【作者】 赵军；

【导师】 董子明； 张曦；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 目前,肺癌的发病率和死亡率在世界范围内均居癌症之首。20世纪90年代与70年代相比,我国肺癌的死亡率上升了111.85%。肺癌的治疗仍以手术切除为主,辅以化疗和放疗,但转移和死亡率仍居高不下,这也是肺癌治疗失败的原因,因此,研究新的肺癌治疗方法显得非常必要。随着免疫学、分子生物学和基因工程技术的不断发展,以免疫治疗为代表的肿瘤生物治疗受到了人们的高度重视,已经成为继手术、化疗和放疗以后的第四种肿瘤治疗模式,它主要通过调动或增强机体内部固有的抗肿瘤能力来抑制肿瘤生长,可以避免当前肿瘤治疗模式的严重不足之处,展示了良好的临床应用前景。肺癌的靶向治疗是生物治疗的一种模式,目前已有研制成功和上市的肺癌的靶向治疗药物,大多是一些单克隆抗体制剂,包括Iressa、Tarceva、Veenat、Sutent和Sorafenib Nexavar等,基本上也都是靶向肿瘤血管上的特异性蛋白分子。虽然这些药物在一定程度上抑制了肺癌的生长,延长了患者的生存期。但是大量的循证医学研究证实,它们的疗效不尽人意。长期使用单克隆抗体还会出现严重的不良反应,并且价格昂贵,这些都限制了它们的临床使用。以肿瘤血管内皮细胞为靶点的主动免疫治疗维持的时间较长,无需频繁的反复给药;且特异性的体液免疫反应或细胞免疫反应副作用较小,所以诱导抗肿瘤血管的主动免疫效应是一种更有效的治疗方法。肿瘤血管内皮细胞处于活跃增殖状态,其细胞膜上表达多种与细胞增殖相关的特异性蛋白分子,是血管内皮细胞增殖相关抗原,如血管内皮细胞生长因子受体Ⅱ(Vascular Endothelial GrowthFactor Receptor,VEGFR-Ⅱ)、整合素av(CD51)、组织因子、Endoglin(CD105)等,这些蛋白分子在体内静止期的血管内皮细胞上却不表达。研究表明,利用体外培养的增殖活跃的异种血管内皮细胞制备疫苗,可以成功打破免疫耐受,诱导抗肿瘤血管生成。近来,用异种血管内皮细胞增殖相关蛋白和DNA分子疫苗相关研究较多,以同种蛋白和DNA分子疫苗也有报道,但由于引起的超敏反应、单一靶点的治疗效果的局限性,备受争议,寻找多靶点治疗且副作用少的主动免疫治疗方法势在必行。本研究对来源于微血管的小鼠脑微血管内皮细胞bEnd.3进行体外培养,制备疫苗,免疫小鼠并观察其对肺癌皮下移植瘤、肺转移癌的抗肿瘤效应。另一方面,本研究应用小鼠bEnd.3制备抗原,冲击树突状细胞(Dendritic Cells,DCs),观察其诱导的体内外特异性抗肿瘤血管生成的免疫反应,并初步探索其机制,试图寻找一种新的抗肿瘤血管治疗方法。人脐静脉血管内皮细胞(Human UmbilicalVein Endothelial Cells,HUVEC)来源于大的静脉血管,多项研究报道异种HUVEC有抗肿瘤血管生成的作用,因此,本研究用异种HUVEC和同种小鼠成纤维细胞(NIH3T3)作对照,来观察不同来源的两个血管内皮细胞的抗肿瘤血管生成作用。第一部分同种bEnd.3细胞疫苗诱导小鼠抗肺癌血管内皮细胞的免疫效应第一章同种bEnd.3细胞的培养及疫苗制备方法:1.体外常规培养小鼠bEnd.3细胞,从人脐带分离培养HUVEC,体外扩增,通过细胞表面标志vWF、CD31和CD144的细胞免疫组化和RT-PCR方法鉴定两种细胞。2.利用RT-PCR方法检测血管内皮细胞增殖相关抗原VEGFR-Ⅱ和整合素av基因在bEnd.3、HUVEC、NIH3T3、肿瘤细胞LLCs和U14的表达。3.bEnd.3、HUVEC、NIH3T3细胞在体外大量扩增后,用0.025%戊二醛固定制备疫苗,疫苗浓度2.5×10~7/ml。结果:1.成功培养和体外大量扩增了小鼠bEnd.3细胞和人HUVEC细胞,它们均分别高表达vWF、CD31和vWF、CD144等血管内皮细胞标志。2.在mRNA水平,bEnd.3细胞和HUVEC细胞同时表达VEGFR-Ⅱ和整合素av基因,NIH3T3细胞不表达VEGFR-Ⅱ和整合素av基因,肿瘤细胞LLCs和U14均只表达整合素av,不表达VEGFR-Ⅱ。3.用0.025%戊二醛成功制备疫苗。第二章同种bEnd.3疫苗诱导小鼠对Lewis肺癌皮下移植瘤免疫效应检测方法:1.小鼠分预防组和治疗组,治疗组又分为血清治疗组和T淋巴细胞治疗组,疫苗分bEnd.3组、HUVEC组、NIH3T3组和PBS组。预防组直接用疫苗免疫小鼠,一周一次,5×10~6个细胞/只,腋窝淋巴结周围皮下注射,共5次,最后一次免疫后一周,皮下注射小鼠Lewis肺癌细胞。治疗组用ELISA检测阳性的免疫小鼠的抗血清和脾T淋巴细胞进行治疗,血清治疗组,在荷瘤一周后,肌肉注射50μl/只,隔日注射一次,共6次。T淋巴细胞组,在荷瘤一周后,肿瘤周围注射1×10~7个脾T细胞,隔日注射一次,共6次。2.观察小鼠肿瘤体积,小鼠生存期;HE染色观察瘤体组织变化。CCK法和流式细胞术对免疫小鼠脾T淋巴细胞CTLs杀伤活性的检测。ELISA法、免疫细胞化学法和Western blot法对免疫小鼠抗血清的特异性反应检测。3.不良反应检测在免疫后2周的小鼠腹壁上划“十”字伤口,观察其伤口愈合情况。结果:1.抑制了小鼠肺癌皮下移植瘤的生长和延长了生存期在小鼠Lewis肺癌皮下移植瘤预防组中,bEnd.3组小鼠肿瘤生长速度缓慢,而且在生长3周肿瘤体积明显减小约20mm~3,之后处于相对停滞状态,与对照组有统计学差异(P＜0.05)。中位生存时间bEnd.3组为90天(终止实验),较对照组明显延长(P＜0.01)。瘤体剥离后HE染色发现,bEnd.3组为脂肪和肌肉组织,无癌细胞,而NIH3T3和PBS组均是癌细胞。T细胞治疗组和血清治疗组中,bEnd.3组小鼠肿瘤生长速度减慢,生存时间较NIH3T3和PBS组长(P＜0.05)。但T细胞治疗组较血清治疗组更能延长小鼠生存时间(P＜0.05)。2.诱导了免疫小鼠脾T淋巴细胞靶向血管内皮细胞的特异性CTLs杀伤活性流式细胞术检测bEnd.3免疫组和HUVEC免疫组CD3~+CD8~+的T细胞百分比高于对照组(P＜0.05),CCK法检测bEnd.3免疫组和HUVEC免疫组在效:靶为25:1时脾T淋巴细胞具有较强的杀伤体外增殖的bEnd.3细胞和HUVEC细胞的CTLs杀伤活性(P＜0.05),而对LLCs肺癌细胞却没有杀伤活性。3.诱导了免疫小鼠血清的特异性抗体产生ELISA结果提示细胞免疫组化结果提示bEnd.3免疫组和HUVEC免疫组抗血清与体外bEnd.3和HUVEC膜蛋白和细胞有特异性免疫反应,并能对体外增殖的bEnd.3细胞和HUVEC细胞起到抑制作用;Western blot检测bEnd.3免疫组和HUVEC免疫组抗血清在220 KD和180 KD处均出现了特异性条带,而HUVEC组在130 KD处有特异性条带,bEnd.3组在130 KD处无特异性条带,两组抗血清与LLCs膜蛋白无特异性反应及条带。4.延迟了伤口愈合bEnd.3组和HUVEC组免疫小鼠的伤口愈合时间较NIH3T3组和PBS组长(P＜0.05),但均能完全愈合。第三章同种bEnd.3疫苗诱导小鼠对宫颈癌U14肺转移癌的免疫效应检测方法:1.小鼠分预防组和治疗组,疫苗分bEnd.3组、HUVEC组、NIH3T3组和PBS组。预防组直接用疫苗免疫小鼠,一周一次,5×10~6个细胞/只,腋窝淋巴结周围皮下注射,共5次,最后一次免疫后一周,尾静脉注射小鼠U14细胞5×10~6个细胞/只。