靶向间皮素的嵌合抗原受体修饰T细胞治疗卵巢癌实验研究

发布时间：2018-06-22 13:10

本文选题：CAR-T + 间皮素　；参考：《第二军医大学》2017年硕士论文

【摘要】：癌症已经严重威胁到了人类的健康,对人类的生存造成了极大的危害。在全球范围内,卵巢癌占妇科最常见癌症的第八位,且死亡人数占妇科常见肿瘤的第七位。每年全世界约有230000名妇女被诊断为卵巢癌,而且超过75%的患者确诊时已经是晚期,卵巢癌的5年生存率低于45%。目前临床上卵巢癌常见的治疗手段包括手术、放化疗等,但这些治疗不能有效的控制肿瘤的复发及进展,也不能有效的延长患者的生存期及改善患者的生存质量。因此我们迫切需要寻求新的治疗手段。近年来生物治疗成为一种重要的抗肿瘤治疗手段,给攻克恶性肿瘤带来了希望。研究表明大多数的肿瘤细胞能够躲避免疫系统的识别,从而限制免疫治疗的抗癌效果。CAR-T(嵌合抗原受体T细胞)技术应运而生,其具有较好的靶向性、杀伤性、增殖性及持久性。大量的临床试验研究也表明该技术在改善恶性血液肿瘤患者生存质量及延长患者生存期方面取得了巨大的成功,CAR-T技术成为了免疫治疗的研究热点。然而该技术在实体肿瘤的研究还不够成熟,未能得到期待的效果,仍然面临许多问题,包括:实体瘤组织特殊的病理学特征、表面缺乏合适的肿瘤特异性抗原,肿瘤周围免疫抑制微环境、共刺激分子的优化组合、自杀基因的设计、回输后的不良反应等。本课题构建并检测了靶向间皮素抗原的嵌合抗原受体T细胞对几种卵巢癌细胞株的体外杀伤作用:1、构建包含两个胞内共刺激区(CD3ζ和4-1BB)的mesoCAR的重组质粒,再通过piggyBac转座子系统转染T细胞,获得第二代mesoCAR-T细胞。其转染效率为30%左右。进一步证明mesoCAR能够在T细胞中高水平稳定表达。流式检测经过间皮素刺激后mesoCAR-T的活化表型的表达情况,结果表明间皮素抗原能使mesoCAR-T细胞活化。2、通过流式及Western Blot检测多种肿瘤细胞株间皮素的表达水平,筛选出五种间皮素不同表达水平的卵巢癌细胞株。通过不同的方式检测mesoCAR-T细胞对间皮素高表达的细胞株及低表达的细胞株的杀伤作用。结果显示mesoCAR-T细胞对间皮素高表达细胞株的杀伤作用明显强于低表达的细胞株,并且能更有效的抑制间皮素高表达细胞株的生长。表明构建的mesoCAR-T细胞对间皮素高表达的卵巢癌细胞株具有更强的杀伤活性及靶向性。3、构建靶向间皮素抗原的嵌合抗原受体T细胞(mesoCAR-T),通过流式检测间皮素抗原刺激后mesoCAR-T细胞的细胞因子分泌的情况,结果发现IL-2、IL-4、IL-6、IL-10、TNF-α、IFN-γ的分泌量明显高于对照组T细胞。同时用流式检测mesoCAR-T细胞与两种卵巢癌细胞共培养后细胞因子的分泌,发现MesoCAR-T细胞与间皮素高表达的细胞株(HO8910)共培养后,IL-2、IL-4、IL-6、IL-10、TNF-α及IFN-γ的分泌量相比间皮素低表达的细胞株(SKOV3)升高。本实验成功的构建了靶向间皮素抗原的嵌合抗原受体T细胞(mesoCAR-T),其结果表明本实验室构建的piggyBac转座酶系统能够很好的将外源基因整合T细胞中,并且构建的mesoCAR-T细胞能够特异性的杀伤间皮素表达的卵巢癌细胞,同时mesoCAR-T和卵巢癌细胞株共培养后能够上调IL-2、IL-4、IL-6、IL-10、TNF-α及IFN-γ的分泌。为进一步体内及临床试验的开展提供了实验基础,也为卵巢癌的治疗提供了新的治疗方法。
[Abstract]:Cancer has been a serious threat to human health and has caused great harm to human survival. On the global scale, ovarian cancer accounts for eighth of the most common cancers in gynecology, and the death toll accounts for seventh of the common gynecologic cancers. About 230000 women all over the world are diagnosed with ovarian cancer every year, and more than 75% of the patients have been diagnosed at the time of diagnosis. The 5 year survival rate of ovarian cancer is lower than that of 45%.. The common treatment methods of ovarian cancer are surgery, radiotherapy and chemotherapy, but these treatments can not effectively control the recurrence and progress of the tumor. It can not effectively prolong the survival time of the patients and improve the quality of life of the patients. Therefore, we urgently need to seek new therapeutic hands. Biologic therapy has become an important antitumor therapy in recent years, which has brought hope to the attack of malignant tumor. The research shows that most of the tumor cells can avoid the identification of the immune system and restrict the anti-cancer effect of.CAR-T (chimeric antigen receptor T cell). A large number of clinical trials have also shown great success in improving the quality of life and prolonging the life of patients with malignant hematological tumors. CAR-T technology has become a hot topic in the study of immunotherapy. However, the study of this technique in solid swelling is not mature enough to be expected. The effect is still faced with many problems, including the special pathological features of solid tumor tissue, the lack