發(fā)布時間：2018-05-20 06:41

  本文選題：痘苗病毒 + 乳腺癌��； 參考：《鄭州大學》2017年碩士論文

【摘要】：背景乳腺癌當前已成為女性最常見的惡性腫瘤之一,其發(fā)病率已上升至女性惡性腫瘤的第一位,嚴重威脅女性的生命健康。隨著現(xiàn)代手術技術的提升以及放療、化療等輔助治療手段的應用,乳腺癌患者的總體生存率得到了較大的改善,但是,每年仍有30%-40%的患者出現(xiàn)復發(fā)和轉移,導致患者死亡[1-3]。因此,在臨床研究中迫切需要研發(fā)出一種有效的乳腺癌治療手段,降低乳腺癌的復發(fā)和轉移。復制選擇性溶瘤病毒(Replication-selectivity oncolytic viruses,RSOV)為腫瘤治療提供了新的治療途徑。其原理在于RSOV可以特異性地在腫瘤細胞中復制,既而溶解腫瘤細胞,卻對正常細胞無影響[4,5]。研究發(fā)現(xiàn)RSOV可憑借多種機制發(fā)揮抗腫瘤作用,包括直接溶解細胞、表達毒性蛋白、誘導細胞凋亡、深入誘導抗腫瘤免疫反應以及阻礙宿主蛋白合成等機制,正是由于多樣化的病毒治療機制,國際上越來越多的研究者開始把目光轉移到溶瘤病毒上來[6,7]。近年來,研究者發(fā)現(xiàn)痘苗病毒N1L蛋白是引起神經毒性的重要因素,同時該基因產物可通過與IKK復合物的靶向結合來抑制NF-k B和IRF3活性,誘導細胞凋亡,從而拮抗宿主細胞的免疫反應[8-10]。因此N1L的刪除不僅可以增加RSOV安全性,更重要的是可以增強機體對其免疫反應,從而有望間接增強機體的抗腫瘤免疫反應。我們實驗室先在野生型Lister痘苗病毒(Vaccinia viruses,VV)的基礎上敲除胸苷激酶(Tymidine Kinase,TK)基因,后續(xù)又敲除N1L基因來降低病毒毒性,同時并期望能夠激起機體更強的免疫反應。本研究通過體外、體內對比研究該病毒對小鼠乳腺癌的治療效果和生物安全性。目的通過觀察溶瘤痘苗病毒VVΔTKΔN1L-RFP和VVΔTKΔN1L-m IL-21對小鼠乳腺癌細胞系JC、TUBO和4T1的體內外治療效果,評價其抗腫瘤療效。方法1.采用MTS法比較兩種病毒在不同濃度下對三株乳腺癌細胞的體外殺傷效果;2.用TCID50法檢測病毒在三株乳腺癌細胞系中的復制能力。用ELISA法檢測兩種病毒感染三種乳腺癌細胞后上清液中m IL-21的水平。3.建立三陰性乳腺癌4T1和Her-2擴增型乳腺癌TUBO原位模型,瘤內注射兩種病毒,檢測其治療作用。結果1.VVΔTKΔN1L-RFP和VVΔTKΔN1L-m IL-21兩種病毒在小鼠乳腺癌細胞中均具有復制能力,低劑量的病毒就對小鼠乳腺癌細胞產生明顯的殺傷效應;2.VVΔTKΔN1L-m IL-21感染的細胞上清中檢測到高表達的m IL-21蛋白。3.在4T1乳腺癌原位模型中,病毒治療無明顯效果(P0.05)。4.在TUBO乳腺癌原位模型中,兩種病毒均能顯著抑制腫瘤生長(t=2.396,P0.05;t=4.008,P0.01),延長荷瘤小鼠生存期(χ2=16.94,P0.01)。結論VVΔTKΔN1L-RFP和VVΔTKΔN1L-m IL-21兩種病毒在小鼠乳腺癌細胞中均具有特異性增殖并殺傷腫瘤細胞的能力,在體內,兩種病毒對Her-2擴增型和三陰性乳腺癌具有不同的治療效果。
[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.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.9

【參考文獻】

相關碩士學位論文 前1條

1 王金禮;新型腫瘤靶向性溶瘤痘苗病毒VV_△TK_△N1L-RFP對小鼠4T1乳腺癌模型的治療作用[D];鄭州大學;2014年

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本文編號：1913579