| Objective:1. To establish an ideal culture system of rat bone marrow mesenchymal stem cells(BM-MSCs) by density gradient centrifugation combined with attachment culturemethod.2. To investigate the targeted differentiation effect of Liraglutide on BM-MSCs invitro，and further to compare the difference with glucagon-like peptide1.3. To observe the effect on gastrointestinal hormone and functions of β together withα cell in islet of type1diabetes rats, which were induced by streptozotocin and thentreated with Liraglutide and BM-MSCs transplantation.Methods:1. Mononuclear cells were acquired according to the density differences among bonemarrow cells by rat lymphocytes separation medium; Then they were further purifiedby changing medium regularly on the basis of discrepant adherent culture property;Finally, isolated cells of passage3were identified with flow cytometer for antigensmarkers of mesenchymal stem cells, CD29, CD90, CD73and CD105, and those ofhematopoietic stem cells, CD34, white blood cells, CD45, their ability to differentiateinto osteogenic and adipogenic cells were also determined under specific culturemedium.2. On the basis condition of high glucose, low glucose with nicotinamide, anothermore10nmol/L liraglutide was added to the medium. After7days, gene measurementof Nestin, PDX-1, GK, GLUT-2, Insulin and Glucagon were tested by Real-time PCR, Insulin and Glucagon protein expression were detected by cell immunofluorescence.We also made glucagon-like peptide1(GLP-1) as control to observe the effect ofBM-MSCs differentiating into islet-like cells.3. Type1diabetes (T1DM) rats were established by injecting60mg/kgstreptozotocin, and they were randomly divided into four groups: Type1diabetesmellitus group (T1DM, n=10), BM-MSCs treatment group (T1DM+BM-MSCs, n=10),Liraglutide treatment group (T1DM+LIRA, n=10), Liraglutide combined withBM-MSCs treatment group（T1DM+LIRA+BM-MSCs，n=10）. Random blood glucoseand general conditions were monitored regularly. After8weeks, HbA1c,24h urinevolume and24h urinary protein excretion were tested. The serum concentration ofinsulin, C-peptide, glucagon, gastrin, cholecystokinin and glucagon-like peptide-1were assayed respectively by ELISA. The expressions of insulin and glucagon inpancreas were measured by immunohistochemistry.Results:1. It was found that isolated cells were highly homogeneous and spindle shape underimicroscope. The Flow Cytometry showed those cells were positive for mesenchymalstem cells surface markers CD29, CD90, CD73and CD105, but negative forhematopoietic stem cell surface markers CD34and white blood cells surface markersCD45. The isolated cells could be both induced into osteogenic and adipogenic cells.2. BM-MSCs showed distinct tendency to form clumps at confluence whenLiraglutide or GLP-1was added into culture medium, and DTZ staining was positive.Real-time PCR showed the mRNA content of Nestin was decreased (P<0.01), PDX-1,GK, GLUT-2, Insulin and Glucagon were increased in Liraglutide induced group andGLP-1induced group compared with high glucose plus nicotinamide induced group(P<0.05), there were no significantly difference on the content of these genes abovebetween Liraglutide induced group and GLP-1induced group （P＞0.05）. Cellimmunofluorescence showed the expression of Insulin or Glucagon protein werenegative in non-induced group or high glucose plus nicotinamide induced group, whilethey were positive in Liraglutide induced group and GLP-1induced group. 3. General index: Compared with T1DM group, the concentration of randomly bloodglucose, HbA1c,24h urine volume and24h urinary protein excretion were significantlydecreased in T1DM+BM-MSCs group, T1DM+LIRA group and T1DM+LIRA+BM-MSCs group（P＜0.01）, and the each index was the lowest in T1DM+LIRA+BM-MSCsgroup (P<0.05).Gastrointestinal hormone: Compared with T1DM group, the serum concentrationof insulin，C-peptide，gastrin and cholecystokinin were significantly increased inT1DM+BM-MSCs group, T1DM+LIRA group and T1DM+LIRA+BM-MSCs group（P＜0.05）; The concentration of glucagon-like peptide1was significantly increased inT1DM+LIRA group and T1DM+LIRA+BM-MSCs group（P＜0.05）, while glucagonwas decreased (P＜0.05); The concentration of glucagon-like peptide1and glucagonin T1DM+BM-MSCs group were no significantly difference with T1DM group (P＞0.05). Compared with T1DM+BM-MSCs group, the serum concentration ofglucagon-like peptide1was significantly increased (P<0.05), glucagon wassignificantly decreased in T1DM+LIRA group (P＜0.05), while the concentration ofinsulin, C-peptide，gastrin and cholecystokinin were no significantly difference withT1DM+BM-MSCs group (P＞0.05); The serum concentration of glucagon wassignificantly decreased (P＜0.05), insulin, C-peptide，gastrin, cholecystokinin andglucagon-like peptide1were significantly increased in T1DM+LIRA+BM-MSCsgroup compared with T1DM+BM-MSCs group (P<0.05). Compared with T1DM+LIRAgroup, the serum concentration of insulin, C-peptide，gastrin and cholecystokinin weresignificantly increased (P<0.05) in T1DM+LIRA+BM-MSCs group, while theconcentration of glucagon-like peptide1and glucagon were no significantly differencewith T1DM+LIRA group (P＞0.05).Correlation Analysis: After8weeks, the serum concentration of insulin waspositively correlated with gastrin（r=0.544，P＜0.01）, cholecystokinin（r=0.710，P＜0.01）and glucagon-like peptide1（r=0.669，P＜0.01），but was negatively correlatedwith glucagon （r=-0.506，P＜0.01）; The serum concentration of glucagon was negatively correlated with gastrin （r=-0.364，P＜0.05）, cholecystokinin（r=-0.433，P＜0.01）and glucagon-like peptide1（r=-0.591，P＜0.01）.Pancreatic immunohistochemistry: Compared with T1DM group, the ratio ofinsulin positive area in islet cells was significantly increased in T1DM+BM-MSCsgroup, T1DM+LIRA group and T1DM+LIRA+BM-MSCs group (P<0.01), and it wasthe highest in T1DM+LIRA+BM-MSCs group（P＜0.01）, there were no significancebetween T1DM+BM-MSCs group and T1DM+LIRA group（P＞0.05）. Compared withT1DM group, the ratio of glucagon positive area in islet cells was significantlydecreased in T1DM+LIRA group and T1DM+LIRA+BM-MSCs group（P＜0.01）, therewere no significance between T1DM+BM-MSCs group and T1DM group（P＞0.05）;Compared with T1DM+BM-MSCs group, the ratio of glucagon positive area in isletcells were significantly decreased in T1DM+LIRA group and T1DM+LIRA+BM-MSCsgroup（P＜0.01）; Compared with T1DM+LIRA group, the ratio of glucagon positivearea in islet cells was significantly decreased in T1DM+LIRA+BM-MSCs group（P＜0.05）.Conclusions:1. It was found that we could get enough BM-MSCs by density gradientcentrifugation combined with attachment culture method at a time.2. BM-MSCs could be further enhanced to differentiate into islet-like cells byLiraglutide, and this effect was the same with glucagon-like peptide1.3. It was found that the therapy of Liraglutide combined with BM-MSCstransplantation could increase the concentration of multiple gastrointestinal hormonesand regulate the β together with α cells function in islet of type1diabetes rats, thus theglucose metabolism could be better improved compared with monotherapy. |
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