【作者】 杨勇；

【导师】 王鹏举；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2024， 硕士

【摘要】 背景和目的癌症是全球亟待解决的问题,食管癌和胰腺癌是常见的消化道恶性肿瘤,预后不良,需要新的靶向治疗策略。溶瘤病毒疗法(Oncolytic virotherapy,OVT)是一种新型癌症疗法,通过病毒将外源基因递送到肿瘤内,最终杀死肿瘤细胞来治疗癌症。相较于其他溶瘤病毒,痘苗病毒(Vaccinia virus,VV)具有很多的优势。它能损伤血管,也能直接裂解细胞并激活免疫系统以破坏肿瘤细胞。猪胰腺弹性蛋白酶(Porcine pancreatic elastase,PPE)是一类丝氨酸蛋白酶,具有强大的抗肿瘤能力。粒细胞-巨噬细胞集落刺激因子(Granulocyte-macrophage colony stimulating factor,GM-CSF)是一种单体糖蛋白,具有多种抗肿瘤作用。我们使用基因工程技术在VV的A49R基因区域插入PPE构建重组溶瘤痘苗病毒VVΔTKΔA49R-PPE-RFP。此外,在VV的胸苷激酶基因(Thymidine kinase gene,TK)区域插入GM-CSF构建重组溶瘤痘苗病毒VVΔTK-GM-CSFΔA49R-PPE-RFP。我们旨在构建治疗效果更好的溶瘤痘苗病毒并探索其对食管癌肿瘤微环境的影响。方法1.利用基因工程改造的方法构建溶瘤痘苗病毒VVΔTKΔA49R-PPE-RFP、VVΔTK-mGM-CSFΔA49R-PPE-RFP(mouse,m);利用 PCR 以及基因测序检测PPE及mGM-CSF是否成功插入;利用ELISA法检测mGM-CSF在小鼠胰腺癌细胞系中的表达水平;利用PCR、qPCR以及酶催化水解反应检测PPE的表达及活性。2.利用 MTS 法和 TCID50 法检测VVΔTKΔA49R-PPE-RFP、VVΔTK-mGM-CSFΔA49R-PPE-RFP对肿瘤细胞的杀伤能力以及在肿瘤细胞中的复制能力。3.通过人食管癌EC9706细胞裸鼠皮下移植瘤模型以及小鼠食管癌mEC25细胞裸鼠和C57BL/6小鼠皮下移植瘤模型来研究VVΔTKΔA49R-PPE-RFP对小鼠食管癌的治疗效果及安全性。4.通过流式细胞术和免疫组化技术探索VVΔTKΔA49R-PPE-RFP在C57小鼠mEC25皮下移植瘤模型中的抗肿瘤机制。5.通过小鼠胰腺癌DT6606和TB11381细胞皮下瘤模型探索VVΔTK-mGM-CSFΔA49R-PPE-RFP对小鼠胰腺癌的治疗效果及安全性。结果1.成功构建了重组溶瘤痘苗病毒VVΔTKΔA49R-PPE-RFP和VVΔTK-mGM-CSFΔA49R-PPE-RFP,且PPE在多种肿瘤细胞中能成功表达并具有生物活性。2.病毒的复制效率和杀伤效果:PPE的插入不影响VVΔTKΔA49R-PPE-RFP在食管癌细胞中复制且显著增强了其对一些肿瘤细胞的杀伤能力。GM-CSF的插入不影响VVΔTK-mGM-CSFΔA49R-PPE-RFP在小鼠胰腺癌细胞系中的复制能力。与对照病毒相比,其对DT4994的杀伤能力有所降低,对DT6606的杀伤能力有所提高,而对TB11381的杀伤能力则无统计学差异。3.动物实验结果表明,VVΔTKΔA49R-PPE-RFP在三种皮下移植瘤模型中均具有强大的抗肿瘤作用。与对照病毒VVΔTKΔA49R相比,除在mEC25裸鼠皮下瘤模型中无统计学差异外,VVΔTKΔA49R-PPE-RFP对肿瘤生长的抑制作用均有显著的提高。4.VVΔTKΔA49R-PPE-RFP治疗后的第7天,脾脏和肿瘤组织中CD8+效应记忆性T细胞(Effective memory T cell,TEM)均有显著升高。5.VVΔTK-mGM-CSFΔA49R-PPE-RFP对DT6606皮下瘤的治疗效果显著的高于VVΔTKΔA49R-PPE-RFP组,但其对TB11381皮下瘤的治疗效果无显著提高。结论1.VVΔTKΔA49R-PPE-RFP是一种具有强大抗肿瘤能力的重组溶瘤痘苗病毒,对小鼠食管癌、胰腺癌皮下瘤模型均有良好的治疗效果,其抗肿瘤机制可能与脾脏及肿瘤微环境内增多的CD8+TEM有关。2.VVΔTK-mGM-CSFΔA49R-PPE-RFP对于小鼠胰腺癌DT6606皮下瘤模型具有较好的治疗效果。更多还原

