| Objectives:The tree shrew model of multiple organ dysfunction syndrome(MODS)was established by blood loss,simulated infection combined with hindlimb extrusion method.Using the preparation method of highly active umbilical cord mesenchymal stem cells(HA-UCMSC)established by our laboratory,the standardized HA-UCMSC was isolated,expanded,characterized and employed for the therapy of the MODS model.The effectiveness of HA-UCMSC treatment was evaluated through blood cell counting,biochemical assays and histological analysis,and the underlying mechanism was further investigated by serum proteomics,so as to provide novel insights into the effective treatment of MODS patients.Methods:1.Establishment of the MODS tree shrew model:Tree shrews were subjected to the combined treatment of bleeding,comminuted fracture of the left femur and lipopolysaccharide(LPS)induction..With 7%of the body weight per tree shew being the total blood volume,30%of the total blood volume was taken from the left rear femoral vein which was followed by 10mg/Kg,20mg/Kg,30mg/Kg,40mg/Kgand 50mg/Kg of LPS administered intravenously,Finally,each group was given a comminuted fracture of the left femoral with the same degree of injury.By monitoring body temperature,48h blood count and blood biochemistry,the mortality rate and pathological changes were observed The optimum dose of LPS(50mg/Kg)was obtained by body temperature monitoring,48h blood cell counting and blood biochemistry assays,and this dose was used for the establishment of the MODS model;model establishment:take 8-month-old healthy adult female tree shrews as the research object,under anesthesia and fixed conditions,blood loss 30%,intravenous injection After LPS 50mg/Kg,the left femur was clamped until the fracture was broken,and the degree of fracture was basically the same according to the anatomical observation.Under conventional feeding conditions,the survival and changes in anal temperature were dynamically observed;48 hours of tree shrew arterial blood was taken for blood cell count and blood biochemical index determination;48 hours after modeling,the heart,liver,spleen,lung,kidney,stomach,and small intestine were taken for measurement.Tissue sections,staining,and observation of histopathological structural changes.Model evaluation criteria:mortality rate>20%within 48 hours,listlessness,significantly increased blood inflammatory cells,elevated C-reactive protein,blood biochemical indicators and pathological tissue structure observations found damage to two or more organs including the heart and liver.2.Preparation and identification of tree shrew UCMSC and HA-UCMSC:(1)Under sterile conditions,the umbilical cords of tree shrew fetuses near the delivery period were collected by laparotomy,the umbilical cord capsule was peeled off,and the residual blood of the umbilical cord was washed away,then transferred to 1.5 m L EP tube,cut up the umbilical cord tissue to a paste.Add 0.5 m L each of Te SR-E8containing 5%Clone R and DMEM containing 10%FBS,and transfer them to LN521-coated HA-UCMSC culture flasks and UCMSC common culture flasks(T75cm~2)to supplement the corresponding culture Shake to disperse evenly on the bottom wall of the bottle;transfer the culture bottle to a 37°C,5%carbon dioxide incubator for cultivation.Observe and record the cell growth under the microscope every 3 days,and change the medium 5m L;when the cells in the tissue block grow to the surrounding,radially covering the bottom of the bottle around the tissue block,digest with 0.25%trypsin(EDTA),Cells were collected,centrifuged and washed;the cells were resuspended in the corresponding culture medium,and inoculated into corresponding T75cm~2culture flasks at a seeding density of 5000 cells/cm~2for subculture to P2 generation;continued subculture and e GFP labeling,The labeled P3HA-UCMSC were centrifuged and washed to prepare a cell suspension at a concentration of 2.5×10~6/m L and stored at 4°C for later use.(2)Use the"OLYMPUS Provi CM20"instrument to observe the changes in the number and degree of cell confluence of two types of cells with the same initial seeding density for 7consecutive days;(3)Flow cytometry analysis of cell cycle and expression of CD73,CD90,CD105,SSEA3,SSEA4,the positive rate of TRA-1-60 cells;(4)Western Blot analysis of the expression levels of Nanog,OCT-4,and SOX2;(5)Induction of the two cells using standardized adipogenic,osteogenic,and chondrogenic induction media For differentiation culture,the induced cells were stained with Oil Red O,Alizarin Red and Alcian Blue staining methods respectively,and the adipogenic,osteogenic and chondrogenic differentiation potentials of the two cells were observed under an optical microscope,and photographed and recorded.(6)Inoculate HA-UCMSC and UCMSC on glass slides coated with LN521 and gelatin respectively(slides are placed in 12-well plates),add standardized three-germ layer induction medium,and perform immunofluorescent staining of marker molecules on the induced cells.Quantitative analysis of its ability to differentiate into three germ layers,in which the endoderm marker molecules are FOXA2 and SOX17,the mesoderm marker molecules are Brachyury(T)and CXCR4,and the ectoderm marker molecules are Nestin and PAX6.