摘要
乳腺癌是女性最常见的恶性肿瘤之一。与人体许多其它恶性肿瘤一样，乳腺癌也是来自自身细胞的突变，因此抗原性较弱，并且肿瘤细胞可产生各种封闭因子使其逃逸机体的免疫监控系统，同时患者的自身免疫系统呈现抑制性优势和癌组织局部的免疫功能低下，使机体的免疫机制已不能有效控制肿瘤的发生、发展，而针对乳腺癌的常规治疗方法如放疗、化疗等对机体的免疫系统有明显的抑制作用。因此，如何调动患者机体自身的防御机制，改善免疫功能状态，探索新的针对乳腺癌的免疫治疗方法，一直是科学家们努力研究的焦点。近年来，树突状细胞(Dendritic Cell，DC)疫苗作为一种新的抗肿瘤免疫治疗手段向人们展示了诱人的前景；但由于外周血中DC的数量仅占白细胞总数的0.1％，且患者的肿瘤细胞常常分泌一些细胞因子而抑制了DC的功能，因此通过体外培养的方式扩增DC，然后回输体内激发机体的抗肿瘤免疫效应，具有重要的实用价值和理论意义。设计肿瘤治疗性疫苗的关键是寻找到肿瘤的特异性靶抗原并将其输送给机体的免疫系统，随后被免疫系统视为异物而被识别，最后激活的免疫系统通过一系列的作用机制将带有肿瘤抗原的癌细胞摧毁。抗原蛋白或多肽在DC内的转运对于抗原的提呈及特异性免疫反应的诱导具有非常重要的作用，因此利用一些载体将肿瘤的抗原成分转染至DC制备瘤苗，已成为肿瘤生物治疗领域备受关注的热点之一。由于病毒性载体存在自身免疫性反应和安全性问题，如外源性病毒DNA注入体内后，可能整合到宿主基因组上，使宿主细胞的抑癌基因失活或癌基因活化从而转化成癌细胞，因此限制了其在临床中的广泛应用；作为非病毒性载体系统的脂质体类物质在体内可自然降解、无免疫原性、可重复使用、无明显的毒副作用等特点，在目前的抗肿瘤疫苗中具有独特的作用。黑色素瘤抗原基因(Melanoma associated antigen gene-A3，Mage-A3)编码的抗原为HLA限制性的肿瘤排斥性抗原，在多种恶性肿瘤组织中表达，在除睾丸和胎盘以外的其他正常组织中均不表达，由于睾丸和胎盘组织中缺乏HLA-Ⅰ类分子的表达，使细胞毒性T淋巴细胞(Cytotoxic T lymphocyte，CTL)无法发挥杀伤作用，同时睾丸和胎盘组织中的滋养细胞间连接十分紧密并表达CD95配体，从而形成了一个物理性和功能性的屏障，可阻止CTL的进入并可引起进入的T细胞发生凋亡，显示了作为肿瘤治疗性疫苗靶抗原的潜在价值。
为此我们首先利用RT-PCR(reverse transcription polymerase chain reaction，逆转录-多聚酶链反应)和SSP-PCR(sequence special primer polymerase chain reaction，序列特异性引物-多聚酶链反应)的方法，分别观察了人乳腺癌MCF-7、卵巢癌SK-OV3及正常血管内皮EC-304等细胞株中MAGE-A3基因及人类白细胞抗原HLA-A2等位基因的表达情况；结果显示：乳腺癌细胞株MCF-7中MAGE-A3及HLA-A2基因均为阳性表达，卵巢癌细胞株SK-OV3中Mage-A3基因呈阳性表达而HLA-A2则为阴性表达，正常血管内皮细胞株EC-304中MAGE-A3为阴性表达而HLA-A2则为阳性表达。随后从HLA-A2表达阳性的健康志愿者外周血中分离获得单核细胞，在含有GM-CSF、IL-4和a-TNF等细胞因子的1640培养基诱导下继续扩增培养，流式细胞仪检测CD1a、HLA-DR、CD80、CD83、CD86的表达率分别由第1天的2.3％、46.2％、18.4％、8.36％、19.8％逐渐升至第3天的14.3％、57.8％、42.7％、9.2％、47.8％及第6天的21.8％、99.0％、63.4％、18.9％、80.6％；其中代表DC成熟程度的CD83及HLA-Ⅱ类分子的表达均有明显的增加。高倍相差显微镜及电镜观察可见形态特异的DC细胞，DC的得率从第1天的2.12％升至第3天的9.89％及第6天的16.31％，DC的纯度从第1天的2.08％升至第3天的61.25％及第6天的90.83％，证实可以从外周血单核细胞中培养扩增出大量较高纯度的DC细胞。
为制备MAGE-A3多肽疫苗，根据网上检索得到MAGE-A3抗原表位的九肽氨基酸序列，人工合成并用荧光素标记MAGE-A3多肽(271-279，Dansyl-FLWGPRALV)，经反相高压液相色谱仪(Reverse Phase High Performance Liquid Chromatography，RP-HPLC)和质谱分析仪(Mass Spectroscopy)检测发现：MAGE-A3多肽的纯度＞95％，分子量为1292.05道尔顿；然后利用一种新型的脂质体类转染试剂BioPORTER reagent作为载体，将MAGE-A3多肽转染至培养第3天的DC细胞(考虑到DC吞噬功能的高峰期及转染试剂要求的转染条件，我们选择培养第3天的DC进行转染)，在转染后的第4、8和24小时倒置荧光显微镜及流式细胞仪分别检测MAGE-A3多肽在DC内的转染效率，激光共聚焦显微镜(laser scanning co-focal microscope，LSCM)观察MAGE-A3多肽在DC内的分布情况，四甲基偶氮唑盐法(MTT)法观察转染试剂对DC活性的影响。结果显示：BioPORTER可有效地将MAGE-A3多肽转染至DC内，MAGE-A3多肽主要分布于DC的细胞膜和细胞浆内，仅有极少量分布于细胞核内。流式细胞仪具有简便、快捷、重复性较好以及准确率较高的特点，可替代传统显微镜方法检测转染效率。在反应体系的溶液PH为7.5的情况下，当DC细胞的密度为5×10~5／ml，多肽与转染试剂的比例(W／V)为4／1，两者形成的复合物与DC细胞孵育8h，获得了较高的转染效率，与同等条件下无转染试剂时比较有显著性增加(14.21％±2.83％Vs 10.73％±2.25％，P＜0.01)，且此条件下细胞的存活率也较高，但与同等条件下无转染试剂时比较无显著性差异(92.58％±4.38％Vs 93.74％±1.94％，P＞0.05)，即对DC不产生明显的细胞毒性，为进一步研究以MAGE-A3为基础的多肽瘤苗，提供了一种较为实用的实验方法。