治疗组先尾静脉注射小鼠U14细胞5×10~6个细胞/只,然后用疫苗治疗小鼠,于第1,3,5,7,9和11天在小鼠腋窝淋巴结周围皮下注射,共6次。2.观察计算小鼠肺表面转移癌结节数;小鼠生存期;HE染色观察转移癌组织变化;CFSE和PI法检测免疫小鼠脾T淋巴细胞CTLs杀伤活性;流式细胞术检测CD3~+CD8~+细胞百分比;ELISA法、免疫细胞化学法和Western blot检测抗血清的特异性反应。结果:1.抑制了小鼠宫颈癌U14肺转移癌的转移和延长了生存期bEnd.3组和HUVEC组左肺癌结节数明显少于NIH3T3组和PBS组(P＜0.05),以预防组更明显;bEnd.3组中位生存时间,预防组可达34天,治疗组26天,而对照组只有19天,预防组生存时间明显延长(P＜0.05)。2.诱导了免疫小鼠脾T淋巴细胞靶向血管内皮细胞的特异性CTLs杀伤活性流式细胞术检测bEnd.3免疫组和HUVEC免疫组CD3~+CD8~+的T细胞百分比高于对照组(P＜0.05),CFSE和PI法检测bEnd.3组免疫小鼠脾T淋巴细胞具有杀伤体外增殖的bEnd.3细胞CTLs杀伤活性,HUVEC组也有一致的结果;对U14细胞却没有杀伤活性。3.诱导了免疫小鼠血清的特异性抗体产生ELISA和细胞免疫组化结果提示bEnd.3组和HUVEC组抗血清与体外bEnd.3和HUVEC膜蛋白和细胞有特异性免疫反应,并能对其体外增殖起到抑制作用;U14小鼠抗血清与bEnd.3膜蛋白有特异性反应发生,Western blot检测bEnd.3抗血清在220 KD和180 KD处出现了特异性条带,在130 KD处无特异性条带,在HUVEC组在220 KD和130 KD处均有特异性条带,在180KD处却无特异性条带;二者与U14膜蛋白无特异性条带产生。第二部分同种bEnd.3细胞抗原致敏DCs诱导小鼠对肺癌血管内皮细胞的免疫效应第一章小鼠bEnd.3抗原制备和小鼠骨髓源DCs培养及鉴定方法:1.收集bEnd.3细胞,反复冻融4次制备抗原。2.体外培养小鼠骨髓来源的DCs,并进行形态学和细胞表面CD11a和CD86表达鉴定。3.抗原以100μg/ml浓度负载DCs。结果:1.根据台盼蓝染色检测细胞冻融效果好,抗原定量检测结果显示1×10~7个细胞能够收获398.98±96.36μg抗原。2.成功培养小鼠骨髓来源的DCs,经鉴定培养至第8天成为成熟DCs。第二章负载bEnd.3抗原的DCs对体外增殖的血管内皮细胞的影响方法:1.分别负载bEnd.3、HUVEC和NIH3T3抗原的DCs培养8天成熟后,与小鼠脾T淋巴细胞混合培养3天,诱导CTLs,CCK法观察T淋巴细胞体外增殖情况。2.将CTLs与靶细胞bEnd.3、HUVEC和LLCs混合培养3天,MTT法观察其体外杀伤作用。结果:1.体外促进了小鼠脾T淋巴细胞的增殖CCK法检测负载bEnd.3抗原和HUVEC抗原的DCs在体外能够促进小鼠脾T淋巴细胞的增殖,负载NIH3T3抗原的DCs也可促进小鼠脾T淋巴细胞的增殖(P＜0.05)。2.体外诱导了内皮细胞特异的CTLs杀伤活性MTT法负载bEnd.3抗原和HUVEC抗原的DCs有明显杀伤bEnd.3细胞和HUVEC细胞的作用(P＜0.05),负载NIH3T3抗原的DCs体外诱导的CTLs对bEnd.3细胞和HUVEC细胞无明显杀伤作用。第三章bEnd.3抗原负载的DCs疫苗诱导小鼠对Lewis肺癌皮下移植瘤免疫效应检测方法:1.负载bEnd.3抗原的DCs培养第8天成熟后,收集制备疫苗。疫苗分为bEnd.3DCs组、HUVECDCs组、NIH3T3DCs组、DCs组和PBS组。直接用疫苗免疫小鼠,一周一次,1×10~6个细胞/只,腋窝淋巴结周围皮下注射,共5次,最后一次免疫后一周,皮下注射小鼠Lewis肺癌细胞。2.观察小鼠瘤体增长速度和生存期;检测免疫小鼠脾T淋巴细胞CTLs杀伤活性和免疫小鼠抗血清的特异性反应。结果:1.抑制了小鼠肺癌皮下移植瘤的生长和延长了生存期负载bEnd.3抗原的DCs培养8天成熟后,制备疫苗,免疫小鼠后,bEnd.3DCs组小鼠肿瘤生长速度缓慢,与NIH3T3DCs组和DCs组比较有统计学差异(P＜0.05)。而NIH3T3DCs组和DCs组肿瘤生长速度又比PBS组慢(P＜0.05)。中位生存时间bEnd.3DCs组为65天,NIH3T3DCs组和DCs组分别为55和57天,较对照组明显延长(P＜0.05)。2.诱导了免疫小鼠脾T淋巴细胞靶向血管内皮细胞的特异性CTLs杀伤CCK法检测bEnd.3DCs组免疫小鼠脾T淋巴细胞具有杀伤体外增殖的bEnd.3细胞CTLs杀伤活性,流式细胞检测结果提示bEnd.3DCs组CD3~+CD8~+的T细胞百分比高于对照组(P＜0.05)。3.诱导了免疫小鼠血清的特异性抗体产生ELISA检测荷瘤前后免疫抗血清特异性结果示,荷瘤前后bEnd.3DCs抗血清与bEnd.3的免疫效应无明显差异(P＞0.05),而荷瘤后的小鼠抗血清中,除PBS组,均能与bEnd.3和LLCs膜蛋白发生反应。Western blot检测bEnd.3DCs组抗血清在220 KD和180 KD处也出现了特异性条带,在130 KD处无特异性条带,HUVECDCs组在220 KD、180 KD和130 KD处均有特异性条带,与LLCs膜蛋白无特异性反应及条带。结论1.同种小鼠bEnd.3细胞疫苗和负载小鼠bEnd.3抗原的DCs疫苗可抑制小鼠Lewis肺癌皮下移植瘤和小鼠宫颈癌U14肺转移癌的生长和转移,延长了小鼠生存期,预防组抑瘤效果优于治疗组。2.抑瘤效应机制是在体内诱导形成靶向增殖血管内皮细胞的CTLs,诱导产生了抗VEGFR-Ⅱ抗体、抗Endoglin抗体和抗整合素av抗体等血管内皮细胞增殖相关抗体,诱导了抗肿瘤血管的细胞和体液免疫反应,抑制肿瘤的生长。3.内皮细胞疫苗能延长伤口愈合时间,但不影响其最终愈合。4.同种bEnd.3疫苗和异种HUVEC疫苗均能诱导抗肿瘤血管的主动免疫反应,二者抑瘤作用无明显差异。5.bEnd.3的两种疫苗形式之间的抑瘤作用有差异,整个血管内皮细胞疫苗较负载血管内皮细胞抗原的DCs疫苗似乎更有优势。更多还原

【Abstract】 Worldwide,lung cancer is the most common cancer in terms of both its incidence and mortality with the highest rates.The incidence of lung cancer went up to 111.85%from the 1970s to the 1990s in China.The present treatment modes of lung cancer,mainly including surgery and supplemental chemotherapy and radiation, couldn’t decrease the mortality rates and its metastasis rates currently.It is necessary for us to seek more rational and effective strategy to treat lung cancer.With the continuous development of immunology,molecular biology and genetic engineering technology,immunotherapy