Preliminary Study On The Expression Of HSP70 And Its Regulation By HSF1 Under Inflammatory Conditions In Grass Carp

In mammals,heat shock protein 70(HSP70)is a multifunctional cytokine.It is produced with large amount by cells in a short time when they are stimulated by stressors.As a molecular chaperone,HSP70 can maintain the survival of cells,and has the function of protecting cells.HSP70 is also involved in the regulation of cellular immunity and plays a key role in immunity.Heat shock transcription factor(HSF)is a key regulator of HSP expression.There are four members of the HSF family,include HSF1,HSF2,HSF3 and HSF4.Among them,HSF1 is the most important member of the HSF family and is responsible for the regulation of the expression of heat shock proteins.Dysfunction of HSF1 is related to various immune diseases and organ development.The study on HSP70 in teleost is limited to the response of cellular HSP70 expression at mRNA levels and protein levels to immune stimulation.However,in vivo,how HSP70 protein responds to immune stimuli has not been studied.In this thesis,specific antibody of grass carp HSP70 was identified,and then was used to explore HSP70 expression in immune tissue by immunohistochemistry(IHC).Results showed that the expression of HSP70 protein was significantly up-regulated in head kidney lymphocytes and mesangial epithelial cells in grass carp kidney infected by Aeromonas hydrophila(A.hy)after 24 hours.HSP70 protein expression was increased in above cells until 72 hours,while that in spleen reached its peak response at 24-hour after A.hy infection and then was decreased to the basal level.However,the expression of HSP70 protein,was undetectable in the liver of grass carp infected by A.hy compared with control group.These results not only indicate that HSP70 rapidly participates in the immune response in grass carp,but also reflect that HSP70 responds differently to the immune stimuli in different organs.We cloned the coding sequence(CDS)of grass carp HSF1,which is 1590 bp long and encodes 529 amino acids.Bioinformatics analysis revealed that HSF1 of grass carp has a DNA binding domain and a C-terminal activator domain.Grass carp HSP70 promoter sequence was cloned,bioinformatics analysis revealed that grass carp HSP70 promoter contains five heat shock elements(HSE).Results showed that under the induction of LPS,HSF1 can significantly up-regulate the expression of HSP70 gene and HSP70 protein,increase the activity of HSP70 promoter.Interestingly,without immune stimulation,expression of HSF1 could promotes the expression of HSP70 genes and proteins,which is different from those in mammals to show that teleost HSF1 may be specific for the transcriptional regulation of HSP70.In summary,this thesis explored the distribution and expression of HSP70 in different grass carp immune tissues infected by A.hy,the regulation mechanism of HSF1 on HSP70 expression at mRNA and protein levels,providing a basis for further elucidation of the immune functions of teleost HSP70 and HSF1.