为观察转染MAGG-A3多肽对DC免疫功能的影响，在MAGE-A3多肽转染8h后的DC悬液中，加入肿瘤坏死因子(a-TNF，终浓度为20ug／ml)继续培养24h作为刺激细胞，以与DC同源的T淋巴细胞作为效应细胞，按效应细胞与刺激细胞20／1的比例混合继续培养6天；流式细胞仪结果表明：MAGE-A3多肽能够显著性提高DC的成熟程度，转染试剂BioPORTER介导的多肽可使DC的成熟程度有进一步的显著性提高(P＜0.05)。
为检测转染MAGE-A3多肽对淋巴细胞分泌功能的影响，我们首先用尼龙毛柱方法对来源于人外周血单核细胞的T淋巴细胞进行纯化，然后分别用各组DC(A组为转染多肽组，B组为游离多肽组，C组为无多肽的T细胞与DC混合组，D组为单独T细胞对照组)与纯化后的T淋巴细胞共同孵育7天，分别于第1，3，5天和第2，4，6天用ELISA方法分别测定培养上清中IL-12和IFN-r的含量变化。结果发现：随着时间的延长，各组细胞上清中IL-12的含量逐渐升高，到第3天时，4个组之间的差异出现显著性意义(F=8.412，P=0.007)，其中A组与B组比较有显著性升高(P＜0.05)，A组与C组、D组比较均有非常显著性升高(均为P＜0.01)，但B组、C组及D组之间比较均无显著性差异(均为P＞0.05)，到第5天时，以上4组之间的差异进一步加大(F=12.223，P=0.002)，且B组与C组、D组比较也有显著性升高(均为P＜0.05)；各组细胞上清中的IFN-r含量也随时间逐渐升高，第4天时，4组之间比较出现显著性差异(F=3.652，P=0.063)，其中A组与B组、C组、D组比较均有显著性差异(均为P＜0.05)，B组与C、D组比较均无显著性差异(均为P＞0.05)，到第6天时，上述4组之间的差异进一步加大(F=5.938，P=0.020)，其中A组与C组、D组之间比较均有显著性差异(均为P＜0.01)，而B组与C组、D组之间比较仍无显著性差异(均为P＞0.05)，A组与B组比较也有显著性差异(P＜0.05)；各时间段C组及D组细胞上清中IL-12和IFN-r含量的比较均无显著性差异(均为P＞0.05)。说明MAGE-A3抗原多肽活化的DC可刺激T淋巴细胞分泌IL-12和IFN-r等细胞因子参与免疫反应，转染试剂BioPORTER可通过增加MAGE-A3抗原多肽转染的效率进一步促进细胞因子的分泌而加强免疫反应。
以密度梯度方法分离与DC不同源的健康人外周血中的淋巴细胞作为同种异体T淋巴细胞；在经紫外线处理后的96孔板中，分别加入以上各组的DC悬液1×10~4，5×10~3，2×10~3，1×10~3细胞／每孔，以自身T淋巴细胞作为对照，每孔设3个复孔，分别加入同种异体T淋巴细胞(1×10~5／每孔)，即配成树突状细胞／T细胞分别为1／10，1／20，1／50，1／100的比例，终体积为200ul，在37℃及5％CO_2培养箱中孵育90h后，四甲基偶氮唑盐(MTT)方法测定DC刺激T淋巴细胞增殖的能力，结果显示：各种比例的转染多肽组，与相应比例无多肽组比较，激发淋巴细胞增殖的能力显著性增强(P＜0.01)，游离多肽组与无多肽组比较也有显著性增强(P＜0.05)；转染多肽组与游离多肽组比较虽有差异但无显著性(P＞0.05)；各实验组最明显的刺激效果均发生在DC浓度相对较高的比例；证实MAGE-A3多肽抗原能够显著性增强DC的抗原呈递功能，转染试剂BioPORTER可进一步增强DC的抗原呈递功能并促进T淋巴细胞的增殖。
为观察各组DC与淋巴细胞作用后T淋巴细胞表型的变化，以与DC同源的T淋巴细胞作为效应细胞，以上述各组不同的DC作为刺激细胞，按照效应细胞与刺激细胞20／1的比例混合继续培养6天，流式细胞仪检测结果显示：转染多肽组、游离多肽组与T淋巴细胞孵育6天后，CD4~+T淋巴细胞的含量有轻度降低，CD8~+T淋巴细胞含量有较为明显的增加；而无多肽组中CD4~+和CD8~+T细胞的含量均有不同程度降低；提示MAGE-A3多肽作用后的DC可能通过促进CD8~+效应T淋巴细胞的分化和增殖而诱导特异性免疫反应。因为发挥细胞毒免疫效应清除肿瘤细胞的主要为CD8~+的T淋巴细胞，而CD4~+T淋巴细胞在免疫反应的激发阶段而不是效应阶段发挥作用。