has emerged as the fourth selection for cancer therapy. Tumor biotherapy,as a representative of the immunotherapy,has been evoked great interest now.Tumor biotherapy mainly transfers or enhances self anti-tumor ability to suppress tumor growth,which can avoid the serious shortcomings of currently tumor therapy modes.It shows a good clinical application prospect.Now,most of the drugs for cancer molecular targeting therapy have been developed successfully and listed,such as Iressa,Sutent,Tarceva,Veenat and Nexavar Sorafenib.These drugs mostly are monoclonal antibodies and are targeted to the specific protein molecules of tumor-derived vascular endothelial cells.To some extent, these drugs can suppress the tumor growth and prolong the patient survival,but their therapeutic effect still unsatisfactory from some evidence-based research studies. Additionally,monoclonal antibodies can result in serious adverse effects and too expensive to afford for most patients if being used for a long term.All of these disadvantages have limited their clinic application.Active immunotherapy of targeting tumor vascular endothelial cells plays an important role in tumor biotherapy.Its therapeutic effects can maintain a long time and less adverse effect.Furthermore,it needn’t repeat dosing frequently and can induce humoral immune or cellular immune response.Anti-angiogenic therapy,which targets tumor vascular endothelial cells,might represent an attractive strategy for the treatment of tumors.Tumor vascular endothelial cells are actively proliferative and there are many specific protein molecular expressing on their membrane surface, which is correlating with cell proliferation,such as vascular endothelial growth factor receptorⅡ(VEGFR-Ⅱ),integrin avβ3,Endoglin and tissue factor,etc.But,they can’t be detected on membrane surface of dormancy endothelial cells in vivo.Many researches have indicated that it can successfully break immune tolerance and induce specific immune response of anti-angiogenesis by using the proliferation activity xenogeneic endothelial ceils as vaccine in vitro.Recently,some studies on the vaccine prepared from proliferation related proteins and DNA molecular of xenogeneic or allogenic tumor vascular endothelial cells have been reported and made some progress.But it has received much concern on its adverse effects,such as hypersensitivity and their single therapy target,etc.Therefore,to decrease side effects and increase multi-targets,it is imperative to discover a new and rational immunotherapy method.In the present study,proliferating micro-vascular endothelial cells(bEnd.3) derived from mouse brain micro-vascular were cultured in vitro.We immunized the mice with a vaccine of glutaraldehyde-fixed bEnd.3 in a transplanted Lewis lung cancer model and in a lung metastasis model of cervical cancer U14.At the same time, we immunized the mice with DCs loading bEnd.3 antigen in a transplanted Lewis lung cancer model.The anti-tumor immune response induced by these different vaccines was evaluated and the mechanism of the anti-tumor effects was discussed preliminarily.The aim of the study was to break tolerance and elicited active immune response