| Ischemia/reperfusion(i/r)injury is a common pathological phenomenon in clinic,which not only seriously affects the process of surgery,but also has a very bad effect on the prognosis of patients.At present,the treatment methods of liver i/r mainly focus on physical methods,drug pretreatment and post injury drug treatment.Although these treatment methods can play a certain role,they all have some defects.Therefore,how to provide a safe and effective protection and treatment for liver i/r patients is a hot topic in the current research.Therefore,a large number of studies have found that ADSCs exo from adipose stem cells can play an effective role in the repair of many tissues and organs,but the role of ADSCs exo from allogeneic sources in liver I/R is not clear.In this study,the protective effect of ADSCs exo on I/R of rat liver was studied by using the i/r injury model in rats,and the mechanism of ADSCs exo was studied preliminarily,which provided a new and effective scheme for the treatment of I/R.Part 1 Isolation and identification of exosomes derived from rat adipose derived stem cells【Objective】The rat adipose derived stem cells(ADSCs)were collected by collagenase digestion method.The exosomes were extracted from cell culture medium and identified,which laid the foundation for the next experiment.【Materials and methods】The adipose tissue(groin)of rats was taken out and treated via type I collagenase.Then adipose derived stem cells were extracted according to the extraction steps.After the culture was stable,the specific markers on the surface of ADSCs were identified,and differentiation ability of ADSCs was detected under different medium that could iduced cell differentiation.ADSCs-exo was collected from the culture supernatant of ADSCs,and then ADSCs-exo were identified by electron microscopy,particle size analysis and western blotting.【Result】1.Microscope observation showed that after culture 4-5 days,the cell arranged in a spiral or radial shape and had high density;after 7 days of culture,the growth morphology of cells was like typical fish,indicating that the isolated cells were ADSCs.2.For ADSCs,flow cytometry revealed that ADSCs were positive for CD90,CD44,CD73 and other positive markers,while there were several negative markers were negative like CD45,CD11b,CD34 and so on.3.ADSCs successfully differentiated into adipocytes,osteoblasts and chondrocytes.4.Electron microscopy showed that ADSCs exo was a typical shape of oval or round small capsule,which was a bilayer lipid membrane with a diameter of50-100nm.5.The particle size analysis revealed that the median volume specific gravity of ADSCs-exo was 164.5;the median diameter distribution of ADSCs-exo was 125.6nm;the actual measured value of particle concentration was 2.4x 10~7/ml.6.Western blotting showed that ADSCs-exo could express CD9,CD63 and TSG101.【Conclusion】ADSCs-exo was successfully isolated in this experiment,and the exosomes met the relevant identification standards,which could be used in the next experimental study.Part 2 Protective effect of ADSCs-exo preconditioning on hepatic ischemia-reperfusion injury in rats【Objective】Hepatic ischemia/reperfusion(I/R)injury is a common clinical pathological phenomenon,which not only seriously affects the process of surgery,but also is extremely adverse to the prognosis of patients.At present,70%rat liver warm ischemia-reperfusion model was established by reversible occlusion of the left middle lobe of liver with non-invasive vascular clamp.The protective effect and mechanism of ADSCs exo preconditioning on liver ischemia-reperfusion were studied by serum enzyme detection,histopathological analysis and Western blotting.【Materials and methods】The rats were randomly divided into sham operation group(sham group),PBS+I/R group and ADSCs exo+I/R group.In sham group,the blood vessels were opened without clamping,and in PBS+I/R group,50%of the blood vessels were injected through portal vein before ischemiaμL PBS,ADSCs exo+I/R group were injected 50mg/L via portal vein before ischemiaμL ADSCs exo suspension.Blood and liver tissues were collected at 2 h,6 h,12 h and 24 h after liver reperfusion,and then the following experiments were carried out:1.Alt,AST,LDH,camp,TNF-a and IL-1 in serum were determined?;2.MDA,SOD and ROS in liver were detected;3.Liver sections were prepared for H-E,TUNEL and immunohistochemical staining;4.Bcl-2,Bax,ERK1/2,p-ERK1/2 and GSK3 were determined?And p-gsk3?And the expression of signal proteins.【Result】1.The results of serum enzyme test in sham group,PBS+I/R group and ADSCs exo+I/R group showed that the levels of alt,AST,LDH and IL-1 in PBS+I/R group at 2 h,6 h and 12 h after reperfusion?The levels of alt,AST and LDH in serum of ADSCs exo+I/R group were significantly lower than those of PBS+I/R group(P<0.05)?Compared with PBS+I/R group,the levels of TNF-a and TNF-a in PBS+I/R group were significantly lower(P<0.05).2.The histopathological results of liver tissue at 6 h and 24 h after reperfusion showed that PBS+I/R group had obvious liver injury,ADSCs exo+I/R group had significantly reduced the degree of liver necrosis(H-E staining),significantly reduced the number of hepatocyte apoptosis(TUNEL staining),and significantly improved the morphology of hepatocyte mitochondria and other organelles(cell ultrastructure under transmission electron microscope).3.The results showed that the levels of MDA and ROS in PBS+I/R group were significantly higher than those in sham group(P<0.05),while the levels of SOD in PBS+I/R group were significantly lower than those in sham group(P<0.05);Compared with PBS+I/R group,the levels of MDA and ROS in liver tissue of ADSCs exo+I/R group were significantly lower(P<0.05),but the level of SOD was significantly higher(P<0.05).4.The level of camp at 2 h and 6 h after reperfusion in the ADSCs exo+I/R group was significantly higher than that in the PBS+I/R group(P<0.05).5.Western blotting results showed that compared with PBS+I/R group,p-ERK1/2and p-gsk3 in ADSCs exo+I/R group decreased at 2 h and 6 h after reperfusion?The expression was significantly increased(P<0.05).【Conclusion】ADSCs-exo can protect and repair liver I/R by increasing the phosphorylation levels of p-ERK1/2 and p-GSK3?to prevent I/R injury.Using ADSCs-exo to prevent liver I/R has a good clinical transformation and application prospect,which provides a new strategy and candidate for the treat of I/R. |
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