(7)Soft agar clone growth experiment:adjust the concentration of He La,MRC-5,HA-UCMSC and UCMSC cells to 2000 cells/m L,mix them with0.7%agarose solution 1:1,and quickly pipette 1 m L into a 6-well plate On the solidified 1.2%soft agar,set a blank control hole at the same time.After standing at room temperature and solidifying,put it in a 37°C,5%CO2 incubator,and observe the growth of each cell clone continuously for 3 weeks.A group consisting of more than 16 cells The cell cluster is 1 clone.3.Animal grouping and HA-UCMSC treatment:(1)Animal grouping:Randomly select 10 normal tree shrews as the control group,and randomly divide the MODS tree shrew model into a model group and a treatment group,with 20 in each group,to observe the mortality rate;The MODS tree shrew model was randomly divided into a model group and a treatment group,and a healthy control group of 6 rats in each group was set up simultaneously for follow-up efficacy evaluation and mechanism research;(2)The treatment group was infused with HA-UCMSC 1.5×10 ~7per Kg,the model group was synchronously injected with the same amount of normal saline,and the control group was not treated.4.Evaluation of the curative effect of HA-UCMSC:72 hours after infusion of HA-UCMSC,blood was collected from the abdominal aorta for blood cell count and blood biochemical detection;the tree shrew was killed by bloodletting,and the heart,liver,spleen,lung,kidney,stomach,and small intestine were collected Tissue sections were stained to observe histopathological changes,and each tissue sample was reserved for mechanism research.5.Mechanism study of HA-UCMSC:(1)Tissue e GFP positive cell tracing:After HA-UCMSC treatment for 72 hours,fresh tissues from heart,liver,spleen,lung,kidney,stomach,small intestine and muscle were immediately placed in"Fusion Solo S.EDGE”chemiluminescent imager stage,trace the distribution of e GFP-positive cells in each tissue;make frozen sections of the tissue,and observe the distribution of e GFP in each tissue under a fluorescence microscope.(2)The heart,liver,spleen,lung,and small intestine were taken for TUNEL and ki67immunofluorescence staining to analyze the apoptosis and proliferation of tissue cells.(3)Western Blot method was used to detect liver IL-1,TNF-αand IL-10,and to analyze the changes in the levels of liver inflammatory factors.(4)q PCR and Western Blot were used to detect the expression levels of genes and proteins(Caspase1 and Gasdermins-D)that mediate pyroptosis in the liver,and evaluate the pyroptosis and repair of liver cells.(5)Immunofluorescent staining of CD133,MPO,CD71 and CD14 was performed on the extramedullary hematopoiesis(EMH)found in pathological tissue observation,and the expression levels of hematopoietic-related factors were analyzed.(6)Separate the serum of each group,and use DIA proteomics sequencing to conduct differential analysis on the proteomic data;through differential protein trend analysis,analyze the proteins that are first up-regulated and then down-regulated and first down-regulated and then up-regulated from the control group to the model group and then to the treatment group Function and regulation of signaling pathways,and calculate the Pearson correlation between these proteins and blood cell counts and blood biochemical indicators,select the proteins with p<0.05 to draw a heat map,evaluate the correlation,and finally screen out the ones that have evaluation for MODS diagnosis and HA-UCMSC treatment potential markers of meaning.Results:1、LPS dose screening and model building methods:(1)Give 10mg/Kg,20mg/Kg,30mg/Kg,40mg/Kg,50mg/Kg doses of LPS to tree shrews with 30%blood loss and left femoral comminuted fracture,24h to 48h after modeling,LPS dosage10mg/Kg,20mg/Kg,30mg/Kg group had no death,40mg/Kg group mortality rate was12.5%,50mg/Kg group mortality rate was 50%,LPS was determined through mortality,histology,and blood index analysis The optimal dosage is 50mg/Kg.(2)The modeling method was as follows:30%blood loss,comminuted fracture of the left hind limb femur,and one-time tail vein injection of LPS 50mg/Kg.2.Evaluation of the MODS tree shrew model:(1)Rectal temperature and mental state:rectal temperature dropped significantly>1°C,the body curled up,the hair was messy and wet,the mental state was listless,and the eyes were dull.(2)Blood cell and biochemical tests showed that compared with the control group,the proportion of neutrophils(NEU%)was significantly increased(P<0.05),and the proportion of lymphocytes(LYM%)was significantly decreased(P<0.05);C Reactive protein(CRP),total bilirubin(TBIL),alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),creatine kinase isoenzyme MB(CK-MB),creatine Kinase(CK)were significantly higher than those in the control group(P<0.05).