against tumor endothelial cells,in order to explore a new anti-angiogenesis approach for cancer immunotherapy.It was reported that human umbilical vein endothelial cells(HUVEC) derived from marco-vascular could induce the anti-angiogenesis effect.Therefore,in the whole experimental process,HUVEC were used as a positive control and mouse fibroblasts(NIH3T3) were used as a negative control to be observed the anti-angiogenesis effect in mice.PartⅠAnti-angiogenesis immunity response of lung cancer induced by aliogenic bEnd.3 cells vaccineChapter 1 Culture of bEnd.3 and preparation of vaccinesMethods:1.Proliferating allogenic bEnd.3 and xenogeneic HUVEC were cultured in vitro. vWF,CD31 and CD144 which were the surface markers of endothelial cells were detected through immunohistochemical and RT-PCR methods.2.The expression of VEGFR-Ⅱand integrin av were detected by RT-PCR method in bEnd.3,HUVEC,NIH3T3 and tumor cells(LLCs and U14).3.The bEnd.3,HUVEC,NIH3T3 cells were proliferated massively and fixed by 0.025%glutaraldehyde and prepared to vaccines.The vaccine’s concentration was 2.5×10~7/ml.Results:1.Proliferating allogenic bEnd.3 and xenogeneic HUVEC were cultured successfully and were proliferated massively in vitro.The two cells have expressed highly vWF,CD31 and vWF,CD144,respectively.2.In mRNA level,bEnd.3 and HUVEC expressed VEGFR-Ⅱgene and Integrin av gene highly,but didn’t express in NIH3T3.LLCs and U14 expressed highly Integrin av gene,but didn’t express VEGFR-Ⅱgene.3.Vaccines could prepare successfully by 0.025%glutaraldehyde.Chapter 2 Detection of anti-tumor effect induced by allogenic bEnd.3 vaccine to a transplanted Lewis lung cancer model Methods:1.Two treatment protocols in a lung transplanted Lewis lung cancer model i.e., preventive and therapeutic ones were used.In the preventive protocol,vaccines were divided into four treatment groups:control(PBS only),bEnd.3(allogenic endothelial vaccine),HUVEC(xenogeneic endothelial vaccine),and NIH3T3 (allogenic fibroblast vaccine) groups(n=15 each),which were given weekly subcutaneous injections of the respective vaccines at the axillary lymph node (5×10~6 fixed cells/0.2 ml/mouse) for 5 consecutive weeks.One week after the last vaccination,a single cell suspension of LLC cells in Hanks’ Balanced Salt Solution was injected subcutaneously(3×10~6 cells/0.2 ml/mouse).In the therapeutic protocol(n=6 each),vaccines were divided into four treatment groups same as the preventive protocol.Mice were divided into serum and T lymphocytes groups derived from the preventive immunized mice and detected positive by ELISA were used to inject into the mice bearing Lewis lung cancer intramuscularly and subcutaneously every other day for 6 times,respectively. Injection dose of serum and T lymphocytes were 50μl/mouse and 1×10~7 cells/0.2 ml/mouse.2.Tumor volume and mice survival were observed after 6 weeks.The changes of tumor tissue were detected by staining with HE.The specific CTLs cytotoxicity activities of spleen T lymphocytes derived from the immunized mice examined by CCK assay.The ratio of CD3~+CD8~+ T lymphocytes was measured by FACS. Antibody specificity was detected by ELISA and immunohistochemical