为了解MAGE-A3多肽疫苗对肿瘤靶细胞的作用，我们应用MTT方法评价各组效应细胞对靶细胞的杀伤作用，流式细胞仪(Annexin V-FITC方法)测定各组效应细胞诱导靶细胞的凋亡率；将各组DC致敏的效应T淋巴细胞(A组为转染多肽组，B组为游离多肽组、C组为无多肽组)分别配成2×10~7／ml，1×10~7／ml，5×10~6／ml，2×10~6／ml等几种浓度，分别取靶细胞和各种不同浓度的效应细胞各100ul加入96孔培养板中，即配成效应细胞：靶细胞(E／T)为100／1，50／1，25／1和10／1的比例，每组设三复孔。并设相应浓度梯度未经DC作用的T细胞与靶细胞混合作为对照孔，各孔终体积均为200ul，每组设三复孔。在37℃及5％CO2培养箱中孵育12h后检测。结果发现：效应细胞对靶细胞的杀伤作用随着效／靶比例(E／T)的增加而增强；转染多肽(A组)和游离多肽(B组)对乳腺癌细胞株MCF-7的杀伤作用均分别显著性强于对卵巢癌SK-OV3及正常血管内皮EC-304细胞株的杀伤作用(均为P＜0.01)；转染多肽组和游离多肽组的杀伤作用均显著性强于无多肽(C组)及单独T细胞对照组(均为P＜0.05)，而后二组之间无明显差异(P＞0.05)；当效／靶比例(E／T)为100／1和50／1时，转染多肽组显著性强于游离多肽组对MCF-7细胞株的杀伤作用(P＜0.05)，当E／T、为20／1和10／1时二者之间无显著性差异(P＞0.05)；而各组效应细胞在不同效靶比例时对SK-OV3及EC-304细胞株的杀伤作用均很低且无明显差异(P＞0.05)。
流式细胞仪测定结果显示：转染多肽组和游离多肽组对乳腺癌细胞株MCF-7的凋亡率均显著性高于对卵巢癌细胞株SK-OV3及正常血管内皮细胞株EC-304的凋亡率(均为P＜0.01)；两组诱导MCF-7细胞株的凋亡率均显著性高于无多肽对照组(均为P＜0.01)；转染多肽组又显著性高于游离多肽组(P＜0.01)；以上三组效应细胞对SK-OV3及EC-304细胞株的凋亡率均无显著性差异(均为P＞0.05)，而无多肽组对三种靶细胞株的凋亡率也无显著性差异(均为P＞0.05)。
上述结果表明：MAGE-A3多肽疫苗诱导肿瘤靶细胞的杀伤和凋亡作用具有MAGE-A3抗原特异性和HLA-A2基因限制性，而对不表达MAGE-A3或HLA-A2基因的其他肿瘤细胞以及正常细胞株均无显著性影响；转染试剂介导的MAGE-A3多肽疫苗可进一步增强对肿瘤靶细胞的杀伤和凋亡作用。
为进一步探讨MAGE-A3多肽疫苗治疗动物乳腺癌的可行性及其作用机制，我们将处于对数生长期的MCF-7细胞株(5×10~6／120ul／只)接种于20只裸鼠背部的一侧皮下建立乳腺癌移植瘤模型，一周后成瘤率为95％(19／20)，随后将成瘤的19只裸鼠随机分为4组：A组5只(BioPORTER介导MAGE-A3多肽瘤苗治疗组)、B组5只(游离MAGE-A3多肽瘤苗治疗组)、C组5只(单独DC治疗组)及D组4只(生理盐水对照组)；分别用以上不同性质的DC细胞(1×10~6／120ul／只)及同体积的生理盐水注入肿瘤对侧的皮下，以后每间隔7天重复相同剂量治疗一次，共4次。连续动态观察各组动物的肿瘤生长情况，观察期末处死所有动物取出瘤体分别天平称重，肿瘤组织进行常规病理切片HE染色观察，并用抗CD8~+的单克隆抗体对肿瘤病理切片进行免疫组化染色，观察CD8~+T细胞在肿瘤组织中的浸润密度变化；结果显示：治疗1周后，A组和B组动物瘤体的大小分别较C组和D组显著性降低(均为P＜0.01)，而A组、B组之间以及C组、D组之间均无显著性差异(均为P＞0.05)；治疗后第2周开始，A组和B组动物瘤体的大小均较C组和D组有进一步显著性降低(均为P＜0.01)，A组与B组比较也开始有显著性差异(均为P＜0.01)，而C组、D组之间仍无显著性差异(P＞0.05)，这种状况一直持续到观察期末；A组和B组动物瘤体的重量均较C组和D组显著性降低(均为P＜0.01)，而抑瘤率均较C组和D组显著性增加(均为P＜0.01)，A组与B组比较也有显著性差异(均为P＜0.01)，而C组与D组之间无显著性差异(P＞0.05)。
免疫组化染色可见大量的CD8~+T细胞在肿瘤组织中浸润，其中A组和B组每高倍视野下CD8~+T细胞的浸润密度分别为72.6±9.76和55.8±3.70，而C组和D组仅分别为14.2±1.92和11.2±3.38，A组和B组均分别显著性高于C组和D组(均为P=0.000)，A组与B组之间也有显著性差异(P＜0.01)，C组和D组之间无显著性差异(P＞0.05)。进一步观察荷瘤动物的生存情况发现：A组和B组动物无一只死亡，4周生存率为100％，明显高于C组及D组的60％(3／5)和50％(2／4)；C组和D组均分别于治疗后的第3和第4周各死亡一只，死亡均与肿瘤的迅速进展相关。由于动物数量太少未作进一步统计学处理。从上述结果初步看出：MAGE-A3多肽疫苗对裸鼠乳腺癌移植瘤有一定的抑制和杀伤作用，但多肽疫苗治疗与生存的关系则有待加大样本量进一步研究。
本实验结果说明：MAGE-A3抗原多肽可促进外周血单核细胞来源的DC成熟，并通过以HLA-A2与多肽复合物的形式将抗原呈递给T淋巴细胞，激活的T淋巴细胞分化增殖进而分泌大量的细胞因子(IL-12、IFN-r等)诱导特异性免疫反应，其中CD8~+T淋巴细胞可能发挥主导的特异性细胞毒作用；MAGE-A3多肽疫苗的作用机制可能为抑制肿瘤细胞的增殖以及促进肿瘤细胞的凋亡，且这种免疫效应具有MAGE-A3抗原特异性和HLA-A2基因限制性，仅对能够同时表达MAGE-A3基因和HLA-A2基因的肿瘤细胞有杀伤和抑制作用，而对不表达MAGE-A3基因或HLA-A2基因的其他肿瘤细胞以及正常细胞株均无显著性影响；MAGE-A3多肽疫苗治疗后，裸鼠乳腺癌组织中CD8~+T细胞的浸润密度有显著性增加，治疗后瘤体的大小和重量均有显著性的减小和下降，对荷瘤动物的生存情况也产生一定程度的积极影响；其中转染试剂BioPORTER通过增加MAGE-A3多肽转染的效率可进一步增强上述效应细胞对靶细胞和裸鼠乳癌移植瘤的特异性免疫效应。这些结果为利用MAGE-A3多肽疫苗治疗乳腺癌提供了一定的实验基础和依据，并对临床乳腺癌的生物治疗有一定的参考价值。
