The Construction Of Orthotopic And Subcutaneous Neuroblastoma Models And The Study Of Tumorigenesis Characteristics

Background:Neuroblastoma(NB)is a common extracranial solid tumor in children.It originates from the embryonic sympathetic renal lineage of the neural crest.It is a special and complex highly invasive malignant tumor.Although the surgical and chemotherapy methods of NB are constantly improving,it is still the main cause of death in children aged 0-5 years old,with a mortality rate of 15%of all children’s tumors.It shows typical biological,clinical,morphological and genetic heterogeneity.Its clinical manifestations are spontaneous tumor regression and no response to standardized anti-cancer therapy,which makes it the focus of clinical transformation research.At present,the research focuses on the exploration of pathogenesis,the discovery of key pathogenic factors and the development of new targeted therapeutic drugs.The establishment and development of animal models are indispensable for the exploration of pathogenic mechanism and the development and screening of new drugs.At present,NB animal model has been widely used,the most characteristic is th MYCN transgenic mouse model,which plays an important role in evaluating the driving factors of tumorigenesis and the role of related genes in tumor progression.It is also used for in vitro research results verification and drug screening.As an experimental tool,it provides a platform and convenience for researchers,but there are few This study is dedicated to explore the difference and application between subcutaneous tumor model and orthotopic tumor model.This study successfully constructed orthotopic tumor model and subcutaneous heterotopic tumor model,to explore whether there are differences between the two models in tumor formation,metastasis,tumor heterogeneity and other aspects,and try to find the reasons for the differences,so as to provide theoretical and technical guidance for the establishment of tumor models in the future According to the characteristics of the two tumor models,combined with the purpose of the study,a more suitable tumor model was selected.Objective:Objective to establish orthotopic adrenal tumor and subcutaneous heterotopic adrenal tumor models in severe combined immunodeficiency NCG mice by using neuroblastoma cell line SH-SY-5Y.To study the tumorigenesis,development characteristics and potential mechanisms of the two NB mouse models,and to explore the biological characteristics of the tumor,so as to select the appropriate animal models according to the follow-up research purposes and provide reference for the establishment of related tumor models And anti-tumor treatment research to provide new treatment ideas and strategies.Methods:SH-SY-5Y cell lines stably expressing Luc-ds Red were constructed.The cell suspensions of 1×10~5/20(?)l and 1×10~5/100(?)l were inoculated into the subcutaneous tissues of the left adrenal gland in prone position and the back near the right hind limb of NCG mice to establish orthotopic and subcutaneous heterotopic tumor models.The weight,tumor growth,tumor metastasis,survival and pathological changes of tumor tissue were observed and recorded.The myeloid derived suppressor cells(MDSCs)in peripheral blood of the two tumor models were detected by flow cytometry,in order to explore the molecular mechanism of the difference between the two tumor models,we digested the tumor of the first batch of model mice into single cells,which were used as the source of tumor cells of the second batch of mice.RT-PCR was used to detect the expression of several key molecules related to the occurrence and development of neuroblastoma.Results:1.SH-SY-5Y cell line expressing Luc-ds Red was successfully constructed.2.Two batches of orthotopic tumor models and subcutaneous heterotopic tumor models of neuroblastoma were successfully constructed,and the tumor formation rate was100%.3.Compared with subcutaneous transplantation tumor model,orthotopic transplantation tumor model has a higher tumor formation rate,tends to implant metastasis,mostly to the mesentery and intestine,the survival time of mice is short,most of them die of bloody ascites,while subcutaneous transplantation tumor tends to vascular metastasis,mostly to the lung,the survival time is relatively long.4.The content of MDSC in subcutaneous and in situ tumor bearing mice was similar at14 days,and the content of MDSC in situ tumor bearing mice was higher than that in subcutaneous tumor bearing mice after 14 days.5.Cyclophosphamide can effectively control the growth and development of Nb.Compared with tumor cells from in situ,tumor cells from subcutaneous origin have more growth advantages after transplantation into mice subcutaneously;compared with tumor cells from subcutaneous origin,tumor cells from in situ origin have more growth advantages after transplantation into situ.6.The results of real-time quantitative PCR showed that compared with in situ tumor,Phox2b(P=0.0002)and GAB2(P<0.0001)in subcutaneous tumor were significantly down regulated.Conclusion:The model of subcutaneous and in situ tumor transplantation was successfully constructed.It was proved that compared with subcutaneous inoculation,in situ inoculation had a faster rate of tumor formation.With its tumor growth environment,it could better simulate the development law of human NB tumor,and provide theoretical and technical guidance for the development and metastasis of neuroblastoma,and could be based on the difference between the two models and the purpose of the study The Nb model is more suitable.