| [Objective]1. To establish and evaluation the Tree shrews T2DM model induced by high sugar, high fat feed urea with the STZ. 2. To observe the therapy and mechanism after UC-MSCs transplantation in type 2 diabetes tree shrews.[Methods]l.In order to separate, culture, amplificate, identificate and mark the tree shrews UC-MSCs, the newborn umbilical cord were collected under the condition of caesarean section in full-term pregnancy tree shrews. The tree shrews UC-MSCs were isolated and cultured in vitro. Morphological observation and the flow cytometry analyzer test were all used to detect positive expression rate of surface antigen, such as CD31, CD34, CD44 and CD90. Observate the differentiational ability of UC-MSCs. Tag the UC-MSCs by GFP and DIR, and identify the positive rate of GFP, and observe the migration and engraftment of UC-MSCs by flow analyzer in alive animals body.2. Construction of Tree shrews T2DM model:48 healthy tree shrews were randomly divided into control group (n=8); T2DM group (n=40). The control group was treated by normal diet, and no special handling; T2DM group was treated by high sugar, high fat feed for 6 weeks, after that, STZ (100 mg/kg) was intraperitoneal injection one or two times. As the successful construction of the model, the general condition, body weight, and by using of Roche glucose meter, FBG was tested in peripheral blood. FINS, triglycerides, c-peptide, total cholesterol, glycosylated hemoglobin levels were tested by automatic biochemical analyzer, and the OGTT was test too. Pancreas, liver and kidney were routine HE staining. ELISA was used to detect level of TNF-alpha and IL-6. Finally, the tree shrews which according with standard of T2DM were used in the further experiments.3. UC-MSCs transplantation was used to treat the T2DM tree shrew:the tree shrews were divided into normal control group (n=8), model control group (n=10) and treatment group (n=26). As to the treatment group, the model will be labeled by UC- MSCs through caudal vein transplantation, model control group was injected volume of physiological saline, and no treatment about the normal control groups. To observe the distribution of GFP and DIR in the pancreas, liver and kidney. Automatic biochemical analyzer was used to test FINS, C peptide, TG and TC after UC-MSCs transplantation. And pancreas, liver and kidney tissues were collected to done the HE staining, and observe its pathological organization structure change after transplantation. Acquisition model control group and treatment group serum samples, through metabonomics analysis method of the metabolic components of two groups of samples and the results are classified statistics, select difference bigger metabolites. Amino Acids and the Derivatives, Organic Acids, Sugars, RNA and 59 factors were analyzed.4. The mechanism of UC-MSCs to the T2DM:With final concentration 40 and 60 tendency for 40 tendency for glucose induced cultivate, and in sixth day, the methylation of area is analyzed in the human UC-MSCs, and the difference of gene pathways in classification.[Results]1. Isolation, identification and marking of The tree shrews UC-MSCs in vitro:The adherent method of culture UC-MSCs was used, a small amount of spindle cell growth was visible around the the original generation UC-MSCs. At the fifth day, the cells shows obvious long spindle, increase in the number, and multiple cell colony were boserved, when the cells grow about 80% alignment, the cells are already post wall at this time. Subculture 3 generation of cells were used in the experiment. The cells were showed markers 100% CD44 and CD90, but endothelial cell marker CD31 and hematopoietic stem cell marker CD34 expression rate is 0.2%. The UC-MSCs were successfully induced in vitro into fat cells and osteogenesis. After GFP marker UC-MSCs cells, hair green fluorescent, fluid identification mark is at a rate of 97.2%. DIR tag cells by observing animals living imager hair dark red fluorescence.2. The tree shrews T2DM diabetes model:(1) generally see more drinks, polyphagia, polyuria, emaciation, slow response, lacklustre, MAO appeared late tail, leg ulcer formation; After intraperitoneal injection of STZ 1～2 times, the control group only 8 animals in good condition, no death,40 animal model group has four death; The change of blood biochemical indicators (2):FBG:model group continued to rise after STZ injection compared with control group (P< 0.05); FINS:model group at 6 weeks peak tendency (8.58 L) compared with control group (P< 0.05), after gradually fell to near the control level (3.56 tendency/L) (P< 0.05).The change trend of C peptide and FINS are similar; HOMA-IR:model group compared with control group after building appear obvious insulin resistance (P< 0.05).TG, TC, since building continue to rise in the model group,12 weeks after stability at a high level, but still higher than control group (P< 0.05). Weight:model group has no obvious change in front of the building, after the high sugar, high fat feed weight gain, in sixth week, compared with the control groups (P< 0.05), and then decreased, and 16 weeks or even lower than the control group (P< 