Study Of Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Islet-like Cells In Vitro And Its Repair Effect Of Damged Pancreas In Rats

Diabetes are divided into type Ⅰ and type Ⅱ in clinic, It's necessary toinject insulin in diabetic' lives, But it is not ideal to inject insulin becauseinsulin can't prevent the development and progress of diseases derived formdiabetes, such as kidney diseases, coronary heart disease, eye diseases and soon. Pancrea islet or pancrea islet cells transplantation is one of the prospectivetherapies on diabetes. However, the lack of donated organs and transplantationreject reaction hinder the application of pancrea islet or pancrea islet cellstransplantation.Enough islet cells will be supplied after the problem of the lack of isletcells resources is resolved and the differentiation of bone marrowmesenchymal stem cells (MSCs) into islet cells is studied, and induction ofstem cells into pancreatic beta cells with function is performed. Not only typeⅠ diabetic but only some type Ⅱ diabetic patients can get an effectivetherapy. Enough islet cells with function will be supplied and transplantationreject reaction can't occur by isolating bone marrow MSCs from patients. Itwill play an important role in therapy of injured pancrea tissues, pancreatitis,especially diabetes, that bone marrow MSCs differentiated into islet-like cellswith function, which has important research value and application prospectionin clinic.1. Materials and methods:1.1 Isolating, culturing, identification and biological characterization ofrat bone marrow MSCs.MSCs were isolated from a-week-old Wistar rats, then cultured andpassaged in vitro. MSCs were induced to differentiate into chondrocyte ininduction medium containing induction factor, such as TGF-β 1,desamethasone, then differentiated cells were identified by toluidine blue stainand immunohistochemistry for collagen Ⅱ . MSCs were induced todifferentiate into adipocyte with desamethasone and insulin and so on. Thedifferentiation cells were identified by sudan Ⅳ stain.1.2 The differentiation of bone marrow MSCs into islet-like cells andidentification of differentiated cellsIn experimental groups, bone marrow MSCs were induced with inductionfactors, like 10ng /ml bFGF, 10ng/ml EGF and 2% B27. A week later, themedia were changed into serum –free media with high glucose, supplementedwith pancreas conditioned media, HGF(10ng/ml), 2% B27, active A, β-mercaptoethanol, and nicotinamide for another week, In control group, nofactors were added into media. Two weeks later, morphology observation,electron-microscopic observation and histochemistry observation like Dithizonstaining was performed. According to the preliminary observation, the passage3 MSCs which were planted into 75ml culture flasks were digested withtrypsin and replanted at 5×106 cells/ml. Then the above induction factorswere added into media, and the cells were induced as the above methods. Theexpression of insulin in induced cells were detected usingimmunohistochemistry and Western blot .1.3 Induction of MSCs into islet-like cells and its repair on injuredpancreas tissues.Injured pancreas tissue model was prepared, and was identified bymorphology and pathology sections. MSCs and bone marrow cells weremarked with DAPI and implanted into experimental group(MSCsimplantation ) and control group (bone marrow mononuclear leukocyteimplantation )1.4 Effect of islet-like cells differentiated from MSCs on therapy ofdiabetic rats.The Wistar rat model of diabetes mellitus by Streptozotocin (STZ) wasprepared. 40 adult Wistar rats were supplied by Laboratory Animal Center ofJilin University, with the body weight of 220±12.2g, After the rats were fedfor one week, urine sugar, blood sugar were detected, respectively, thendiabetes model was prepared as follows: STZ solution at the dose of 75mg/kg bodyweight was injected into rats by i.p. . 6 days later, urine sugar andblood sugar were detected again. Method of blood sugar determination: A dropof blood of rats' tail veins was dropped on the test paper for blood sugar, thevalue of blood sugar was obtained from blood sugar analyzer. Method of urinesugar determination: one part of the test paper was emerged into urine for 2seconds, then compared with standard controls within30-60 seconds and readthe value of urine sugar.The rats were divided into 2 groups including the experimental andcontrol groups (17 rats each group )The rats in experimental group were anesthetize with 10% ChoralHydrate (30mg/kg ), then induced islet-like cells were transplanted intocapsule of kidney. In control group, passaged bone marrow MSCs weretransplanted into the same location, and chinical syndrome was observed. Tendays later, the therapy effective was observed by urine sugar, blood sugra, therate of death.1.5 StatisticsT test between two groups was performed