【Abstract】 ObjectiveCancer is a global issue that urgently needs to be addressed.Esophageal cancer and pancreatic cancer are common gastrointestinal malignancies,with poor prognosis.New targeted treatment strategies are needed.Oncolytic virus therapy(OVT)is a novel cancer therapy that delivers exogenous genes to tumor cells through viruses,ultimately killing the tumor cells to treat cancer.Compared to other oncolytic viruses,vaccinia virus(VV)has many unique advantages.It can cause vascular damage,directly lyse cells and activate the immune system to destroy tumor cells.Porcine pancreas elastase(PPE)is a type of serine protease with strong anti-tumor capabilities.Granulocyte-macrophage colony stimulating factor(GM-CSF)is essentially a monomeric glycoprotein with various anti-tumor effects.We use genetic engineering technology to insert PPE into the A49R gene region of VV to construct recombinant oncolytic vaccinia virus VVΔTKΔA49R-PPE-RFP.In addition,GM-CSF was inserted into the thymidine kinase gene(TK)region of VV to construct recombinant oncolytic vaccinia virus VVΔTK-GM-CSFΔA49R-PPE-RFP.We aim to construct a more effective oncolytic vaccinia virus and explore its impact on the tumor microenvironment of esophageal cancer.Methods1.Oncolytic vaccinia viruses VVΔTKΔA49R-PPE-RFP and VVΔTK-mGM-CSFΔA49R-PPE-RFP(mouse,m)were constructed using genetic engineering techniques.PCR and gene sequencing were used to check if PPE and mGM-CSF were successfully inserted;ELISA was used to detect the expression of mGM-CSF in mouse pancreatic cancer cells;PCR,qPCR and enzyme catalyzed hydrolysis reactions were used to detect the expression and activity of PPE.2.The cell killing effect and replication efficiency of VVΔKΔA49R-PPE-RFP and VVΔTK-mGM-CSFΔA49R-PPE-RFP in various cell lines were detected using the MTS and TCID50 methods.3.To study the therapeutic effect and safety of VVΔTKΔA49R-PPE-RFP on mouse esophageal cancer through subcutaneous transplantation tumor model of human esophageal cancer EC9706 cells in nude mice and mouse esophageal cancer mEC25 cells in nude mice and C57BL/6 mice.4.The anti-tumor mechanism of VVΔTKΔA49R-PPE-RFP in C57 mouse mEC25 subcutaneous tumor model was investigated using flow cytometry and immunohistochemistry.5.To explore the therapeutic effect and safety of VVΔTK-mGM-CSFΔA49RPPE-RFP on mouse pancreatic cancer by subcutaneous tumor models of DT6606 and TB11381 cells.Results1.Successfully constructed recombinant oncolytic vaccinia virus VVΔTKΔA49RPPE-RFP and VVΔTK-mGM-CSFΔA49R-PPE-RFP.PPE was successfully expressed with biological activity across various tumor cell lines.2.The replication efficiency and killing effect of the virus:The insertion of PPE does not affect VVΔTKΔA49R-PPE-RFP replicates in esophageal cancer cells and significantly enhances its killing ability against some tumor cells.The insertion of GM-CSF does not affect the replication ability of VVΔTK-mGMCSFΔA49R-PPE-RFP in mouse pancreatic cancer cell line.Compared with the control virus,its killing ability to DT4994 was decreased,while that to DT6606 was increased,and there was no statistical difference in the killing ability to TB11381.3.Animal experiments have shown that VVΔTKΔA49R-PPE-RFP has strong antitumor effects in all three subcutaneous transplant tumor models.Compared to the control virus VVΔTKΔA49R,the inhibitory effect of VVΔTKΔA49R-PPE-RFP on tumor growth was significantly improved except in the mEC25 nude mouse subcutaneous tumor model.4.On the seventh day after treat with VVΔTKΔA49R-PPE-RFP,CD8+effector memory T-cells(TEM)was significantly elevated in the spleen and tumor tissue.5.Compare to VVΔTKΔA49R-PPE-RFP,the therapeutic effect of VVΔTK-mGMCSFΔA49R-PPE-RFP is better on DT6606 subcutaneous tumor model but has no significant improvement on TB11381 subcutaneous tumor model.Conclusion1.VVΔTKΔA49R-PPE-RFP is a recombinant oncolytic vaccinia virus with stronger antitumor capabilities,demonstrating significant therapeutic effects on subcutaneous tumor models of mouse esophageal and pancreatic cancer.Its anti-tumor mechanism may be related to the increase of CD8+TEM in the spleen and tumor microenvironment.2.VVΔTK-mGM-CSFΔA49R-PPE-RFP shows good therapeutic effects on DT6606 subcutaneous tumor models.更多还原