Breast cancer is one of the most common malignant tumors in women.Like many other malignant tumors, breast cancer derived from mutation of itsown cells with a weak antigenicity. The cancer cells can escape from themonitoring system of the body with variety of blocking factors. At the same time,the immunorepressive predominance and the low immunoreaction of somecancer cells, make the development of knubs out of control. Even more,general treatments such as radiotherapy and chemotherapy, do obvious harm tothe immune system. Therefore, experts put eyes on the new immunotherapy ofbreast cancer that can wake up patients' defensive system so as to improve theirimmune function. Dendritic Cell (DC) vaccine shows an attractive foreground asa new immunotherapy. However, the percentage of DC in peripheral blood isjust 0.1% of leucocyte, and cancer cells always excrete some cytokines whichrestrains DC, it means that DC cultured in vitro would be very important forexciting anti-cancer immunological effect when infused in vivo. The key point ofdesigning oncotherapy vaccine is to find out the specific target antigen, and sendit to the immune system, so as to be recognized as the foreign object by immunesystem, that the activated immune system would kill the cancer cell with tumourantigen at last by some very mechanism. Antigen albumen or polypeptide is very important for presentation of antigen and induction of specific immunoreaction,so transfecting antigen to DC for preparing the tumour vaccine by carrier hasbeen one of the hotspots of tumour biotherapy. Because of the immunity reactionand security, making full use of viral carrier is restricted. Taking exogenousDNA for example, when infused in vivo, it could be integrated into the hostgenome, which make the anti-cancer genes of host cells inactive, or cancer genesactive, then would be transformed into cancer cells. Working as in-viral carrier,liposome can be degraded naturally in vivo, without any immunogenicities,obvious toxicities,or side effects, but can be cycle using, which makes the uniqueeffects in cancer antigen carrier vaccine.
