Contrasting Experiment Research About Mesenchymal Stem Cells Orientational Induction And Differentiation Into Myoblasts

Mesenchymal stem cells (MSCs) is early phase cells in middle germiallayer growth. They can not only differentiate creating blood substance, stromacells et al, but also differetiate many kinds tissue outside creating bloodsubstance by separation and culture, Particularly the cells of tissue coming frommiddle germinal layer and neuroectodermal. In the special inducingcircumstance, it can differentiate into adipose cell, fibroblast cell, muscle cell,nerve cell, cartilage cell. At the same time, MSCs is easy transfect and expressto exogenetic gene. So it become ideal target cell for treatment and genetreatment. It is very important to develop deeping research in orientationaldifferentiation and ensure better apply to clinical about MSCs. When skeletalmuscle is seriously injured, the muscle cells can not regeneration. After repaired,the contraction function is poor deeping on cicatrix.So mesenchymal stem cellorientation and differentiation into myoblasts is very important in tissure repairafter trauma. In recent the research discover 5-Aza can induce Mscs differentiateinto myoblast. But the differentiation mechanism is not clear. And 5-Aza hasmajor toxic as chemotherapy medicine. It confine to apply. Due to the up reason,we separately utilize 5-Aza and different period serum of injured muscle animalto induce MSCs to muscle cell and to do constrasting analysis at the same time,to deeply realize the regular and feature about orientational differentiation aboutMSCs. It provide grounds for mesenchymal stem cells orientational inductionand differentiation into perfect muscle cells in function and appearance byartificial method in external. At the same time, discuss the feasibility of the newinducing medicine about serum of injured muscle animal model. Look down themain work:1. Experiment research about separation, culture, identify of mesenchymalstem cells: Extract bone marrow of 4 weeks wistar rats. Separated and purifiedcells by percoll density gradient and centrifugation method combined withsticking wall screening method. After separated and purified and then werevaccinated in culture plate after 24h.At the time of changing culture fluid, therewere a little sticking wall cell under the culture flask. It express short shuttle shape,scattered distribution. After some days, cell increased apparently and appearfusiform and colony. It is longitudinal arrangement or radiate shape distributionand gradually mix to slice appearing well-distributed long shuttle shape. Byflowing cell instrument analysis, CD29,CD44,CD166 of cell superficialmolecule positive rate high to 90%, but the expression of CD34,CD45 is negative.CD34 is 0.11%, So it can be proved that percoll density gradient andcentrifugation method combined with sticking wall screening method can gethighly purified MSCs and can transmit descendant.2. Influences of 5-azacytidine on mesenchymal stem cells proliferation anddifferentiation into myoblasts: Bone marrow-derived MSCs of 4 weeks Wistar ratswere separated and purified, After induction by 5-Aza with differentconcentrations. Then culture the cell by changing culture fluid. Continuouslyobserve the changing of appearance by micrscope. The growth ability of cell wasassayed with methyl thiazolyl tetrazolium (MTT) method. The expression ofskeletal muscle actin and myosin was determined with reverse transcriptionpolymerase chain reaction (RT-PCR) , western blot method after inducingmesenchymal stem cells on the level of mRNA and protein. Results indicate: Thecell proliferation was not affected by 0and1μmol?L-15-Aza. 20-30μmol?L-15-Azainduced the toxic effect on proliferation of MSCs. 30μmol?L-15-Aza can inducethe cell die. There were expressions of skeletal muscle actin and myosin treatedwith 3-12μmol?L-15-Aza. Induced by 6μmol?L-1 5-Aza, proliferation of cell isapparent after 6-12d. The body of cell become longer and coarse, refraction isbetter. Proliferation of cell is relatively slow in 15d. Some cells become longerfusiform and appear 2-3 cell fusion each other. myotubu-like cells were found.Draw an conclusion: 5-Aza affects the expression of regulatory gene to the stemcells and regulate MSCs to orientationally differentiate into myotube-like cells.Massive dosage Aza has apparently toxic.3. Influences of the different period serum of muscle injured animal modelon messenchymal stem cells proliferation and differentiation into myoblasts.Frist build up the model of muscle injured animal. Obtain remarkably differenceabout CK activity. Pathology show muscle cell denaturation and necrosis.Injured animal model successed. Then the different period serum of muscleinjured animal model continuously induct MSCs. After 10days of serumcontinuous inducting. The growth ability of cell was determined with methylthiazolyl tetrazolium (MTT) method. At the same time, observe the changing ofappearance by micrscope. Extract RNA MSCs after induction.The expression ofskeletal muscle actin and myosin was determined with reverse transcriptionpolymerase chain reaction(RT-PCR) , western blot method after inducingmesenchymal stem cells. No matter what shape change, all expresselectrophorosis belt about gene part of actin and myosin. The serum of musclenoinjured animal model and the serum of the second,the fifth and the tenthmuscle injured animal model. Everyday chang it. But it isnot result. Imitatingnature body, it proved that the different period serum of injured animal modelcontinuously induct MSCs which can differentiate to myoblasts. But single andsteady serum is no this function. When the body is injured, physiology factorcontinuously chang which can stimulate MSCs to orientation induction. At thesame time, it proved that the serum of injured animal model is new inductor.4. Preliminary research about MSCs after orientational induction anddifferentiation transplanting to muscle injured animal:Inducting MSCs intomuscle cell by 5-Aza and continuous serum induction into body of muscleinjured animal. We realized by the observation that two method all can developto muscle structure. But the later develop mature and completed. It is welldistributated in space. Cicatrix is little. The result express the serum group issuperior to the 5-Aza group.Compared with the experiment results. Draw an conclusion: Serumcontinuous induction can make MSCs orientational induction and differentiation.The ability and differentiation degree of serum group superior to the 5-Azagroup. The mechanism and results which 5-Aza and the serum of muscle injuredanimal model orientationally induce MSCs into skeletal muscle cells aredifferent. 5-Aza is a kind of methylating restraining reagent. It can initiaterelated gene of myoblast differentiation. Induced method is simple, poor array oftime and space. Array cell differentiation is nomature. The induced method ofserum is a kind of regulation to the stem cell differentiation imitate organismnature environment by the continuously feedback of organism total information.Differentiation of stem cell have strongly array of time and space, completedinduced method, intensivly sequence. Cell differentiation is mature. In short,external manual inducement of stem cell orientationally become ideal muscleseed cell and achieving functional replace is very completed works.