Therapy And Mechanism Research On The Transplantation Of UC-MSCs In Metabolic Syndrome Tree Shrews

Objectives:1. To establish the model of the metabolic syndrome (metabolic syndrome, MS) and its evaluation techniques of tree shrew through combining feeding of MS Diet and indution of streptozotocin (streptozotocin, STZ).2. Evaluating the curative effect of UC-MSC (umbilical cord mesenchymal stem cells, UC-MSCs) transplantation to treat MS, To explore the mechanism of action from inflammatory factors, cytokines, transcription of the insulin signal transduction pathway related genes, metabolomics.Methods:1. The culture, identification and lambing of tree shrews UC-MSCs:In vitro, the cord was obtained via caesarean section from healthy full-term pregnancytree shrews under sterile conditions. Then UC-MSCs was got by adherent culture method to isolated, culture and expansion in vitro. Cell morphological observation, flow cytometry analyzer test and the differentiation of fat, osteogenic and cartilage cells were used to identify the UC-MSCs. In vitro, GFP and DAPI was used to lamb tree shrew UC-MSCs, and to observe the positive rate of GFP and DAPI lambing by fluorescence microscopy.2.Ta establish the MS Modeof tree shrew:50 healthy male tree shrews were randomly divided into normal control group (n= 10) and MS model group (n= 40). Tree shrews in normal control group were just fed with normal diet. But the MS model group were fed with MS Diet, STZ(80 mg/kg) was given by intraperitoneal injection in week 5. The general conditions, body weight, fasting blood glucose(FBG), fasting insulin (FINS) content, OGTT, TG, TC, LDL-C, HDL-C and HbAlc were tested in all the groups during the modeling time.Pancreas, liver and kidney were sectioned to perform HE staining and then observing its organizational structure. Collecting serum samples from normal control group and model group, metabolic component changes was detected by metabolomics analysis, then screening travelheterosexual major metabolites. The tree shrews finally determined in accordance with MS standard, and they would include on further experiments.3. UC-MSCs transplantation to treat tree shrews of MS:The tree shrews of MS model group were randomly divided into model control group (n= 10) and treatment group (n= 20). The GFP and DAPI lambed UC-MSCs were transplanted into the treatment group by the tail vein, and the model control group injected with equal volume of saline, and normal control group received no treatment. After the UC-MSCs transplantation,testing the general conditions, body weight, FBG, FINS, OGTT, TG, TC, LDL-C, HDL-C and HbAlc in all the groups. Pancreas, liver and kidney were sectioned to perform HE staining and then observing its organizational structure. Collecting serum samples from each group,and metabolic component changes was detected by metabolomics analysis, then screening the metabolites with travelheterosexual major.4. The mechanismof umbilical cord mesenchymal stem cells to treat MS:Serum samples were collected in the model group, model control group and treatment group, cell growth factors and inflammatory factors were tested by antibody chip technology, then screening the factors with travelheterosexual major. Take the normal control group’s, model contol group’s and treatment group’s liver tissue and muscle tissue to obtain tissue RNA by liquid nitrogen grinding method. By RT-PCR method, observing genes IR, GLUT4, mTOR, MAPK which were related to the insulin signal transduction pathway transcript expression in the liver tissue and muscle tissue.Results:1. UC-MSCs culture, identification and labeling in vitro of tree shrews:Afetr 24 hours, UC-MSCs were seen around the umbilical cord tissue with adherent method cultured. The fourth generation of UC-MSCs were spindle, spiral growth; UC-MSCs can be successfully differentiate into bone, cartilage and fat-like cell in vitro. GFP-labeled UC-MSCs exhibited bright green fluorescence positive expression with the rate of 98%under the fluorescence microscopic; DAPI-labeled UC-MSCs exhibited bright green fluorescence positive expression with the rate of 98%under the fluorescence microscopic.2. The establishment of MS model in tree shrews:Model group tree shrew showed hair turned gray, water intaking increased, urine outputing increased, unresponsive, activity decreased and other symptoms. After 2 intraperitoneal injection STZ,8 animals were in good condition of the control group; 3 animals died in model group. Blood biochemical index changes:FBG of model group continued to rise after STZ injection compared with the control group (P<0.05);FINS of model group rose in peak, comparison with the control group (P<0.05) in week 4, and then gradually to near the level of the control group; HOMA-IR of model group showed obvious insulin resistance compared with the control group (P<05). TG, TC and LDL of model group rise constantly, it stabilization at a high level compared with control group (P<0.05) after 8 weeks. HDL of model group rose to peak in week 4, compared with control group (P<0.05), then gradually decreased to the control group levels; Weight of model group increased in the MS diet weight gain, but their weight gradually decreased after 4 weeks, or even lower than the control group (P<0.05) in week 10. The area under the curve of OGTT:Cmpareing 10th week with control group (P<0.05), showed that there was the presence of abnormal glucose tolerance in model group. HbAlc:it’s less than lOg/L in normal control group, and, more than 16g/l in model group at week 10, indicating that elevated blood sugar is formed within the previous 2-3 months.3. After UC-MSCs transplantation to MS tree shrews and its biochemical indexes: ① After 4 weeks of DAPI, GFP-labeled UC-MSCs were