| Background and objective:Sepsis is one of the common complications and causes of death after severe burns.It is the difficulty and key point in clinical treatment of severe burns.Wound infection is the leading cause of burn sepsis:after burn,the protective barrier of skin is destroyed,microbes invade from the wound,and the necrotic tissue provides a suitable growth environment for the pathogens,which makes the wound prone to invasive infection and further aggravates can cause sepsis.It is confirmed that the incidence and mortality of burn sepsis are significantly related to the size of wound and degree of burn.In the early stage of sepsis,the main manifestations of inflammation and immune response are systemic excessive inflammation,tissue necrosis and organ dysfunction caused by the"inflammatory storm"is the main cause of death.The immune status of sepsis gradually changed to compensatory anti-inflammatory response syndrome and immunosuppression based on anti-inflammatory reactions.The body is prone to secondary infection and injury,which is also the main cause of death in late sepsis.Therefore,the wound inflammatory response and immune status of body play a key role in the treatment and prognosis of burn sepsis.Dendritic cells(DCs)are the only antigen-presenting cells that can activate the primary T cells in the body,and there are a variety of subpopulations of DCs settle in skin tissue.Studies have confirmed that sepsis can lead to changes in the quantity and function of DCs,which is an important part of immunosuppression.However,at present,there are few reports about the changes of DCs and cytokines in burn wounds and their relationship with the immune state of the body.We have done some experimental research on this topic.But at present,there are few reports about the changes of DCs and cytokines in the burn wounds and immune organs and their relationship with the immune state of the body.This project will conduct an experimental study on this issue,provides help for treatment of burn wounds with sepsis and the regulation the immune status.Methods:1.Grouping and treatment of animals:Male SPF BalB/c mice of 8 to 12 weeks were randomly divided into three groups:control group(shame:20%TBSA skin at the back contacted with water bath at 37°C for 9 seconds),burn group(burn:20%TBSA skin at the back exposed to a 90°C water bath for 9s),scald and infection group(sepsis:20%TBSA skin at the back,90°C water bath for 9s,after the wound was cooled,50ul of Pseudomonas aeruginosa with a concentration of 1×10~6CFU/ml was injected subcutaneously at in the wound center).2.Observe the appearance and behavior characteristics of the three groups of mice,record the survival rate of the mice every 24 hours until an week,and record the body weight of the mice every 24 hours,record 96 hours.3.Detect the serum levels of aspartate aminotransferase,alanine aminotransferase,urea nitrogen,and serum creatinine in three groups of mouse peripheral blood to compare the liver and kidney function.4.Take three groups of mice’s organ tissue(heart,liver,spleen,lungs,kidneys).After grinding,centrifuge to take the supernatant.Dilute at a ratio of 1:10 and draw100ul of the diluted solution and spread evenly on the blood culture plate.After being cultured at 37°C incubator for 24 hours,two individuals independently counted colony formation and recorded the results.5.Observe the histopathological sections of the dorsal and dermal tissues of the three groups of mice.6.The skin tissues,liver and spleen of the three groups of mice were taken at24h,48h and 72h respectively.The mRNA expression level of IL-6,IL-10,IL-12,IL-15 and TNF-αin the above tissues were detected by qRT-PCR.7.Peripheral blood,wound skin tissue,spleen and lymph nodes(bilateral subphrenic and bilateral inguinal lymph nodes)of three groups of mice were collected at 24h,48h,and 72h to prepare single cell suspensions.CD4~+T cells,CD8~+T cells,CD11c~+DCs,and CD11c~+CD86~+DCs in the above mentioned tissues were detected by cytological analysis.Results:1.The general condition,independent activity and response to external stimuli of the mice with sepsis were significantly worse than those in the control group and the burn group.Body weight continued to drop significantly,decreased by about 13%at72h,the death rate was 60%to 80%.The other two groups were generally in good condition;the body weight was stable and the death rate was not abnormal.2.The epidermis of the sepsis group have a large number of necrotic adhesions,the skin appendages such as hair follicles are basically necrotic.The collagen fibers in the dermis are disordered,a large number of vacuoles were formed.Muscle fibers of the muscularis muscles disappeared,the cells disintegrated and necrosed,a large number of polymorphonuclear cells infiltrated.3.The bacteria quantification in each organ of the sepsis group was significantly higher than that of the other two groups,and the difference had significant statistical significance,among them,the content of the spleen was the highest,about 10 times the lowest one,content of the heart.The organs of mice in the control group and the burn group have low levels of bacteria.4.The liver and kidney function of mice in the sepsis group decreased significantly,ALT,the liver function index,increased to 3~4 times of the control group,and the AST was 2~3 times of the control group.BUN and Scr the renal function index,increased to 2~3 times of the control group.5.The expression of cytokine mRNA in the mice tissues of the sepsis group increased significantly,and the expression level of IL-6,IL-12,TNF-A and IL-15increased obviously in 48 hours,and there was a significant difference from the control group.The expression of IL-6 in the spleen was 5-fold higher in the control group,and the highest level in the skin could reach 10 times that of the control group.The expression level of IL-10 in the wound tissue and liver tissue increased continuously,about teice as much as the control group,and the expression level in the spleen tissue was relatively stable,about 3 times that of the control group.6.The proportion of T cells in the peripheral blood,spleen and lymph nodes of mice infected with scald was significantly decreased,and the radio of CD4~+T cells/CD8~+T cells continued to decrease and reached a low level at 72 h.The proportion of T cells in the burn group was between the other two groups,and there was no significant change in the ratio of CD4~+T cells/CD8~+T cells.The proportion of T cells in the skin tissue of the sepsis group continued to increase,which was significantly higher than that of the control group.The ratio of CD4~+T cells/CD8~+T cells reached a maximum at 24 h,which was approximately twice that of the control group and decrease continued at 48 h and 72 h,there was no statistical difference with the control group.T cells in the burn group continued to increase,but were lower than those in the sepsis group,and the ratio of CD4~+T cells/CD8~+T cells had no significant changes.7.The percentage of dendritic cells in the peripheral blood of the burn group and the sepsis group continued to decrease,and the sepsis group decreased more significantly.The difference between the two groups and the control group was statistically significant.The proportion of CD86~+dendritic cells was higher at 24 h and 48 h than in the control group,up to 30%at 48 h and 20%at 72 h.The proportion of dendritic cells in the wounded skin of burn group and sepsis group continued to increase,and the differences were statistically significant.The proportion of CD86~+dendritic cells was consistent with that of peripheral blood,reaching the highest at 48h and decreasing at 72 h,to a lower level.The dendritic cells in the spleen of the sepsis group were higher than those of the control group at 24h and 48h.There was no statistical with the control group at 72h.The proportion of CD86~+dendritic cells was the highest at 24h,which was about 30%,which decreased to 20%or less at 72h.The proportion of dendritic cells in the sepsis group was lower than that in the control group,and the difference was statistically significant.The proportion of CD86~+dendritic cells also decreased continuously,and were significantly lower than the burn group and the control group at 48h and 72h.Conclusion:1.The burn wound sepsis mouse model established in this experiment is stable and effective.2.The rapid development of early inflammatory reaction in burn wound sepsis mice including cell immune and non-specific immune response,After 48 hours,cellular immunity weakened,dendritic cell differentiation was impaired,pro-inflammatory cytokine expression levels decreased,but anti-inflammatory responses continued to increase.3.The cellular immunity in the spleen tissue of burn wound sepsis mice was stable at24h and 48h,and there was obvious cell immune barrier after 72h,and the differentiation disorder of dendritic cells appeared at 48h. |
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