Antigens encoded with Melanoma associated antigen gene-3 (MAGE-A3)is the HLA restricted tumour-rejection antigens which express just in malignanttumor. They also express in none of the normal tissues but testis and placentas.Cytotoxic T lymphocyte (CTL) didn't have any lethal effects because of noexpression of HLA-I in testis and placentas. At the same time, the close connectsbetween trophocyte in testis and placentas which express CD95 ligand, cominginto a physical and functional barrier, which prevent the entry of CTL and causethe apoptosis of T-cell. All of these shows the potential in target antigen of cancerremedial vaccine.
So, the RT-PCR(Reverse Transcription Polymerase Chain Reaction) andthe SSP-PCR(Sequence Special Primer Polymerase Chain Reaction) were firstused apart to observe the expression of MAGE-A3 and histocompatibilityleukocyte antigen-A2 (HLA-A2) in human breast cancer cell line MCF-7,ovarian cancer cell line SK-OV3 and normal vascular endothelial cell lineEC-304. It showed that Mage-A3 and HLA-A2 genes were positive-expressed inbreast cancer cell MCF-7. In ovarian cancer cell line SK-OV3, MAGE-A3 geneswere positive-expressed while HLA-A2 negative-express. The complete reverseresult showed in the normal vascular endothelial cell line EC-304.
Then we separated the mononuclear cell from peripheral blood of healthyvolunteers who expressed HAL-A2-positive, amplified in the 1640 culture medium with GM-CSF, IL-4, a-TNF and the other cytokines. The resultsuggested that detected by flow-cytometer, the percentages of CD1a, HLA-DR,CD80, CD83 and CD86 increased step by step, from 2.3%, 46.2%, 18.4%, 8.36%,19.8% by the first day to 14.3%, 57.8%, 42.7%, 9.2%, 47.8% by the third daythen 21.8%, 99.0%, 63.4%, 18.9%, 80.6% the sixth day. It showed that CD83and HLA-Ⅱmolecules both increased which stood for the maturity of DCs. DCswith specific shape can be seen in high-multi-microscope and electric microscope.The yield of DCs increased from 2.12% by the first day to 9.89% by the thirdday then to 16.31% the sixth day, while purity of DCs increased from 2.08% bythe first day to 61.25% by the third day then to 90.83% the sixth day. Itconcluded that DCs with high purity can be amplified with mononuclear cellfrom the peripheral blood.