(3)Histopathological structural changes:cardiomyocyte necrosis can be seen in the heart tissue;severe degeneration of liver cells,loose cytoplasm and light staining of the cytoplasm,and vacuoles in the cytoplasm can be seen in the liver;the number and volume of white pulp in the spleen are severely reduced and the number of lymphocytes is large Severe reduction in area;severe thickening of alveolar walls,widening of alveolar spacing,and infiltration of granulocytes in the lungs;edema of the small intestinal mucosa,loose arrangement of connective tissue in the lamina propria,and widening of the spacing of small intestinal glands;no significant changes in kidney and gastric tissue.The above results showed that the model had obvious dysfunction and tissue damage of 5 kinds of tissues and organs,which met the MODS standard.3.Cultivation and identification of tree shrew UCMSC and HA-UCMSC:(1)Growth morphology:Compared with UCMSC,HA-UCMSC have higher nucleoplasmic ratio and smaller volume;(2)Proliferation ability:observed in 12-well plates Within 7 days,the density of HA-UCMSC was always higher than that of UCMSC.On the 7th day,the count of HA-UCMSC was 8.6×10~3,while that of UCMSC was 7.3×10~3.The degree of cell fusion of HA-UCMSC(90.8%)higher than that of UCMSC(86.7%);(3)cell cycle:G0/G1 cells of HA-UCMSC and UCMSC accounted for 73.2%and 60.8%respectively;(4)surface antigen markers:CD90,The positive cell rates of CD73 and CD105 were 97.7%,96.4%,97.2%and 98.4%,96.5%,96.8%,respectively,all of which had low expression of CD34 and CD45,HA-UCMSC were 1.56%and 0.67%,UCMSC were 1.47%and 1.32%.HA-UCMSC also expressed embryonic stem cell(Embryonic stem cell,ESC)-related surface markers SSEA-3(51.6%),SSEA-4(38.7%)and TRA-1-60(21%);UCMSC did not express SSEA-3 and TRA-1-60,SSEA-4 was 31.3%;(5)Differentiation potential:the adipogenic,osteogenic and chondrogenic differentiation abilities of HA-UCMSC were significantly higher than those of UCMSC(P<0.05),and induced three germ layers Differentiation found that the ability of HA-UCMSC to differentiate into three germ layers was higher than that of UCMSC.Among them,there was no significant difference in the expression of mesoderm marker molecule Brachyury(T)and ectoderm marker molecule PAX6(P≥0.05),and the expression levels of other marker molecules were average.Significantly higher than UCMSC(P<0.05).(7)e GFP-labeled cells:When the optimal MOI value of HA-UCMSC was 150,the infection efficiency was 80%,and the cell morphology and proliferation ability were not affected.4.Efficacy evaluation of HA-UCMSC:(1)After continuous observation for 30days,the mortality rate in the treatment group was 10%,and the mortality rate in the model group was 55%.HA-UCMSC effectively reduced the mortality rate of MODS tree shrews.(2)Compared with the model group,the treatment group had a good mental state,no redness and swelling in the femoral wound,only subcutaneous congestion,and elevated body temperature;(3)After HA-UCMSC treatment for 72hours,the model group The mortality rate was 50%,and the mortality rate in the treatment group was 16.7%;(4)After HA-UCMSC treatment for 72 hours,there was no significant difference in the changes of blood cell count and biochemical indicators(P≥0.05);(5)In the model group,heart,liver,spleen,lung,Small intestinal tissue structure disorder,degeneration and necrosis,vacuolar degeneration of cardiomyocytes,extramedullary hematopoiesis(EMH)in the liver,indistinct red and white pulp in the spleen and necrosis,thickening of the lung septum,small intestinal edema,renal and gastric tissue The structure was normal.In the treatment group,the heart,small intestine and liver were repaired.The liver had EMH.The boundary between the red and white pulp of the spleen was obvious but necrosis could be seen.The structure of the lungs was not obviously repaired.The results showed that HA-UCMSC treatment prevented disease progression and promoted the repair of damaged heart,liver,spleen,and small intestine tissue,while the structure of lung tissue did not significantly improve.5.The mechanism of HA-UCMSC in treating MODS tree shrews:(1)Chemiluminescence imaging tracing of e GFP-labeled cells found that a certain number of e GFP-positive cells were distributed in the heart,liver,lung,kidney,small intestine,and muscle tissue,and frozen sections of e GFP The observation of positive cells revealed that there were e GFP-positive cells in the heart,liver,spleen,lung,kidney,and stomach,suggesting that HA-UCMSC were involved in tissue damage repair;(2)The intensity of TUNEL staining in the lung tissue was not significantly different from that in the model group(P>0.05),while other tissues were significantly lower(P<0.05).(3)ki67 immunofluorescence staining of heart,liver,spleen,lung,and small intestine tissues showed that the fluorescence intensity was significantly increased(P<0.05)except for lung tissue(P>0.05).(4)The levels of pro-inflammatory factors TNF-αand IL-1 in the liver were lower than those in the model group(P<0.05);the levels of anti-inflammatory factor IL-10 were higher than those in the model group(P<0.05).