transplanted into the body, we can observe blue and green fluorescent cells in pancreas, liver and kidney interstitial portions, suggesting that UC-MSCs may be located in MS tree shrews pancreas, liver and kidney. Instead, the model group of organs and cells, no blue-green fluorescence expression. ② The general situation:after UC-MSCs treatment, the symptoms of polydipsia, polyphagia and polyuria are reduced, its weight and activity increased. ③ Blood biochemical index:The treatment group: compared withe the model group, FBG, TG, TC, LDL, HOMA-IRdecreased significantly (P<05); HDL and FINS were significantly increased (P<0.05).4. Organizational structure change:Model group tree shrew:Hepatic cells showed fatty degeneration seriously, the volume of cells increased, the nucleus is pushed aside by fat vacuoles, part of hepatic cells were hydropic degeneration, and it accompanied with inflammatory cells infiltration; Some of the glomerular showed atrophy and necrosis, basement membrane thickening, glomerular clearance increased, and it accompanied with inflammatory cells infiltration, fat vacuoles appeared in interstitial portions; Pancreatic islet showed atrophy and necrosis, the number of pancreatic isle reduced significantly, and the volume reduced and it’s irregular, the surrounding tissue boundaries is unclear, accompanied by inflammatory celsl infiltration. The treatment group:2 weeks after the end of treatment, the treatment group tree shrew liver cells is substantially the same as the control group, only a small part of hepatic cellsr showed fatty degeneration, cell morphology rules, reducing inflammatory cell infiltration; 4 weeks after the end of treatment, there was basically no difference between the treatment group tree shrew liver cells and normal control group, no inflammatory cells infiltration; there was no glomerular atrophy and necrosis, no inflammatory cells infiltration, no obvious fat vacuoles in interstitial; Pancreas islets were oval, and the number was increased compared with the model group, with clear boundaries and decreased inflammatory cells infiltration.5. The genes expression of IR, GLUT4, MAPK, mTOR in liver and muscle tissues: After UC-MSCs treatment, we can see in liver tissue that compared with the model group, the amount of mRNA quantity of IR, GLUT4, mTOR increased significantly, and MAPK reduced. After UC-MSCs treatment, we can see in muscle tissue that compared with the model group, the amount of mRNA quantity of IR, GLUT4, MAPK increased significantly, and mTOR reduced.6. Analysis of inflammatory cytokines:Model group:Interleukin 16 (IL16), interleukin-1β (IL-1β), macrophage inflammatory factor 1δ (MIP-1δ), macrophage colony stimulating factor (MCSF), granulocyte colony stimulating factor (G-CSF), matrix metalloproteinase 1 (TIMP-1), a recombinant human chemokine (1-309), platelet-derived factor (PDGF-BB) were lower than the control group, the activation of eosinophil chemotaxis factor (Eotaxin), matrix metalloproteinase 2 (TIMP-2) were significantly higher than the control group. The treatment group:IL-1β, MIP-1δ, MCSF, Eotaxin, G-CSF, TIMP-1,1-309 were significantly higher than the model group IL16, TIMP-1, PDGF-BB decreased compared with the model group.7. Analysis of cell growth factors:Model group:stem cell factor (SCF) decreased compared with the control group, growth differentiation factor (GDF-15), epidermal growth factor receptor (EGF-R), osteoclast inhibition because (OPG), recombinant bovine insulin-like growth factor binding protein-2 (IGFBP-2) were significantly higher than the normal control group. The treatment group:SCF, GDF-15 were significantly higher than the model group, EGF-R, OPG, IGFBP-2 were lower than the model group.Conclusions:1. In this study, UC-MSCs of tree shrews were isolated and cultured by adherent culture method, the cells were spindle and spiral-like growth in morphologically.; High expression of CD44, CD29, did not express CD34; induced to differentiate into fat, osteogenic and cartilage cells in vitrol. Its identification accorded with the biological characteristics of UC-MSCs.2. Using GFP and DAPI successfully lambed UC-MSCs of tree shrews with the rate of 98%.3. MS Diet combined with intraperitoneal injection of STZ to construct t MS model of tree shrews, showing obviouslywith the symptoms of polydipsia, polyphagia, polyuria, weight loss, FBG, TG, TC, LDL and OGTT increased, decreasing of FINS, tissue sections results showed fatty degeneration of the live, kidney glomerular atrophy and focal necrosis of pancreatic tissue injury. They meeted the evaluation standard of MS, and the model rate is as high as 90%.4. After UC-MSCs treatment, the MS tree shrews with the symptoms polydipsia, polyphagia, polyuria, weight loss to improved, FBG, TG, TC, LDL and OGTT reduced, FINS increased. Tissue sections showed that the liver, kidneys, pancreatic tissue damage improved. By cell labeling technique, we observed that UC-MSCs can planting in liver, kidney and pancreas tissue.5. After UC-MSCs treatment, we can see that pro-inflammatory cytokines and stem cells growth factor expression increased in MS tree shrews, indicating that UC-MSCs can regulate the immune response and inflammation, repair of damaged tissue cells by accommodatong the inflammatory cytokines and cell growth factors.6. After metabolomic analysis, compared with normal control group, MS tree shrews’s content of glucose, lactate, methanol and alanine decreased, but the content of urea increased, indicating that metabolic disorder of sugar, protein and other. After UC-MSCs treatment, MS tree shrews’s content of glucose, lactate, urea and alanine decreased, but the content of methanol increased, indicating that sugar, protein and other metabolic disorders were improved.