Response And Mechanism Of Insect Primary Cells To Microenvironment Ex Vivo

Insect cell lines are important experimental materials for the study of insect and agricultural biology in vitro.They are excellent vectors for the propagation of insect viruses in vitro,and can be used for high-throughput drug screening,as well as research and development and production of new biological insecticides.They have great scientific and economic value.Most existing cell lines derived from insect ovaries have been established by ex vivo fragmentation,culture,and passage of ovarian tissue,leading to uncertainty about their origin and cell type.In addition,in the process of ex vivo culture of primary cells,cells often appear proliferation stagnation phenomenon can not be normal passage,resulting in the establishment of cell line failure.Up to now,the mechanism of ex vivo transformation of insect primary cells is still unclear,which leads to problems such as low cell transformation rate and difficulty in building insect lines.In this study,the ovarian tissues of pupae such as Mythimna separata and Spodoptera exigua were used as the research materials.According to the tissue culture time ex vivo,combined with the structure and development rules,the main cell types of the primary cells were identified.The proliferation and death of these cells ex vivo were investigated.At the same time,the research of insect cell culture conditions in vivo and ex vivo microenvironment were compared,and test its response to the external pressure,combined with the transcriptome sequencing technology,analyzes the expression differences significant pathways and key genes,in insect cells ex vivo molecular mechanism of response to environmental changes in the environment has carried on the preliminary analysis.The results can be seen in the following paragraphs:(1)The primary cells isolated from the pupae ovarian tissue of Mythimna separata were somatic cells from the lumen cells between the lamina propria and the ovariole proper in the growth region of the ovarian tube.Ex vivo,most proliferating cells were distributed in the growth area and reproductive area of the ovarian tube.With the extension of ex vivo culture time,the number of cells in mitosis in the ex vivo tissue was significantly reduced,and the proliferating ability was significantly decreased.As a result,after a brief increase in the number of cells that have left the tissue,they do not have the ability to proliferate continuously.(2)Insects ex vivo culture of dissolved oxygen content is significantly higher than in insects,primary cell in an airtight,after long-term culture bottle original raise all sorts of metabolic activity consumes large amounts of oxygen,nutrient solution in the dissolved oxygen content decreased significantly,gradually close to the body's oxygen environment,at the same time because of some of the metabolic activity in cells produced many metabolites make cultivation environment acidification,the p H in the medium also gradually decreased.(3)After the ovarian tissue is isolated ex vivo,due to the stimulation of high concentration of oxygen in the culture environment ex vivo,a large number of reactive oxygen species(ROS)are produced in the cells,leading to cell damage.Mitochondrial membrane potential(MMP)decreased,which destroyed the intracellular redox balance and stimulated autophagy to protect itself through autophagy reaction,but did not cause cell apoptosis in the short term.Cells undergoing autophagy may promote self-transformation and become immortalized cell lines by activating anti-apoptotic and tumorigenesis mechanisms in the cells.(4)The transcriptional level of primary cells cultured for a period of time in ovary of S.exigua was compared with that of primary cells cultured for a period of time ex vivo.The pathways with significant changes mainly included lysosomal pathway,phagocytic pathway,autophagy pathway,antioxidant pathway,apoptosis pathway and so on.The expression of superoxide dismutase(SOD)in the antioxidant system was significantly down-regulated,catalase(CAT)was significantly up-regulated,the expression of Atg8,a marker gene for autophagy,was also significantly increased,and the expression of TOR,a major inhibitor of autophagy pathway,was significantly down-regulated.The expression level of apoptosis inhibitor protein DIAP1 in apoptosis pathway was also significantly increased,suggesting that cells may activate anti-apoptotic mechanism in response to oxidative stress.In this study,we preliminarily discussed the response of insect cells to environmental changes caused by the changes of culture environment,and preliminarily inferred the change rules of the signal pathway of primary cells ex vivo from the transcriptional level,which provided an important reference for further research on the mechanism of insect primary cell transformation.