methods. The effect on anti-sera derived from the immunized mice to proliferation of target cells was examined by CCK assay also.At last,activities of anti-VEGFR-Ⅱantibody and anti-integrin av antibody in anti-sera were detected with Western blot.3.Effect of vaccination on wound healing:a cross wounds of 7-mm diameter each were inflicted on the upper back of C57BL/6 mice 2 week after the last vaccination.Effect of vaccination on wound healing was observed. Results:1.The tumor growth in a transplanted Lewis lung cancer model was surpressed and survival of mice was prolonged.In the preventive group and therapeutic group,endothelial vaccination induced a significant inhibition of growth of transplanted Lewis lung cancer in mice(P＜0.05).The tumor volume of 100%of mice immunized with bEnd.3 vaccine and 90%of mice immunized with HUVEC decreased to 20 mm~3 3 weeks after the challenge with tumor cells and arrived tumor-free at the end of test(90 days).Compared with control,it was significant statistically(P＜0.05).The median survival time of bEnd.3 group and HUVEC group were 90 days,which was more prolonged significantly than controls(P＜0.05).Tumors grew slower in mice of T lymphocytes and serum treatment groups than controls(P＜0.05).But the mouse survival of T lymphocytes treatment groups could be prolonged more than serum treatment groups(P＜0.05).2.The specific cytotoxic T iymphocytes immune response targeting to tumor vascular endothelial cells were induced.To confirm the functional activity and specificity of the cellular immunity activated by endothelial vaccination,spleen T lymphocytes isolated from immunized mice used as effectors in CCK assay against endothelial or tumor targets.BEnd.3-induced CTLs at a 25:1 ratio showed a significant lytic activity against bEnd.3 when compared to the CTLs of control mice.HUVEC-induced CTLs showed same lytic activity to HUVEC.However,immune effects of both groups showed no significant lytic activity against LLC cells when tested under the same conditions.The ratio of CD3~+CD8~+ spleen T lymphocytes in bEnd.3 immunized mice was higher than controls(P＜0.05).3.The specific antibody immune response in anti-sera derived from mice immunized was induced.Antibody specificity detected by ELISA and immunohistochemical methods showed the anti-sera of mice with endothelial cells had specific immune response with endothelial cells,but not tumor cells.The anti-sera could also inhibit the proliferation of endothelial cells in vitro.Western blot results showed that anti-sera of mice receiving bEnd.3 or HUVEC vaccinations precipitated membrane proteins of the respective endothelial cells,which both appeared as specific bands of approximately 220 KD and 180 KD and didn’t appear as specific bands of approximately 130 KD in bEnd.3 but in HUVEC.The specific bands couldn’t see in target LLC cells.4.Wound healing time was delayed.Wound healing time was delayed in bEnd.3 and HUVEC groups compared with NIH3T3 group and PBS group(P＜0.05).Chapter 3 Detection of anti-tumor effect induced by allogenic bEnd.3 vaccine to a lung metastasis model of cervical cancer U14Methods:1.Two treatment protocols in a lung metastasis model of cervical cancer