摘要
目的:探讨Survivin的显性负突变体SurvivinT34A的重组腺病毒治疗小鼠Lewis肺癌(Lewis lung Cancer,LLC)的效果及作用机制。方法:采用Gateway技术分别构建、制备高滴度的人源的SurvivinT34A重组腺病毒(Ad-hSurvivinT34A,Ad-HS)和鼠源的SurvivinT34A重组腺病毒(Ad-mSurvivinT34A,Ad-MS)。在体外试验中,采用Hoechst染色和流式细胞术检测感染重组腺病毒的小鼠LLC细胞的促凋亡作用。在体内实验中,绘制肿瘤生长曲线和荷瘤小鼠的生存曲线观察Ad-HS和Ad-MS的抗LLC肿瘤效果;TUNEL荧光显色检测肿瘤组织内凋亡情况;通过CD31免疫组化染色,观察肿瘤组织内新生血管抑制情况;通过Cr51释放法检测脾T细胞对肿瘤细胞的特异杀伤活性。结果:制备的高纯度和高滴度的Ad-HS和Ad-MS在体外均能够有效诱导抗LLC肿瘤效应,其中异种重组腺病毒(Ad-HS)的效果更好,但差异无统计学意义。主要表现为治疗组肿瘤细胞凋亡率增加,肿瘤组织内微血管密度降低,激发肿瘤特异性的细胞免疫反应,杀伤LLC肿瘤细胞,因此治疗组荷瘤小鼠肿瘤生长缓慢,生存期延长。结论:异种Ad-hSurvivinT34A重组腺病毒对LLC细胞有较强的抑制作用。
Objective: To investigate the therapeutic effect and mechanism of recombinant adenovirus containing Survivin T34 A, a dominant negative mutant of survivin, on Lewis lung cancer(LLC) in mice. Methods: High titer recombinant adenovirus(Ad-hSurvivinT34 A, Ad-HS) and mouse-derived recombinant adenovirus(Ad-mSurvivinT34 A, Ad-MS) were constructed and prepared by gateway technique. In vitro, Hoechst staining and flow cytometry were used to detect the apoptotic activity of LLC/c in LLC cells of mice infected with recombinant adenovirus. In vivo experiments, tumor growth curve and survival curve of tumor-bearing mice were drawn to observe the anti-LLC/c effect of recombinant adenovirus(Ad-HS, Ad-MS);terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to detect apoptosis in tumor tissue;CD31 immunohistochemical staining was used to observe the inhibition of neovascularization in tumor tissue;and Cr51 release assay was used to detect the specific killing activity of spleen T cells to tumor cells. Results: The prepared recombinant adenovirus(Ad-HS, Ad-MS) with high purity and titer could effectively induce anti-LLC effect in vitro, especially the heterologous recombinant adenovirus(Ad-HS). However, this effect was not statistically significant. The main manifestations were the increase of apoptotic rate of tumor cells, the decrease of microvessel density in tumor tissue, the stimulation of specific cellular immune response and the killing of LLC tumor cells in the treatment group. Therefore, the growth of tumors in the treatment group was slow and the survival time was prolonged. Conclusion: The recombinant adenovirus of heterogeneous Ad-hSurvivinT34 A could effectively inhibit LLC cells.
作者
张鹏
王春婷
黄雪梅
谢康
Zhang Peng;Wang Chunting;Huang Xuemei;Xie Kang(Department of Head and Neck Surgery,Sichuan Cancer Hospital&Institute,Sichuan Cancer Center,School of Medicine,University of Electronic Science and Technology of China,Chengdu 610041,Sichuan,China;State Key Laboratory of Biotherapy and Cancer Center,West China Hospital,Sichuan Universityy Chengdu,610041,Sichuan,China;Second Department of Hematology and Oncology,Hospital(T.C.M)Affiliated to Southwest Medical University,Luzhou,646000,Sichuan,China)
出处
《肿瘤预防与治疗》
2020年第2期111-118,共8页
Journal of Cancer Control And Treatment
基金
四川省应用基础研究项目(编号:2017JY029 9)
CSCO-默克雪兰诺肿瘤研究基金(编号:Y-MT2016-009)~~
