Development Of TRAP System To Analyze The Translatome Changes Of Paraventricular Nucleus And Pituitary Induced By LPS

Lipopolysaccharide(LPS),also known as endotoxin,is the main substance causing Gram-negative bacteria to cause inflammation in the host,often used to establish a systemic inflammation model.Systemic inflammatory response is a common pathological process.Excessive inflammatory response can cause severe organ failure.Therefore,studying the mechanism of the immune system in the inflammatory response would guide the prevention and control of infectious diseases.A large number of studies have proved that the immune system and the neuroendocrine system form a neural-endocrine-immune network to coordinate and maintain the body's homeostasis.The hypothalamus-pituitary system is the core system that regulates the neuroendocrine response.The system integrates the neuroendocrine function,Participate in various biological processes.The axons of a large number of neurons in the paraventricular nucleus(PVN)of the hypothalamus project to the posterior pituitary gland to control the hormone secretion of the pituitary gland.A systematic study of the differences in gene expression at the translatome level between PVN and pituitary in the inflammatory response is of great significance to reveal the role of the neuroendocrine system in the inflammatory reaction.The effect of translation regulation on protein abundance accounts for 54%of all regulation,so studying translatome can help people better understand various physiological regulation processes better.The main purpose of this study is to develop a set of tools that can be used to analyze the information of translatome,and analyze the differential gene expression of PVN and pituitary at translatome under the stimulation of LPS.The research content of this project mainly includes the following aspects:1. Developing the primary r AAV-TRAP system,and verify the feasibility of the system at the cell level. In the early stage,Translating Ribosome Affinity Purification(TRAP)was combined with Ribosome Profiling(Ribo-seq),and we tried to construct c DNA library with footprinted m RNA fragments.However,this method requires an initial cell volume of at least 7.5×10~6 cells,which cannot meet the requirement of experiments in vivo.Therefore,it was decided to use the traditional SMART library construction method.It was proved that the SMART library construction method can reduce the initial amount of cell to 2×10~3 by experiments,which meets the requirements of experiments in vivo.2. Developing the second generation r AAV-m APP-TRAP system. At the cellular level,it was verified that TRAP can successfully purify the m RNA being translated,and the method of establishing a c DNA library of oligo cells was determined.Subsequently,the initial r AAV-TRAP system was appropriately modified.On the premise of ensuring the function of the TRAP system,a m APP sequence containing a presynaptic membrane protein sequence was added to the N-terminal of the fusion protein Fc NBL10,The TRAP system could be located from PVN to the pituitary by this sequence.With the help of this system,the translatomic information of the PVN and pituitary can be analyzed.3. It was proved that r AAV-m APP-TRAP could be successfully expressed in mice by observing the fluorescence of vibratome section of mouse brain tissue. Subsequently,we analyzed the differential genes experience of the PVN and pituitary at translatomic level by the LPS treatment.Both the PVN and pituitary activate a large number of inflammation-related endocrine pathways treated by LPS:the oxytocin signaling pathway,thyroid hormone signaling pathway,glucagon signaling pathway and estrogen signaling pathway are activated,and the hormones such as oxytocin(PRL)and inflammatory factors such as IL-1?and TNF?are up-regulated in the PVN;the thyroid hormone synthesis pathway,oxytocin signaling pathway,gonadotropin releasing hormone signaling pathway,cortisol synthesis pathway and prolactin signaling pathway are activated,the oxytocin(PRL),thyroid stimulating hormone releasing hormone(TRH)and IL-17 and other immune related substances are up-regulated in the pituitary.Other inflammation-related pathways co-activated in PVN and pituitary include circadian rhythm,heat production,and fat metabolism and so on.In this study,we use transtomcic technology to analyze the translatomic information of the PVN and pituitary in the inflammation induced by LPS,and the information would help to understand the regulating function of PVN and pituitary in the endocrine and immune process,and providing a theoretical basis for preventing and controlling bacterial inflammation.