表达mIL-21的溶瘤痘苗病毒对小鼠乳腺癌的治疗作用

发布时间：2018-05-20 06:41

本文选题：痘苗病毒 + 乳腺癌　；参考：《郑州大学》2017年硕士论文

【摘要】：背景乳腺癌当前已成为女性最常见的恶性肿瘤之一,其发病率已上升至女性恶性肿瘤的第一位,严重威胁女性的生命健康。随着现代手术技术的提升以及放疗、化疗等辅助治疗手段的应用,乳腺癌患者的总体生存率得到了较大的改善,但是,每年仍有30%-40%的患者出现复发和转移,导致患者死亡[1-3]。因此,在临床研究中迫切需要研发出一种有效的乳腺癌治疗手段,降低乳腺癌的复发和转移。复制选择性溶瘤病毒(Replication-selectivity oncolytic viruses,RSOV)为肿瘤治疗提供了新的治疗途径。其原理在于RSOV可以特异性地在肿瘤细胞中复制,既而溶解肿瘤细胞,却对正常细胞无影响[4,5]。研究发现RSOV可凭借多种机制发挥抗肿瘤作用,包括直接溶解细胞、表达毒性蛋白、诱导细胞凋亡、深入诱导抗肿瘤免疫反应以及阻碍宿主蛋白合成等机制,正是由于多样化的病毒治疗机制,国际上越来越多的研究者开始把目光转移到溶瘤病毒上来[6,7]。近年来,研究者发现痘苗病毒N1L蛋白是引起神经毒性的重要因素,同时该基因产物可通过与IKK复合物的靶向结合来抑制NF-k B和IRF3活性,诱导细胞凋亡,从而拮抗宿主细胞的免疫反应[8-10]。因此N1L的删除不仅可以增加RSOV安全性,更重要的是可以增强机体对其免疫反应,从而有望间接增强机体的抗肿瘤免疫反应。我们实验室先在野生型Lister痘苗病毒(Vaccinia viruses,VV)的基础上敲除胸苷激酶(Tymidine Kinase,TK)基因,后续又敲除N1L基因来降低病毒毒性,同时并期望能够激起机体更强的免疫反应。本研究通过体外、体内对比研究该病毒对小鼠乳腺癌的治疗效果和生物安全性。目的通过观察溶瘤痘苗病毒VVΔTKΔN1L-RFP和VVΔTKΔN1L-m IL-21对小鼠乳腺癌细胞系JC、TUBO和4T1的体内外治疗效果,评价其抗肿瘤疗效。方法1.采用MTS法比较两种病毒在不同浓度下对三株乳腺癌细胞的体外杀伤效果;2.用TCID50法检测病毒在三株乳腺癌细胞系中的复制能力。用ELISA法检测两种病毒感染三种乳腺癌细胞后上清液中m IL-21的水平。3.建立三阴性乳腺癌4T1和Her-2扩增型乳腺癌TUBO原位模型,瘤内注射两种病毒,检测其治疗作用。结果1.VVΔTKΔN1L-RFP和VVΔTKΔN1L-m IL-21两种病毒在小鼠乳腺癌细胞中均具有复制能力,低剂量的病毒就对小鼠乳腺癌细胞产生明显的杀伤效应;2.VVΔTKΔN1L-m IL-21感染的细胞上清中检测到高表达的m IL-21蛋白。3.在4T1乳腺癌原位模型中,病毒治疗无明显效果(P0.05)。4.在TUBO乳腺癌原位模型中,两种病毒均能显著抑制肿瘤生长(t=2.396,P0.05;t=4.008,P0.01),延长荷瘤小鼠生存期(χ2=16.94,P0.01)。结论VVΔTKΔN1L-RFP和VVΔTKΔN1L-m IL-21两种病毒在小鼠乳腺癌细胞中均具有特异性增殖并杀伤肿瘤细胞的能力,在体内,两种病毒对Her-2扩增型和三阴性乳腺癌具有不同的治疗效果。
[Abstract]:Background at present, breast cancer has become one of the most common malignant tumors in women. The incidence of breast cancer has risen to the first of female malignant tumors, which seriously threatens the life and health of women. With the improvement of modern surgical techniques and the application of radiotherapy and chemotherapy, the overall survival rate of breast cancer patients has been greatly improved. However, 30 to 40% of the patients still have recurrence and metastasis every year, resulting in death of patients [1-3]. Therefore, there is an urgent need to develop an effective treatment for breast cancer in clinical research to reduce the recurrence and metastasis of breast cancer. Replication of Replication-selectivity oncolytic virus (RSOV) provides a new approach to tumor therapy. The principle is that RSOV can specifically replicate in tumor cells, dissolve tumor cells, but have no effect on normal cells [4]. It has been found that RSOV can play an anti-tumor role by several mechanisms, including the direct dissolution of cells, the expression of toxic proteins, the induction of apoptosis, the further induction of anti-tumor immune response and the obstruction of host protein synthesis. Because of the variety of virus treatment mechanisms, more and more researchers have begun to focus on the tumor virus [6]. In recent years, researchers have found that the vaccinia virus N1L protein is an important factor in neurotoxicity, and that the gene product can inhibit the activity of NF-k B and IRF3 and induce apoptosis by targeting IKK complex. Thus antagonize the immune response of host cells [8-10]. Therefore, the deletion of N1L can not only increase the safety of RSOV, but also enhance the immune response to N1L, which is expected to indirectly enhance the anti-tumor immune response. We first knocked out the thymidine kinase Tymidine Kinase TKV gene on the basis of wild type Lister vaccinia virusesVV, and then knocked out the N1L gene to reduce the toxicity of the virus and hope to stimulate a stronger immune response. The therapeutic effect and biological safety of the virus on mouse breast cancer were studied in vitro and in vivo. Objective to observe the therapeutic effect of VV 螖 TK 螖 N1L-RFP and VV 螖 TK 螖 N1L-m IL-21 on mouse breast cancer cell line JCG-TUBO and 4T1 in vitro and in vivo, and to evaluate the antitumor effect of VV 螖 TK 螖 N1L-RFP and VV 螖 TK 螖 N1L-m IL-21. Method 1. MTS assay was used to compare the killing effect of two viruses on three breast cancer cells in vitro. The replication ability of the virus in three breast cancer cell lines was detected by TCID50 assay. The level of m IL-21 in supernatant of three kinds of breast cancer cells infected by two viruses was detected by ELISA assay. The in situ model of 4T1 and Her-2 amplified breast cancer TUBO was established. Two kinds of viruses were injected into the tumor and their therapeutic effects were tested. Results both 1.VV 螖 TK 螖 N1L-RFP and VV 螖 TK 螖 N1L-m IL-21 have the ability to replicate in mouse breast cancer cells. The high expression of m IL-21 protein 路3 was detected in the supernatant of mouse breast cancer cells infected by low dose of 1.VV 螖 TK 螖 N1L-RFP and VV 螖 TK 螖 N1L-m IL-21. In the in situ model of 4T1 breast cancer, there was no obvious effect of virus therapy. In the TUBO breast cancer in situ model, both of the two viruses could significantly inhibit the growth of tumor and prolong the survival time of tumor-bearing mice (蠂 ~ 2 ~ (2) ~ (16.94) (P ~ (0.01). Conclusion both VV 螖 TK 螖 N1L-RFP and VV 螖 TK 螖 N1L-m IL-21 have the ability to proliferate and kill tumor cells in mouse breast cancer cells. In vivo, the two viruses have different therapeutic effects on Her-2 amplified breast cancer and triple negative breast cancer.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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