Experimental Study Of Tumor Cell Death Mediated By Recombinant Virus And Its Macropinocytosis Behavior

For the past few years,viral vector has been widely applied in gene therapy and vaccine development by virtue of the molecular mechanism of entering into other cells for infection by virus transmiting genome.Therefore,virus vector optimization and transformation has become R&D focus of relevant treatment means.Virus recombinant construction technology and its internalization cellular entry ability are two key research directions for further developing into viral vector.Currently,pancreatic cancer which has been listed as one of the top ten malignant tumors shows a growing tendency of its morbidity and mortality.However,no effective measures have been taken for good therapeutic methods and effectiveness,so it is often called "king of cancer.With the deepening studies on cancer,gene therapy and cell therapy have become effective new-type treatment methods.Therefore,this research has recombinantly constructed adenovirus and poxvirus,evaluated their pancreatic cancer inhibiting ability and virulence.Next,this paper analyzed the internalization cellular entry ability of viruses according to the size and endocytosis mode,discussed explore the potential of further developing two viruses into viral vectors.First,adenovirus as an oncolytic virus has a certain ability of suppressing tumor growth,and plays a role of further killing tumor by acting as a viral vector and carrying other foreign genes.Secondly,poxvirus without direct killing effect on tumors can also be used as a viral vector carrying other foreign aid genes to kill tumors.Third,viropexis mode is determined by its particle size,receptor selection,host cytotaxis,and transmission pattern.Generally,adenovirus has a partical size of 70-90 nm,but the poxvirus is large with a diameter of up to 300 nm.Virus size is a key index for endocytosis mechanism study.However,due to virus species preference,high infectivity and other characteristics,it is hard to conduct extensive experiments now by using viruses.In this reserach,mesoporous silica nano particales(MSNs),a nanomaterial with flexible and controllable size,uniform heigh and strong internalization ability,was selected as a substitute for virus endocytosis to synthesize MSNs with different sizes.TEM was used for size verification.The cellular entry of MSNs with different sizes was investigated to determine the size scope of the internalized substances by tumor cell macropinocytosis.According to the results,three MSNs were successfully synthesized in this research,with cell size of 53 nm approximating to adenovirus,270 nm approximating to poxvirus and 420 nm.By the research which can be preliminarily concluded that adenovirus can be endocytosed into cells by means of clathrin-mediated endocytosis and macrocytosis.To sum up,this research has found recombinant adenovirus with good pancreatic cancer inhibiting effect.The recombinant vaccinia virus toxicity to cells significant reduced after missing two gene fragments,and the virus diffusion level reduced significantly reduced in cells.Adenovirus can be endocytosed into cells by means of clathrin-mediated endocytosis and macrocytosis.But poxvirus can only enter cells by macropinocytosis means.The results of this research have provided significant experimental data support and design references for the further developing adenovirus and poxvirus as virus vectors.Meanwhile,nanomaterial is used as substitute of virus for macropinocytosis research,which can deepen the understanding of the mechanism on pancreatic cancer cell endocytosis of large substances,better understand the process of virus entering pancreatic cancer cells,and avoid the limitation to use virus for experiments,thus making studies on recombinant virus vectors with better killing effect more efficient.