using lysis software.2. Results2.1 Isolating, culturing, identification of rats bone marrow MSCsFew of long spindle-shape cells attached on the flasks after MSCs wereseparated using percoll for 24h. 72h later, many cells attached on the flasks,cells shapes were round, oval, spindle-shape and so on, When cells wereconfluent, cells were passaged with 0.25% trypsin. Most of cells were spindleshape after being passaged. The cells didn't express CD45, CD44, andexpressed CD34 slightly using flow cytometry, which suggested the cellswere a population of undfferentiated childish stem cells.2.2 multipotency observation of rats bone marrow MSCsDifferentiation of bone marrow MSCs into ChondrocyteAfter induction factors were added, cells attached on the flask with cellcluster aggregation, and cells were triangle and polygon. In control group,toluidine blue metachromatic staining of undifferentiated MSCs was notsignificant. In experimental group, MSCs changed into polygon and cytoplasmwas blue-purple by toludine blue metachromatic staining after being iuducedfor 7 days, MSCs changed into polygon or round and cytoplasm wassignificant blue-purple and toluidline blue metachromatic staining wasobvious, and nuclei were clear. The expression of collagen type II was positiveby immunochemistry.Differentiation of bone marrow MSCs into adipocyteSmall lipid droplet appeared after being induced for 48h. 2 weeks later,lipld droplets increased. long spinde-shapke cells changed into round andpolygon. In experimental groups, after sudan Ⅳ staining, lipid droplets werered, and nuclei were blue.2.3 Differentiation of bone narrow MSCs into islet-like cells andidentification of differentiated cells.During the process of induction, the morphology of MSCs changedgradually. At first, most of cells were long skinde-shape, few cells weretriangle or round A week leter, cells number increased with cell clusteraggregation. Two weeks later, some cells suspended.Dithizon staining showed bright red color for most of induced cells, andthe expression of insulin was positive by double-immunofluorescence.Uninduced cells didn't express insulin. In experimental group, inducedislet-like cells expressed insulin, uninduced cells didn't express insulin byWestern blot.2.4 Induction of bone marrow MSCs into islet-like cells in vitro and itsrepair on injured pancreas tissues.The survival rate of the experimental group was 80% after differentiatedislet-like cells were transplanted into rats in experimental group and neurosispancrea tissue recovered basically. Islet cells recovered gradually, but fewinflammation cells were observed around the blood vessels in tissue pathologysections. DAPI marked cells were blue under laser scanning confocalmicroscopy. In control group, all the rats died with a great deal of liquids inthe abdoment and black and attached organs.2.5 Therapy effect of islet-like cells derived from bone marrow MSCs ondiabetic rats.Preparation of diabetic rats model: STZ solutions was injected into ratsby i.p., for 3 days later, they appeared syndrome of drining more water andeating more. A week later, most of rats appeared urorrhagia besides the abovesyndrome. After being detected, blood sugar and urine sugar after injectionsignificantly higher than those before injection.After differentiated islet-like cells were transplanted into diabetic rats fora week, the diabetic syndrome was relieved. Two weeks later, the level ofblood sugar decreased, but there was no difference between the experimentaland control groups. The level of urine sugar decreased and there wassignificantly different compared with the control group. The survival rate ofthe experimental group was 80% and that of the contral group was 45%3. Conclusion(1) Bone marrow MSCs can be obtained, and passaged in vitro.The purebone marrow MSCs can be separated by both percoll seperation method andattaching screen method.(2) In vitro bone marron MSCs were induced to differentiate intoadipocyte, chondrocyte and islet-like cells.(3) Islet-like cells differentiated from bone marrow MSCs can secretinsulin, which expressed insulin by immunohistochemistry and Western blot.(4) Islet-like cells differentiated from bone marrow MSCs can decreasethe level of blood sugar and urine sugar of diabetic rats, so it was suggestedthat it is possible to treat the diabetes using differentiated islet-like cells frombone marrow MSCs.4.Significance(1) It was suggested that the obtained cells was a population ofundifferentiated childish stem cells by the differentiation of MSCs into manykinds of cell, and the expression of surface markers of MSCs detected by flowcytometry.(2) The method of differentiation of bone marrow MSCs into islet-likecells was mastered.(3) The results showed that induced islet-like cells can repair the injuredpancrea tissue and have a potential for therapy on diabetes.