Primary Study Of SLC35E2B Gene In Mycobacterium Infection And Its Effect On Macrophage

Background:Solute Carrier(SLC)35 family is mainly located on the endoplasmic reticulum and Golgi body membrane,which is responsible for transporting ribonucleotides and participating in the nucleotide metabolism process.Our previous study found a high frequency mutation of SLC35E2B gene in patients with leprosy.This study aims to clarify the expression and localization of SLC35E2B gene in mycobacterium infected skin lesions,and explore the regulatory role of SLC35E2B gene in macrophage polarization and autophagy at cellular level,as well as the influence of SLC35E2B gene defect on macrophage polarization,autophagy and phagocytic functions.Furthermore,our study aims to understand the effect of SLC35E2B gene on macrophage function and provide a theoretical basis for the pathogenesis of mycobacterium infected skin disease.Objective:Clarifying the expression and localization of SLC35E2B gene in mycobacterium infected dermatosis;Investigating the effect of SLC35E2B gene on macrophage function.Methods:1.The expression of SLC35E2B in mycobacterium infected skin lesions was detected by immunohistochemical staining;SLC35E2B was co-stained with CD68 and S100 by immunofluorescence staining,and the localization of SLC35E2b in mycobacterium infection skin disease was preliminaryexplored.2.RAW264.7Macrophages were cultured and silences of SLC35E2B gene in RAW264.7 macrophages were induced by siRNA.MRNA and protein expression levels of polarization marker molecules and autophagy related molecules in the two groups were detected by qRT-PCR and Western-blot.The phagocytic function of macrophages was detected by neutral red method and FITC-dextran uptake experiment.Result:1.Immunohistochemical results showed that the expression of SLC35E2B in mycobacterium infected skin lesions was significantly higher than that in healthy controls,and the difference was statistically significant.Immunofluorescence staining showed that SLC35E2B and CD68 expression overlapped.2.After SLC35E2B gene silencing,qRT-PCR detection showed that the M1-type polarization marker was significantly decreased in the mRNA level of macrophages,while the M2-type polarization marker was significantly increased;Western-blot analysis also found that the protein level of M1-type marker molecule was significantly decreased,and the protein level of M2-type marker molecule was significantly increased.3.After SLC35E2B gene silencing,qRT-PCR and Western-blot detection showed no significant difference in the expression of autophagy-related factors on mRNA and protein levels in macrophages.4.Neutral red method and FITC-dextran uptake experiment showed no significant difference in phagocytic function of macrophages after SLC35E2B gene silencing.Conclusion:SLC35E2B was highly expressed in mycobacterium infected skin diseases and mainly localized in macrophages.S1C35E2B gene silencing can inhibit the polarization of macrophages to M1 type and promote the polarization of macrophages to M2 type,without affecting the phagocytosis and autophagy function of macrophages.