U14 i.e., preventive and therapeutic ones were used.In the preventive protocol,vaccines were divided into four treatment groups(n=10 each),control(PBS only),bEnd.3 (allogenic endothelial vaccine),HUVEC(xenogeneic endothelial vaccine),and NIH3T3(allogenic fibroblast vaccine) groups,which were given weekly subcutaneous injections of the respective vaccines at the axillary lymph node (5×10~6 fixed cells/0.2 ml/mouse) for 5 consecutive weeks.One week after the last vaccination,a single cell suspension of cervical cancer U14 cells in Hanks’ Balanced Salt Solution was injected into the tail veins of mice(5×10~6 cells/0.2ml /mouse).In the therapeutic protocol,vaccines were divided into four treatment groups same as the preventive protocol,which were injected with tumor cells (day 0) prior to vaccination on days 1,3,5,7,9,and 11.2.Lung metastasis was evaluated macroscopically by counting metastatic nodules that were clearly visible on the lung surface.Mice survival was observed after 17 day.The changes of tissue were detected by staining with HE.The specific CTLs cytotoxicity activities of spleen T lymphocytes derived from the immunized mice examined by CCK assay.The ratio of CD3~+CD8~+ T lymphocytes was measured by FACS.Antibody specificity was detected by ELISA and immunohistochemical methods.The effect on anti-sera derived from the immunized mice to proliferation of target cells was examined by CCK assay also.At last,activities of anti-VEGFR-Ⅱantibody and anti-integrin av antibody in sera were detected with Western blot.Results:1.Lung metastasis of cervical cancer U14 was inhibited and survival of mice was prolonged.In lung metastasis model of cervical cancer U14 experiment,metastatic nodules of left lung surface in bEnd.3 and HUVEC group were less than controls significantly(P＜0.05),especially in preventive group.The median survival time of bend.3 group was 34 days in preventive group and 26 days in therapeutic group,whereas 19 days in negative control.It showed significant statistically(P＜0.05).2.The specific cytotoxic T lymphocytes immune response targeting to tumor vascular endothelial cells were induced.In CFSE and PI assay,bEnd.3-induced CTLs and HUVEC-induced CTLs at a 30:1 ratio showed a significant lyric activity against bEnd.3 and HUVEC when compared to the CTLs of control mice(P＜0.05).However,immune effects of both groups showed no significant lyric activity against U14 cells when tested under the same conditions.The ratio of CD3~+CD8~+ spleen T lymphocytes in bEnd.3 immunized mice was higher than negative controls(P＜0.05).3.The specific antibody immune response in anti-sera derived from mice immunized was induced.Antibody specificity detected by ELISA and immunohistochemical method showed the anti-sera of mice immunized with endothelial cells had specific immune response with endothelial cells,but not tumor cells.Furthermore,the anti-sera of mice immunized with U14 cells had immune response with bEnd.3. The anti-sera of mice immunized with endothelial cells also could inhibit the proliferation of endothelial cells in vitro.Western blot results showed that anti-sera of mice receiving bEnd.3 or HUVEC vaccinations precipitated membrane proteins