Effect Of Regulation Of CH25H Expression On Mtb Survival In Macrophages

Objective: To investigate the effect of Mycobacterium tuberculosis infection on cholesterol ester in mouse macrophages.To investigate the changes of CH25 H in macrophages of mice infected with Mycobacterium tuberculosis.To construct CH25 H overexpression and interference expression vector to explore the role of CH25 H in Mycobacterium tuberculosis infection.Using bioinformatics software to analyze the structure and function of CH25 H.Methods:(1)RAW264.7 cells were divided into blank control group and infection experimental groups where the amount of bacteria and the cell number ratio is 5:1,10:1 and 20:1.The mixture of RAW264.7 cells and Mycobacterium tuberculosis was incubated for 1,3,5 and 7 days,respectively.After oil red O staining,the cell morphology of each group was observed under a microscope.Using enzymatic method to determine the level of intracellular Total cholesterol,free cholesterol and cholesterol ester of each group.(2)RAW264.7 cells were divided into blank control group and infection experimental groups where the amount of bacteria and the cell number ratio is 5:1,10:1 and 20:1.The levels of CH25 H in the cells were measured by RT-PCR and elisa after incubation for 3 days.The changes of cell structure in infected cells were observed by electron microscopy.(3)Reverse transcription PCR was used to clone human CH25 H gene from human peripheral blood mononuclear macrophages cells.The reverse transcription product was used as template,CH25 H gene was amplified by PCR method.The p CD513B-1 plasmid and CH25 H gene connected after double enzyme digestion and the recombinant plasmids were constructed.Then recombinant plasmid was transformed into Escherichia coli stabl3.Positive recombinant plasmids were selected by ampicillin screening and digestion.Screened positive recombinant plasmids were sent to the company for sequencing.(4) Selecting two target sequences based on CH25 H gene m RNA sequence.According to each target sequence,the corresponding sh RNA was designed according to the principle of si RNA design.A pair of single-stranded oligodeoxynucleotides was designed for each sh RNA.The double-stranded DNA of sh RNA was cloned into Pglv3/H1/GFP+Puro plasmid and transformed into Escherichia coli stabl3,and sent to the company for sequencing.(5)The recombinant plasmids were e transfected into THP-1 cells,green fluorescence was appeared in the cells and represent successful transfection after 30 h.Expression level was then detected by RT-PCR.The successfull transfected cells were infected by Mycobacterium tuberculosis which the bacteria and the cell number ratio is 10:1.The cells were acid-fast stained after infected with Mycobacterium tuberculosis for 48 h.Rsidual cells were lysed,then cultivated and colony count were done to determine the intracellular bactericidal activity.(6)By using bioinformatics software and website to analysis its physical and chemical properties;signal peptide;spatial structure;epitopes.Results:(1)Then the amount of bacteria and the cell number ratio of 20:1 were incubated one day,large number of lipid droplets can be seen in the cell,and its intracellular cholesterol ester content was(65.91%±2.45%).Incubated for seven days,a large number of cells were lysed,lipid droplets overflow.With the time and the amount of bacteria increasing,the content of intracellular cholesterol ester is increased.(2)The level of CH25 H in Mycobacterium tuberculosis infected macrophages increased at both transcriptional level and protein level,and was proportional to the amount of intracellular Mycobacterium tuberculosis.(3)CH25H overexpression and interference expression plasmid were sucessfully constructed.After 30 h of transfection,green fluorescence was observed.The overexpression and interference expression positive were significantly different from those in the blank control group.Compared with the blank group,the relative content of cholesterol ester in CH25 H overexpression group increased,while in the interfering expression group was decreased.The colony count showed that the intracellular bacterial loading of CH25 H was increased and the amount of bacteria in the interfering expression group was decreased.(4)The full gene is 1 378 bp in length,coding 272 amino acids.The protein had unstable physical chemical characteristics and had four transmembrane region and six phosphorylation sites.Conclusion:(1)Mycobacterium tuberculosis can induced cholesterol ester increased in macrophage.The amount of bacteria and cholesterol ester were positively correlated,and it is time-dependent.(2)Levels of CH25 H in Mycobacterium tuberculosis infected mouse macrophages was increased at both transcriptional level and protein level.Electron microscopy results show that the degree of organelle lesion is proportional to the bacterial concentration.(3)Successfully constructed CH25 H overexpression and interference expression vector,and found that CH25 H can promote the survival of CH25 H cells.(4)Understand the basic structure and function of CH25 H gene and its encoded protein.