For preparing MAGE-A3 polypeptide vaccine, 9-peptide amino acidsequences with MAGE-A3 epitope which searched in net, synthesized artificiallyand then marked by fluorescein, analysed by reverse phase high performanceliquid chromatography (RP-HPLC) and mass spectroscopy(MS), were foundthat the purity of polypeptide reached more than 95%, the molecular weight is1292.05. Then we transfected MAGE-A3 polypeptide to the third-day-DCs withBioPORTER as the carrier. The distributing of MAGE-A3 mainly in cellularmembrane and intracytoplasm of DCs were detected by laser scanning co-focalmicroscope(LSCM). The transfection efficiency of MAGE-A3 in DCs weredetected by fluorescence microscope and flow cytometer by the fourth, eighth,and twenty-fourth hour. And the transfection reagent infection to DCs' activitywas observed by MTT (4,5-dimethyiazol-2-diphenyl tetrazolium bromide).Wefound that BioPORTER can transfect MAGE-A3 polypeptide to DCs effectively.Transfection efficiency detected by fluorescence microscope and flow cytometewas positive correlation, correlation coefficient was 0.796(P＜0.01). Flowcytometer is convenient, quickly, well repeatability and nicety compared withconventional microscope. In the condition that PH of reaction system was 7.5,density of the DC suspension was 5×10~5/ml, proportion of polypeptide and transfection reagent was 4/1, higher transfection efficiency was obtained by thecomposite cultivated 8 hours with DCs. The average transfection efficiency ofMAGE-A3 polypeptide with BioPORTER was more higher than withoutBioPORTER(14.21%±2.83% versus 10.37%±2.25%, P＜0.01) under thisconditions. Even more, it did no obvious cytotoxicity to DCs, the average celllivability was not different from each other (92.58%±4.38% versus93.74%±1.94%, P＞0.05) under the same conditions. All the results provided anapplied experiment method for DC vaccine based on MAGE-A3 antigenpolypeptide.
To observe the infection of DC immunity function transfected withMAGE-A3 polypeptide, we added the tumor necrosis factor to the DCssuspension in 8 hours of MAGE-A3 polypeptide transfection, cultivating asstimulus cells for 24 hours. On the other hand, T lymphocyte as effect cells,mixed with 1/20 stimulus cells to cultivate for 6 days, it indicated by flowcytomete that MAGE-A3 polypeptide could accelerated the DC maturation, DCswith transfection reagent would more mature than that with free antigen load(P＜0.05).
To detect the affection of MAGE-A3 polypeptides on secretary functionof T lymphocytes which derived from peripheral blood mononuclear cells, the Tlymphocytes were purified with nylon wools firstly. And then the T lymphocyteswere mixed with DCs which delivered polypeptides for 7 days. The content ofIL-12 were detected in the first day, third day and the fifth day respectively byELISA. And the content of IFN-r were detected in the second day, the fourthday and the sixth day respectively also by ELISA. The results showed that theIL-12 contents in supernatant of each culture media increase by degrees,contents of DC group with polypeptide antigen was more than that withoutpolypeptide antigen (P＜0.01), contents of DC group with transfection reagentwas more than that with free antigen load (P＜0.05). The IFN-r contents insupernatant of each group were also increased gradually. Till the fourth day,compared the DC/MAGE3-BIO group with the DC/MAGE group, the DC group without MAGE-A3 polypeptide and single T cells, the IFN-r content insupernatant of culture media increased obviously(P＜0.05). Till the sixth day, thedisparity enlarged (P＜0.01), while the last three group had quiet disparity(P＞0.05). It means that DCs which activeited by MAGE-A3 antigenpolypeptide, can stimulate T cell to excrete IL-12, IFN-r which can excite thespecific immunoreaction. BioPORTER can accelerate the secretion of cytokine toenhance immunoreaction by improving the transfection efficiency of MAGE-A3antigen polypeptide.