(5)Caspase1 and Gasdermins-D in liver tissue were significantly increased in the model group and decreased significantly after treatment(P<0.05).(6)Immunofluorescent staining of hematopoietic-related marker molecules in liver hematopoietic lesions revealed that the surface marker CD133 of endothelial progenitor cells was expressed in the treatment group,but not in the model group;Both were significantly higher than those in the model group(P<0.05),and the monocyte surface marker CD14 was only expressed in the treatment group(P<0.05).(7)Serum protein group analysis results:a total of 1405 proteins were obtained in the control group,model group and treatment group;all proteins were clustered according to their expression levels,and the control group,model group and treatment group could be divided into The three groups showed that there were significant differences in protein expression patterns among the control,model and treatment groups;compared with the control group,174 proteins were up-regulated and 57 proteins were down-regulated in the model group,and these differential proteins were mainly enriched in cell division,In terms of functions such as platelet aggregation and ADP metabolism,it mainly regulates signaling pathways such as glycolysis/gluconeogenesis,tight junctions,and pyruvate metabolism;compared with the model group,40 proteins were up-regulated and 56 proteins were down-regulated in the treatment group.Differential proteins are mainly enriched in immunoglobulin complexes,actin cytoskeleton cell-substrate adhesion and other functional aspects,and mainly regulate signaling pathways such as adhesion,necroptosis,adhesion pathway,protein digestion and absorption TNF signaling pathway.The trend analysis of differential proteins showed that there were 8 trends,including proteins with two trends of first down-regulation and then up-regulation and first up-regulation and then down-regulation;after GO enrichment analysis,it was found that the first down-regulation and then up-regulation were mainly enriched in oxidoreductase activity,oxygen In terms of transport protein activity,oxygen binding,riboflavin reductase activity and other functional aspects,KEGG enrichment found that these proteins mainly regulate protein digestion and absorption,riboflavin metabolism and other signaling pathways,and were first up-regulated and then down-regulated by GO enrichment analysis The main enrichment is in the functions of transmembrane transporter activity and ATPase activity.After KEGG enrichment,it is found that these proteins mainly regulate the signaling pathways related to infectious diseases.Among them,COL1A2 protein is mainly enriched in protein and digestion and absorption pathways,suggesting tissue repair,and Blvrb protein is enriched in riboflavin metabolism pathway,indicating the existence of early immature red blood cells produced by EMH,avoiding oxidative damage of EMH in the process of hematopoiesis,and promoting Erythrocyte maturation,compared with model group EMH,HA-UCMSC treatment is beneficial to the regulation of hematopoietic homeostasis in EMH.The correlation analysis of these two trends of proteins and blood indicators shows that 25 proteins such as HBG,HBA,GSN,A1BG,Blvrb,and COL1A2 that are first down-regulated and then up-regulated,and AZGP1,HP,and PRBC that are first down-regulated and then up-regulated can be used as a diagnosis And candidate marker proteins for judging the course of MODS and evaluating the curative effect of HA-UCMSC on MODS.Conclusions:1、The tree shrew MODS model was successfully established by femoral vein blood loss of 30%of systemic blood volume,left femoral comminuted fracture,and tail vein injection of 50 mg/Kg LPS 1 m L.The model manifested as:listlessness,inflammatory response,liver and heart dysfunction,and damage to the tissue structure of five organs.2.Establish a method for the preparation and identification of tree shrew umbilical cord HA-UCMSC,which has higher nucleoplasmic ratio,proliferation activity,and differentiation potential than UCMSC,and can express ESC surface markers SSEA-3,TRA-1-60,and SSEA-4 and Nanog,OCT4,SOX2.3.It is confirmed that HA-UCMSC can reduce the mortality rate of MODS tree shrews,improve the mental state,reduce the inflammatory response,and promote the damage repair of heart,liver,spleen,small intestine and other tissues.4.It is found that HA-UCMSC can home to various tissues of MODS tree shrews,and promote the repair of damaged tissue by promoting the proliferation of damaged tissue cells,reducing apoptosis.Serum proteome analysis found that HA-UCMSC exerted a therapeutic effect by promoting the up-regulation of 40proteins and the down-regulation of 56 proteins,and regulating the corresponding signaling pathways through these molecules.5.Found 28 proteins such as HBG,GSN,A1BG,Blvrb,SERPINA1,C1R,C1S,FBLN1,IGLV3-25,Adipoq,ALB,TREH,TTR,Ca3,COL1A2,AZGP1,HP,PRBC,etc.can be used as diagnostic and determination MODS Candidate marker proteins for the process and evaluation of the efficacy of HA-UCMSC in the treatment of MODS. |
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