth of tumor angiogenesis and the metastasis of tumor更多还原