of proper tumor specific antigen on the surface, the immunosuppressive microenvironment around the tumor, the optimal combination of CO stimulatory molecules, the design of suicide gene, the adverse reaction after the retransmission, and so on. The in vitro killing effect of primary receptor T cells on several ovarian cancer cell lines: 1, a recombinant plasmid containing two intracellular co stimulatory regions (CD3 zeta and 4-1BB) was constructed, and T cells were transfected through the piggyBac transposon system to obtain second generation mesoCAR-T cells. The transfection efficiency was about 30%. Further demonstrated that mesoCAR could be in the high level of T cells. The expression of the activated phenotype of mesoCAR-T after mesothelin was detected by flow test. The results showed that the mesothelin antigen could activate the mesoCAR-T cells to activate.2. The expression level of mesothelin in a variety of tumor cell lines was detected by flow and Western Blot, and five kinds of ovarian cancer cell lines with different levels of mesothelin were screened. The cytotoxicity of mesoCAR-T cells to mesothelin high expression cell lines and low expression cell lines was detected in the same way. The results showed that the killing effect of mesoCAR-T cells to mesothelin high expression cell lines was stronger than that of low expressed cell lines, and could effectively inhibit the growth of mesothelin high expression cell lines. The results showed that the constructed mesoCAR- was constructed. T cells have a stronger killing activity and targeted.3 for the ovarian cancer cell lines with high expression of mesothelin, and construct a chimeric antigen receptor T cell (mesoCAR-T) targeting mesothelin antigen. By flow cytometry, the cytokine secretion of mesoCAR-T cells after the stimulation of mesothelin antigen was detected. The results showed that IL-2, IL-4, IL-6, IL-10, TNF- a, IFN- gamma were found. The secretion of T cells was significantly higher than that of the control group. At the same time, the secretion of cytokines in the co culture of mesoCAR-T cells and two kinds of ovarian cancer cells was detected by flow cytometry. The secretion of IL-2, IL-4, IL-6, IL-10, TNF- alpha and IFN- gamma in the co culture of MesoCAR-T cells with the high expression of mesothelin (HO8910) was found to be higher than that of the cells with the low expression of mesothelin (SKOV3). High. This experiment successfully constructed the chimeric antigen receptor T cell (mesoCAR-T) targeting mesothelin antigen. The results show that the piggyBac transposing enzyme system constructed in this laboratory can integrate the exogenous gene in T cells well, and the constructed mesoCAR-T cells can specifically kill the ovarian cancer cells with the expression of mesothelin, and mesoCAR The co culture of -T and ovarian cancer cells can increase the secretion of IL-2, IL-4, IL-6, IL-10, TNF- alpha and IFN- gamma. It provides an experimental basis for further in vivo and clinical trials and provides a new treatment for the treatment of ovarian cancer.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31

【参考文献】