0.05). OGTT area under curve:8,16 weeks increase (P< 0.05) compared with control group, model group exists abnormal glucose tolerance. HbAlc: the control group, under stable tendency for 5/L specification does not have spontaneous diabetes is made before the mold, and 16 weeks/L above model group 7 tendency, that formed after 2,3 months have diabetes. Cytokines (3):the experimental group 6 weeks and 8 weeks of peripheral blood qing TNF alpha and IL-6 levels higher than the control group (P< 0.05).3. The UC-MSCs transplantation treatment:(1) DIR, GFP respectively labeled UC-the third and fourth weeks of MSCs transplantation to the body, can in the pancreas, liver and renal interstitial department respectively observed in deep red and green fluorescent cells, UC-MSCs can be located in T2DM tree shrew in the pancreas, liver and kidney. Transplant model of the control group did not see cells expressing green fluorescent.(2) general situation:after treatment, appear more drinks, polyphagia, polyuria, were improved, increased activity, tail, leg ulcer lesions. The change of blood biochemical indicators (3):the UC-six weeks after MSCs transplantation treatment, FBG, IR, TG, TC, HOMA IR compared with before treatment decreased significantly (P< 0.05). FINS, C peptide:4 weeks after treatment with lower than still have before treatment (P< 0.05), but in 6 weeks after treatment rising trend (P< 0.05).4. Histopathological changes:the model group, the liver, liver apparent confusion, lobular damage, liver cell clear vacuoles degeneration, swelling, visible point necrosis, collect abbacy inflammatory cells infiltration. Kidney:mesangial proliferation renal capsule ball cavity decreases, and bleeding blood clot, renal tubular lipid droplets deposition with inflammatory cells infiltration, pancreas islet atrophy, reduced, distribution is sparse, islet volume decreases, and form highly rules, the nucleus pycnosis, part of the nucleus is missing, the control of the liver, kidney, pancreas without the above changes. Treatment group:the liver:a neat liver and liver cell degeneration, necrosis, inflammatory exudation reduce; Kidney:glomerular bleeding blood, inflammatory cells infiltration reduction, cystic cavity without atresia, interstitial department did not see fat droplets appear; pancreas islet morphology is complete, cell degeneration, necrosis, compared with model group.5. Metabonomics analysis it is found that Urea (Urea), Taurine (Taurine), Lactate (Lactate), Glucose (Glucose) distinguish between multiple metabolites on the sample making an important contribution. After treatment than before treatment concentration, urea, lactate and glucose concentration decreased, while the rise in the concentration of taurine.6. Compared to normal group and 60 tendency/1 glucose change has 2304 genes, there are 1292 high gene methylation,432 genes in low methylation,580 gene methylation.The genetic pathway analysis, found that the changes of related genes mainly gathered in Insulin signal is shown in figure 1 (a-B), has 15, including gene methylation increases gene methylation downgrade have 3.Compared to normal group and 40 tendency/L glucose change has 1979 genes, there are 1029 high gene methylation,409 genes in low methylation,541 gene methylation.And found that the changes of gene mainly gathered at the Notch signal (figure 2 a-B), including gene methylation raised seven,4 gene methylation downgrade.[Conclusion]1. The adherent method successfully won the tree shrew were isolated and cultured UC-MSCs cells, UC-MSCs markers CD44 and CD90 expression rate was 100%, and the endothelial cell marker CD31 and hematopoietic stem cell marker CD34 expression rate was 0.2%, the fat cells of a class differentiation in vitro and osteogenesis cells.2. Using GFP, DIR tag tree shrews UC-MSCs cells, mark rate of 97.2%-98%.3. High sugar, high fat feed joint STZ intraperitoneal injection of tree shrews T2DM model was established successfully, showing obvious more drinks, food, polyuria, symptoms of weight loss, blood biochemical examination and shown obvious pointer to hyperglycemia, hyperlipidemia and insulin resistance. Accord with standard of the diagnosis of T2DM, and ChengMo rate was 90%.4. Based on the above tags of UC-MSCs tracer, can be observed that cells can engraftment in the liver, kidney, and pancreatic tissue, can reduce blood sugar, blood fat, adjust the secretion of insulin, C peptide, for the damage of liver, kidney, action of insulin.5.After UC-MSCs transplantation, the expressional level of some metabolites, such as glucose, lactate, urea were down-regulated, and the taurine was up-regulated. These changes were speculated to protect the pancreatic cells, promote the secretion of insulin, increase the using of glucose, reduce the glucose in the blood, repair the injury of liver, kidney and pancreatic, and alleviate the pathological changes.6.By using chip technology, the up-regulation of gene methylation in the 40 mmol/L glucose environment by UC-MSCs, and the Notch pathway was inhibited, but while the in the 60 mmol/L glucose environment, the insulin signaling pathway was inhibited. |
PDF Full Text Request