To evaluated stimulating effects of DCs on the allogeneic T lymphocytes,density gradient method was used to separate the lymphocytes of the healthymen's peripheral blood which had the different origin from the DCs. The96-pore plate disposed with ultraviolet radiation, added apart with the DCsuspension of transfected polypeptide, 1×10~4, 5×10~3, 2×10~3 and 1×10~3 cells perpore, compared with the self T-lymphocytes, 3 ambo-pores in one. Variantlymphocytes of the same race were added to the ambo-pores(1×10~5 per pore),that meant the proportions of DCs and T cells were 1/10, 1/20, 1/50 and 1/100,end with 200ul in volume. Cultivated in incubator for 90 hours, 37℃, 5%CO_2,the MTT showed that all transfected polypeptides were more better stimulatingT lymphocyte to proliferate (P＜0.01). DC group with transfection reagent wouldbe more proliferous than that with free antigen load (P＞0.05). The more DCsconcentration was, the more obvious the stimulate would be. MAGE-A3 antigenpolypeptide would enhance the presentation of DC polypeptide. BioPORTERwould enhance that even more while accelerate the proliferation of Tlymphocyte.
Observing the colony fission alleosis of CD4~+ and CD8~+ T lymphocytesaffected with DC, T lymphocytes which were the homologies of DCs, worked aseffector cells, mixed with 1/20 stimulus cells to cultivate for 6 days. It indicatedby flow cytometer that the contents of CD4~+ T lymphocytes of theDC/MAGE-BIO group and DC/MAGE group reduced slightly, while that of CD8~+ T lymphocytes increased sharply. Contents of CD4~+ and CD8~+ Tlymphocytes without polypeptide reduced by some degrees. It clued us that DCsstimulatived with MAGE-A3 polypeptide would accelerate the differentiationand proliferation of CD8~+ T lymphocytes so as to induce the specificityimmunoreaction. Because the main ingredient of eliminating tumor cells withcytotoxic immunological effects was CD8~+ T lymphocytes, while CD4~+ Tlymphocytes worked on the excitation moment of immunoreaction but noteffector moment.
For finding out that how the MAGE-A3 polypeptide vaccines killstumour target cells, MTT was used for evaluating the infection that each effectorcell group worked on proliferation of target cells. The apoptosis of target cellsinduced by each effector cell group were determined by flow cytometer (kitAnnexin V-FITC, PI-PE). The groups of T-DC/MAGE-BIO, T-DC/MAGE, T-DCwere prepared for 2×10~7/ml, 1×10~7/ml, 5×10~6/ml and 2×10~6/ml concentration,100ul target cells were mixed with 100ul different effector ceils in 96-pore plate,which meant the proportions of effector cells and target cells were 100/1, 50/1,25/1 and 10/1, three pores in one group. T cells without DCs stimulation mixedwith target cells at the same concentration gradient as the control group, 200uleach pore, three pores in one group. Cultivated in incubator for 12 hours, 37℃,5% CO_2, it showed that lethal effects of MCF-7 with MAGE-A3 andHLA-A2-positive, which affected by effector cells transfected with MAGE-A3polypeptide, was more distinct than the SK-OV3 with MAGE-A3-positive,HLA-A2-negative and EC-304 with MAGE-A3-negative, HLA-A2-positive.Result demonstrated the significant difference (P＜0.01). Lethal effects enhancedby the proportion of E/T. Lethal effects of MAGE-A3 polypeptide group wasmore significance than the control groups of without polypeptide, while therewas no significant difference in the two control groups(P＞0.05). Lethal effects ofpolypeptide group with transfection reagent would be more significant than thatwith free polypeptide (P＞0.05). While the Lethal effects of each effector cell group which acted on SK-OV3 and EC-304 was too low to show the significantdifference (P＞0.05). When tumor vaccine of MAGE-A3 antigen polypeptidemediated with BioPORTER in the proportion of 100/1 and 50/1, lethal effectswere more significant difference than that of free MAGE-A3 antigen polypeptide(P＜0.05). While there was no significant difference in the proportion of 20/1and 10/1 (P＜0.05). There was no significant difference between the two controlgroups (P＞0.05).
According to the results hereinbefore, we detect the target cells apoptosisinduced by effector cell group alone in the proportion of 100/1. It showed by flowcytometer that mortalities of MCF-7 cell line affected by MAGE-A3 polypeptidevaccine were distinct higher than that of SK-OV3 and EC-304 cell lines(P＜0.01).Mortalities of MCF-7 cell lines induced by polypeptide groups were higher thancontrol groups without polypeptide (P＜0.01). Significance of polypeptide groupsmediated by transfection reagent were higher than the free polypeptide groups(P＜0.01). Fluorescence microscope also suggested that MAGE-A3 polypeptidevaccine would accelerate the apoptosis of MCF-7. There was no significantdifference between the mortalities of SK-OV3 and EC-304 induced by three DCvaccines (P＞0.05), the same with that of the three cell lines induced by the groupwithout antigens (P＞0.05). These findings demonstrate that lethal and apoptosiseffects of tumor cells induced by MAGE-A3 polypeptide vaccine showed theMAGE-A3 antigenic specificity and HLA-A2 gene restriction. It meant that theeffects were indistinctive for the normal cells and other tumor cells whichexpressed no MAGE-A3 genes or HLA-A2 genes. MAGE-A3 polypeptidemediated by transfection reagent would more induce the lethal and apoptosiseffects of tumor target cells.