of the respective endothelial cells,which appeared as specific bands of approximately 220 KD and 180 KD in bEnd.3 and approximately 220 KD and 130 KD in HUVEC.The specific bands couldn’t see in target U14 cell s.PartⅡAnti-angiogenesis immunity response of lung cancer induced by DCs loading allogenic bEnd.3 cells antigenChapter 1 Preparation of bEnd.3 antigen and culture of DCsMethods:1.Whole cell lysates of bEnd.3,HUVEC or NIH3T3 were prepared by four rapid freeze-thaw cycles.2.DCs were cultured from mice bone marrow in vitro and identified by morphology and surface markers i.e.,CD11a and CD86 by FACS.3.The antigen concentration of loading DCs was 100μg/ml.Results:1.It proved that the cells were lysed completely after freeze-thaw method by trypan blue staining.Quantitative detection of antigen showed that 1×10~7 cells could get 398.98±96.36μg antigen.2.DCs were cultured successfully from mice bone marrow in vitro and were mature at day 8.Chapter 2 Anti-angiogenesis response induced by DCs loading bEnd.3 antigen in vitroMethods:1.Freshly isolated DCs were incubated with the respective cell lysate for 72 h at a final protein concentration of 100μg/ml.Spleen T lymphocytes isolated from non-immunized mice mixed with DCs loading antigens for 3 days at 20:1 and 40:1.Spleen T lymphocytes proliferation in vitro was observed.2.CTLs induced by DCs loading antigens mixed with target cells for 3 days and cytotoxic activity were detected by MTT assay.Results:1.Proliferation of Spleen T lymphocytes was promoted in vitro.Spleen T lymphocytes proliferation assay showed that DCs loading antigens of bEnd.3,HUVEC and NIH3T3 antigen could make the T lymphocytes proliferate in vitro(P＜0.05) at 20:1 and 40:1 compared with DCs group and PBS group.2.The specific cytotoxic T iymphocytes immune response targeting to tumor vascular endothelial cells were induced in vitro.The cytotoxic activity detected by MTT assay showed that bEnd.3DCs-induced CTLs and HUVECDCs-induced CTLs at a 30:1 ratio had a significant lytic activity against bEnd.3 and HUVEC when compared to the NIH3T3DCs-induced CTLs group,DCs group and PBS group mice(P＜0.05). However,immune effects of both groups showed no significant lytic activity against LLC cells when tested under the same conditions(P＞0.05).Chapter 3 Anti-angiogenesis in a lung transplanted Lewis lung cancer model induced by DCs loading bEnd.3 antigen in vivoMethods:1.DCs loading antigens were collected at day 8 as vaccine.Mice were divided into five treatment groups:bEnd.3DCs,HUVECDCs,NIH3T3DCs,DCs and PBS group and given weekly subcutaneous injections of the respective vaccines at the axillary lymph node(1×10~6 fixed cells/0.2 ml/mouse) for 5 consecutive weeks. One week after the last vaccination,a single cell suspension of LLC ceils in Hanks’ Balanced Salt Solution was injected subcutaneously(3×10~6 cells/0.2 ml /mouse).2.Tumor volume and mice survival were observed after 6 weeks.The changes of tissue were detected by staining with HE.The specific CTLs activities of spleen T lymphocytes derived from the immunized mice examined by CCK assay.The ratio of CD3~+CD8~+ T lymphocytes was measured by FACS.Antibody specificity was detected by ELISA