云南白族地区乳腺癌EphA2表达检测及以T淋巴细胞为载体的Caspase-3靶向治疗的研究

Bai nationality is one of the main peculiar minorities in Yunnan, of which the women are the important force. Breast cancer is one of the most common malignant tumors and has become the number one killer among women carcinomas, which greatly affects human health and life. It is estimated that 1.2 million of female will be diagnosed of breast cancer, with 0.5 million death annually in the world. In recent years the incidence and mortality of breast cancer are increasing rapidly, with an annual rate of approximately 0.2～0.3％. And more and more young women were affected by this disease. The treatment of the breast cancer is a hard nut to crack in the world at present, and the research on a new targeted drug is still a hot academic issue. Immunotoxins (ITs) is a new targeted agent of the highly specific nature. Recent evidence has shown that the EphA2 receptor is overexpressed in a number of human malignancies, including that of breast, prostate, melanoma and of ovary as well as in Glioblastoma multiform, and is associated with their development and progression.Thus, the EphA2 receptor may represent a very attractive target for immunotoxin therapy. For the ideal toxin-based targeted therapy for caner, one of the most important issues to be considered is how to target only tumor tissue effectively, killing cancer cells selectively, and to protect the surrounding normal tissues from side effects. In the present study, we will first examine the expression of EphA2 receptor and its ligand EphrinAl in breast cancer tissue from Yunnan Dali Bai, Han and other minority women. Then we construct an adenoviral vector encoding EphrinA1-PE38KEDL, a recombinant immunotoxin based on the EphrinA1 ligand and a truncated form of Pseudomonas exotoxin A, which could specifically target EphA2 receptor-overexpressing tumor cells. We also confirm the production of EphrinA1-PE38KEDL in the mesenchymal stem cells (MSCs) derived from the bone marrow via adenoviral transduction and the effects of these EphrinA1-PE38KEDL-carrying MSCs on the proliferation and apoptosis of the EphA2-overexprssing breast cancer cells in vitro. And then, utilizing the tumor tropism of MSCs, the MSCs that were transiently transduced with these virus will be transplanted into the animal models of breast cancer, and the anticancer effects in vivo would be observed. The aim of our study is to obtain reliable evidence that genetically modified MSCs may function as a novel therapeutic vehicle for targeted delivery of immunotoxin to malignant tumor and explore a new strategy for the site-specific treatment of breast cancer.

白族是云南的一种主要世居少数民族，而女性是这支生力军的重要组成部分。乳腺癌是女性最常见的恶性肿瘤之一，其发病呈较快增长和年轻化趋势，严重威胁妇女的身心健康。乳腺癌的治疗仍是世界性难题，新的靶向性药物研究仍是学术热点。免疫毒素是近年来新兴的一种特异性靶向治疗药物。最近发现EphA2在多种恶性肿瘤中过度表达，可能是一个新的肿瘤标志物。本研究首先检测白族地区乳腺癌中EphA2及其配体EphrinAl的表达，利用骨髓间充质干细胞（MSCs）对肿瘤组织有趋化性的特点，以EphrinAl为导向分子，以绿脓杆菌外毒素活性片段PE38KEDL为毒素分子，通过腺病毒介导，建立分泌重组免疫毒素EphrinAl-PE38KEDL的MSCs，体外观察其对乳腺癌细胞增殖和凋亡的作用，然后植入乳腺癌动物模型，观察分泌重组免疫毒素的MSCs对乳腺癌的治疗作用，探讨骨髓MSCs作为靶向毒素基因治疗载体的可行性。

乳腺癌是女性最常见恶性肿瘤之一，其治疗仍是世界性难题，最近发现EphA2在多种恶性肿瘤过度表达，可能是一个肿瘤标志物。本项目收集大理大学附属医院2012-2018年女性乳腺癌标本486例，免疫组化和原位杂交显示EphA2、EphrinAl主要表达于肿瘤细胞和血管内皮细胞，其蛋白阳性率分别为74.49%、74.07%，mRNA阳性率为72.43%、68.31%，均与患者年龄和病理类型不相关，而与肿瘤大小、淋巴转移、临床分期和组织学分级相关（P<0.05）；EphA2和EphrinAl蛋白、EphA2和EphrinAl mRNA的阳性分布基本一致；EphA2、EphrinA1各自蛋白与其相应mRNA的阳性表达相关，但表达水平不完全一致。接着以EphrinAl为导向分子、人活性型Caspase-3为治疗因子，定向克隆入质粒；经体外同源重组构建携带靶向促凋亡基因EphrinAl- Caspase-3的重组腺病毒载体pAd-EphrinAl- Casp3；以HEK 293细胞包装和扩增，获得重组腺病毒rAd-EphrinAl- Casp3。以适度MOI病毒感染T淋巴细胞，建立稳定分泌靶向促凋亡蛋白EphrinAl-Caspase-3的T淋巴细胞。Ad- EphrinA1- Caspase-3-T淋巴细胞感染上清能促进体外EphA2阳性乳腺癌细胞凋亡，强烈抑制其生长，中和抗体能拮抗该作用。建立裸鼠乳腺癌模型，与PBS组或T淋巴细胞组相比，Ad-EphrinA1-Caspase-3-T细胞组的肿瘤体积小（P<0.05），第2天就有EphrinAl-Caspase-3表达，第8天达到高峰，随后分泌量逐渐降低，可能与T细胞自身凋亡及数量下降有关；培养结束，肿瘤组织Ki67阳性率明显降低，绿色荧光标记EphrinAl-Caspase-3-T细胞散在分布。可见，乳腺癌中EphA2、EphrinAl高表达，并与肿瘤发生发展、侵袭转移及恶性程度有关；重组EphrinAl-Caspase-3对体外乳腺癌细胞有靶向性杀伤作用，对体内乳腺癌生长有抑制作用，EphA2有望作为乳腺癌早期诊断及预后判断标志物，作为治疗新靶向；但T淋巴细胞作为重组EphrinAl- Caspase-3载体靶向治疗肿瘤的可行性有待进一步研究。该项目属于基础理论研究，成果主要是论文，已发表8篇，其中SCI收录1篇。