CIK细胞作为载体介导重组溶瘤痘病毒rVV-CCL5-SSTR2-Luc+靶向治疗大肠癌的实验研究

Colorectal cancer threaten the healthy of human beings; Vaccinia virus is hotspot in research field of oncolytic virus; Vaccinia virus delivered by intravenous injection can be cleared by immune system, so delievery method of vaccinia virus has been limitid to intratumoral injection mostly, which limited its application in colorectal cancer; Deliver CIK-mediated vaccinia virus by intravenous injection by using CIK cells as vehicles has clinical application prospects in colorectal cancer treatment; CCL5 and SSTR2 have strong anti-tumor effects. In the present study,a vaccinia virus which coexpresses CCL5 and SSTR2 genes - rVV-CCL5-SSTR2-Luc+(rVV) - will be constructed, CIK-mediated rVV will be injected intravenous by using CIK cells as vehicles for targeted colorectal cancer treatment, in this case, rVV can effectively avoid being cleared by immune system, and will be delivered accurately into tumor, after CIK play its roles by killing tumor cells, rVV will be released into tumor cells and play their roles of killing tumor cells secondary time. In addition, high expression level of CCL5 in tumor cells will induce T, NK cells targetting to tumor cells; Meanwhile, the high expression level of SSTR2 in tumor cells will help precise orientation of Octreotide to tumor, which will benefit for the inhibiting of tumor growth and inducing tumor cell apoptosis; CIK-mediated rVV will play the immune / oncolytic double effects. The study aimed to evaluate the therapeutic effect of CIK-mediated rVV in targeted colorectal cancer threatment based on in vitro and in vivo experiments, the mechanisms behind tumor-killing effects will also be explored. This study may be beneficial to estabish the experimental and theoretical basis for the application of CIK-mediated rVV in targeted colorectal cancer threatment.

大肠癌严重危害人类健康；痘病毒是溶瘤病毒研究热点；静脉注射痘病毒可被免疫系统清除，因此多限于肿瘤内注射，限制了其在大肠癌的应用；将CIK作为载体介导痘病毒经静脉注射治疗大肠癌具临床应用前景；CCL5和SSTR2具强力抗肿瘤作用。本研究拟构建重组溶瘤痘病毒rVV-CCL5-SSTR2-Luc+（rVV），以CIK为载体介导其经静脉注射治疗大肠癌，不仅有效避免rVV被免疫系统清除，将其准确输送至肿瘤，而且当CIK进入体内杀伤肿瘤细胞后，释放出的rVV将发挥二次杀伤肿瘤细胞作用。并且，CCL5高表达将定向诱导T、NK细胞靶向杀伤肿瘤；SSTR2高表达将精确定向并诱导奥曲肽到达肿瘤部位抑制肿瘤生长、诱导肿瘤细胞凋亡；发挥免疫/溶瘤双重协同抗肿瘤作用。研究拟通过体内/外实验评估CIK作为载体介导rVV治疗大肠癌的疗效，探讨其发挥溶瘤效应的作用机制，为应用CIK介导rVV治疗大肠癌奠定实验和理论基础。

大肠癌严重危害人类健康；痘病毒是溶瘤病毒研究热点；静脉注射痘病毒可被免疫系统清除，因此多限于肿瘤内注射，限制了其在大肠癌的应用；将CIK作为载体介导痘病毒经静脉注射治疗大肠癌具临床应用前景；CCL5和SSTR2具强力抗肿瘤作用。本研究成功构建了重组痘病毒真核表达载体pSC65-CCL5-SSTR2-Luc及其对照pSC65-Luc，经同源重组WR痘病毒，获得了重组溶瘤痘病毒rVV-CCL5-SSTR2-Luc+（rVV）及其对照 rVV-Luc+。确定感染复数50（MOI=50）和感染时间12h（T=12h）为CIK细胞作为载体荷载rVV的最佳实验方案。CIK细胞介导重组溶瘤病毒rVV治疗肿瘤的体外实验结果表明，与对照病毒组或未荷载溶瘤病毒的CIK细胞阴性对照组NC相比，rVV感染可增高肿瘤细胞CCL5和SSTR2表达水平；rVV感染可提高肿瘤细胞系增殖抑制率，不同肿瘤细胞系抑制率达峰时间不尽相同。对CIK细胞介导rVV治疗肿瘤作用的研究结果表明：痘病毒主要通过激活肿瘤细胞凋亡发挥抗瘤作用，而并不引起细胞周期的改变。rVV及其对照病毒均能在一定程度上引发肿瘤细胞的凋亡，且rVV引发细胞凋亡的作用更显著、作用持续时间更长。对CIK 细胞介导rVV发挥溶瘤作用的机制研究结果表明明，p53、p16、Kras、Erk1/2蛋白并不是rVV发挥溶瘤作用的主要作用靶点。在大部分肿瘤细胞中，rVV可能是通过激发促凋亡蛋白酶caspase-3和凋亡促进因子Bax的表达升高，促进肿瘤细胞凋亡。部分肿瘤细胞中应激性地激发了自身抗凋亡因子survivin和Bcl2的表达上调。在部分肿瘤细胞中， rVV可通过降低VEGFA蛋白表达来阻断肿瘤血管生成，进而发挥抑瘤作用。并且，感染rVV可提高肿瘤细胞免疫检查点共抑制分子PD-L1的表达水平。本研究通过体外实验评估了CIK作为载体介导rVV治疗大肠癌的疗效，并探讨了其发挥溶瘤效应的作用机制，为后续开展应用CIK介导rVV治疗大肠癌的体内研究和临床前应用奠定了实验和理论基础。