Feasibility and mechanism of breast cancer treated by MAGE-A3polypeptide vaccine were studied further for founding a breast cancertransplantation tumor model of naked-mice by inoculating MCF-7 cell lines inexponential phase of growth to one-side back subcutaneously of 20 naked-mice (5×10~6 /120ul/mouse). Production rate of tumor was 95% (19 in 20) after oneweek. The 19 naked-mice were divided into 4 groups in random that 5 mice withMAGE-A3 tumor vaccine mediated by BioPORTER in group A, 5 mice with freeMAGE-A3 tumor vaccine in group B, 5 with single DCs in group C and 4 in D asthe control group with normal saline, 1×10~6/120ul/mouse DCs or normal salinewere infused into the opposite back of tumor subcutaneously in every seven days,for 4 weeks. Developments of tumor were under continuous dynamicobservations until the mice were executed at the end of the observation.
The results shows that the bulk of knubs in group A and B both reducedobservably compared with group C and D (P＜0.01) ,while there was nosignificant difference between group A and B, the same with group C and D(P＞0.05) after one week. The condition changed after two weeks, the bulk ofknubs in group A reduced observably compared with group B(P＜0.01).and thechanges among other groups were the same as before. Such state was continueduntil the end of date. The weight of knubs in group A and B both reducedobservably compared with group C and D (P＜0.01). The weight of knubs ingroup A reduced observably compared with group B (P＜0.01), While there wasno significant difference between group C and D (P＞0.05). Which meant thattumor inhibition ratio increased observably in group A and B. The order oftumor inhibition ratio is: group A＞group B＞group C and group D.
And then stained the knub slices with anti-monoclonal antibody of CD8~+.The results of infiltration densities variety of CD8~+ T cells in tumor tissuessuggested that lots of CD8~+ T cells were infiltrated in tumor tissues by staining.Infiltration densities of CD8~+ T cells were 72.6±9.76 in group A and 55.8±3.70 ingroup B, while 14.2±1.92 in C and 11.2±3.38 in D under per high power field.Infiltration densities of group A and group B were observably higher than C andD respectively by statistics (P=0.000), group A were higher than groupB(P＜0.01),but with no significant difference between group C and D (P＞0.05).Subsistences of the cancer bearing mice in four weeks were that 100% in group A, 100% in B, 60% in C(3 in 5) and 50% in D(2 in 4). After the treatment, twomice died in the third and fourth week respectively in group C, while the othertwo also died the third and fourth week respectively in in group D, whichinterrelated with the knubbly augment. The further statistics treatment didn'ttake because of the small sample size. The present studies have assessed theinhibition and lethal effects of MAGE-A3 polypeptide vaccine for the breastcancer transplanted tumor of naked-mice. Further research would be taken inlarge-sample mice to study the connection between therapeutics of polypeptidevaccine and subsistences.
It concluded from the experiment that MAGE-A3 antigen polypeptide canaccelerate the DCs maturated, which came from mononuclear cells of peripheralblood, and submited the antigen to T lymphocyte via HLA-A2 and polypeptidecompounds. The active T lymphocytes then divided, proliferated, and excretedlots of cytokine (IL-12, IFN-r) to start the specificity immunoreaction. CD8~+ Tlymphocytes educed the magistral specificity cytotoxic, which mechanism couldbe to restrain cells proliferating and accelerate cells to apoptosis. Theimmunological effects were MAGE-A3 antigen specific and limited by HLA-A2genes. It meant that the effects were indistinctive for the normal cells and othertumor cells which expressed no MAGE-A3 genes or HLA-A2 genes. We found inthe breast cancer tissues of naked-mice cured with MAGE-A3 polypeptidevaccine, infiltration density of CD8~+ T cells increased sharply. The bulk andweight of knubs both reduced, which affected affirmative to the subsistences ofanimals bearing cancer. The increase of transfection efficiency of MAGE-A3polypeptide mediated by BioPORTER would enhance the specificityimmunological effects of effector cells, which acted on tumor target cells andbreast metastatic tumor of naked-mice. Experiments of curing breast cancerwith MAGE-A3 polypeptide vaccine could be based on these resultshereinbefore.
引文
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