Experimental Research Of CIK Cells-Mediated Recombinant Vaccinia Virus RVV-CCL5-SSTR2-Luc+ In Targeted Colorectal Cancer Treatment In Vitro

Colorectal cancer,including colon and rectal cancer,is one of the most common gastrointestinal malignancies.Surgical resection is the most effective and most important means of treating colorectal cancer;however,the on set of colorectal cancer is concealed,and there is no special clinical manifestation at the early stage,so when the colorectal cancer patients are found to be mostly advanced.As an integral part of tumor comprehensive treatment,bioimmune therapy can be used independently,but also with the combination of surgery and radiotherapy and chemotherapy,with high efficacy,specificity and small adverse effects,has become one of hot topics in the field of cancer treatment research.Science's selection of the six most promising scientific fields in 2013 included tumor immunotherapy.Among them,the application of oncolytic virus in tumor immunotherapy has attracted much attention.Vaccinia virus has wide host range;large genome can accommodate the large fragment of exogenous DNA,suitable for expresses many genes;its immunogenicity can enhance the host immune destruction of the tumor;they can travel systemically through the blood,vaccinia virus can target transport to tumor tissue.Recombinant vaccinia virus has deleted TK can selectively replicate with in and lyse cancer cells,so the purpose gene can bepreferably expression in the local of tumor.Thus the Vaccinia virus is an ideal gene therapy vector in gene therapy of cancer and will have a promissing prospects in cancer gene.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.CCL5 can chemotaxes a variety of white blood cells such as T cells,monocytes,NK cells,eosinophilic/alkaline granulocytes infiltrating into tumor tissue,and strengthen the antitumor immune response of the host,which is involved in the development,progression and outcome of tumor play an important role in becoming a new research hotspot.Somatostatin(SS)is a widely distributed hormone in the human body,through the target cell membrane somatostatin receptor(SSTR)mediated,negative regulation of a variety of physiological functions.SSTR has five subtypes,of which type 2 receptor(Somatostatin receptor 2,SSTR2)is considered the most closely related to the tumor.Somatostatin analogue(SSA)can inhibit tumorigenesis through SSTR2,induce tumor cell apoptosis,and participate in immune system regulation,which is of great concern in tumor therapy.In this study,a vaccinia virus which coexpresses CCL5 and SSTR2 genes rVV-CCL5-SSTR2-Luc+(rVV)was 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 eukaryotic expression vector pSC65-CCL5-SSTR2-Luc was constructed and the rVV was obtained by homologous recombination.The best experimental protocol of CIK as carrier load rVV was determined by in vitro experiments.This provide strong support to evaluate the efficacy of CIK as a vector-mediated rVV in the treatment of colorectal cancer and to explore its mechanism of oncolytic effect,and lay a solid experimental and theoretical basis for further in vivo research and clinical research.Research results:1.Successfully constructed eukaryotic expression vector of recombinant vaccinia virus pSC65-CCL5-SSTR2-Luc and its control pSC65-Luc.Recombinant vaccinia virus rVV-CCL5-SSTR2-Luc+ and its control rVV-Luc+ was obtained by homologous recombinant method.2.Determine the multiplicity of infection 50(MOI = 50)and infection time 12h (T = 12h)as the best experimental protocol of the CIK cells load rVV.