and immunohistochemical methods.At last,activities of anti-VEGFR-Ⅱantibody and anti-integrin av antibody in anti-sera derived from the immunized mice were detected with Western blot.Results:1.The tumor growth in a transplanted Lewis lung cancer model was surpressed and survival of mice was prolonged in vivo.3 weeks after incubation with LLCs,tumors grew slower in mice immunized with bEnd.3DCs and HUVECDCs vaccines than NIH3T3DCs groups, DCs groups and PBS groups(P＜0.05).But the tumors of NIH3T3DCs group and DCs group grew slower than PBS groups’(P＜0.05).The median survival time of bEnd.3DCs group,NIH3T3DCs group and DCs group was 65 days,57 days and 55 days,whereas 19 days in PBS group,which showed significant statistically(P＜0.05).2.The specific cytotoxic T lymphocytes immune response targeting to tumor vascular endothelial cells were induced in vivo.The cytotoxic activity detected by CCK assay showed that bEnd.3DCs-induced CTLs and HUVECDCs-induced CTLs at a 30:1 ratio had a significant lytic activity against bEnd.3 and HUVEC when compared to the CTLs of NIH3T3DCs group,DCs group and PBS group mice(P＜0.05).The ratio of CD3~+CD8~+ spleen T lymphocytes from bEnd.3 immunized mice was higher than negative controls(P＜0.05).3.The specific antibody immune response in anti-sera derived from mice immunized was induced in vivo.Antibody specificity detected by ELISA indicated that antibody specificity to endothelial cells had no marked difference before and after the incubation with LLC cells.But antibody specificity in the serum,excluded PBS group could make response with endothelial cells and LLC cells.It suggest the important function of DCs.Western blot results showed that anti-sera of mice receiving bEnd.3DCs or HUVECDCs vaccinations precipitated membrane proteins of the respective endothelial cells,which both appeared as specific bands of approximately 220 KD and 180 KD and didn’t appeare as specific bands of approximately 130 KD in bEnd.3DCs but in HUVECDCs.The specific bands couldn’t see in target LLC cells.Conclusions1.Allogenic bEnd.3 vaccine and DCs vaccine loading allogenic bEnd.3 antigen can surpress the tumor growth and metastasis in a transplanted Lewis lung cancer model and a lung metastasis model of cervical cancer U14 and prolong survival of mice.This suppressive effects is more effective in preventive groups than therapeutic group.2.The anti-tumor effect is attributed to obtain endothelial cell specific CTLs and induce production of antibodies that can react with VEGFR-Ⅱ,intergrin av or endoglin proteins etc expressing on tumor endothelial cells in mice.The endothelial vaccine induces an active specific cellular and humoral immune to anti-angiogenesis.3.Although the endothelial vaccines can delay the wound healing time,the wound can heal completely at last.4.Allogenic and xenogeneic vaccines can both induce anti-angiogenesis active immune response.5.Compare DC vaccine loading endothelial cells antigen with the whole endothelial cell vaccine,the latter has more effective anti-tumor effect than the former.It indicates that the whole endothelial cell vaccine can yet be regarded as a promising research direction of anti-angiogenesis.更多还原