| Objective: Bone marrow mesenchymal stem cells (MSCs) todifferentiate into muscle cells, bone cells, cartilage cells, fat cells, nervecells and other cell differentiation potential,Its source separation, easier totrain, highly amplified in vitro, autograft tissue matching and the problem ofimmune rejection. MSCs with the above-mentioned advantages, it causedwidespread concern, especially in tissue repair, the use of its uniqueadvantages in the treatment of organ and tissue repair with a lot of work.Thisexperiment to study bone marrow mesenchymal stem cells under differentconditions, separation, purification, passage, identification and induced thegrowth conditions, derived cultured bone marrow mesenchymal stem cellculture conditions.Methods:(1)The separation and training of MSCs.(2) MSCs appraisal(3)The MSCs go down to the future generation training.(4)Different plantdensity on the influence of MSCs growth.(5)Different concentration tirebovine serum the influence of MSCs to grow.(6)Future generation cellsgrowth curve.(7)5-Aza-CR induction MSCs differentiation into muscle cells.(8)Induction muscle differentiation related protein expression (9)BrdUidentifying.(10)Model of the construction of the rat.(11)Bone marrowstromal allograft stem cells.Result:(1)The separation of MSCs training Take off the neck to deathand6weeks old SD rats only1,75%ethanol for5min sterilization, removethe rat bilateral tibial and femoral, to cut ends long bone, exposed bonemarrow cavity,5ml syringe rinsed repeatedly get single-celled levitationliquid, move into the cell culture in the bottle. According to MSCs andimpurities of the cells to stick wall between the purification stem cells within24h after bottle training after the first change fluid, microscopically cells begin to stick wall deformation, the generation of stem cells, polygonal,triangular spindle, into fibrous, impurities, cells form a round or not stick tothe wall. Training after48h, microscope to stick wall cell number issignificantly increased, some of which are colony sample growth. Training4~6d, stick wall cell division proliferation obvious, visible more split phase,with the increased number of cells,2d growth within the group of space tendto be narrow stem cells, it appears vortex kind, into fiber appearance colonycells.(2)The identification of MSCs to stem cells Appraisal is still a difficultproblem, at present there is no unified, authoritative standard. The presentsituation is, most people using combined application of a variety of antibodyisolation and identification MSCs, generally USES the exclusive method orpush on the method of inverse identification. The most common mesenchymalstem cells positive markers are: SH-2, SH-3, SH-4, CD29, CD44, CD54,CD73, CD105, CD166, etc,Negative markers are: CD14, CD34, CD45,CD34, CD31, VWF etc, this experiment choice CD29,CD44as positivemarker, the CD34, CD45as negative marker to immunohistochemical methodto detect cells specific antigen.(3)The MSCs go down to posterity10to14days later, the cell density increases gradually, confluent. Go down to posterityinoculation cell distribution even, with spindle primarily.1and2, thegeneration of MSCs uniform form, a long spindle, impurities, cells go down toposterity, change with liquid drop. The train to P8(the eight generations) cells,stem cells are still alive, but slow growth of cells, form started to change andrefraction the gender is poorer, began to take off the wall.(4)Different plantingdensity on the influence of MSCs growth. In106/ml density vaccination ODvalue when the largest, living cell number, cell activity is better, moreconducive to the growth of MSCs.(5) Different concentrations of tire bovineserum MSCs growth impact. In the10%~15%of the womb bovine serumconcentrations than MTT method to detect the color OD490and SI supreme,the most suitable serum concentrations MSCs growth.(6) Cell growth curveThe cell growth curve is S form, therefore, after staying time, logarithmicgrowth period, platform of three periods and P1(generation) cell proliferation faster, the faster than other period.(7)5-Aza-CR induction MSCsdifferentiation into muscle cells for5-Aza-CR induced the two generations ofMSCs,7d began to find stem cells gradually become the body of the bulky,10d see small cells increases, length,13d visible part of the cells are touchingeach other, to15d-17dvisible have kind of muscle tube sample structureappear.(8) By5-MSCs Aza-CR after induction muscle differentiation relatedprotein expression induction of cells after4d detection, did not find cellsexpress muscle cells specific antigen, induction of cells after12d, the cellsexpress desmin protein obvious positive,5-Aza-CR after induction14dexpress myosin a significantly positive.(9) BrdU colony and appraisal on thethree generations of stem cells to contain BrdU40μ g/ml mark culturemedium, colony48h, observation visible cell growth is good, suspendeddeath cells less, further spread to immunohistochemical method of detectioncolony situation, visible part of the nuclei are tan, explain colony success.(10)Bone marrow mesenchymal stem cells allograft will induce the stem cells afterBrdU with calibration, surgery look down rats implanted inside bladder, takeout after four weeks rat bladder to immunohistochemical method detection,transplantation in living cells rat bladder can continue to survive and evendistribution.Conclusion:(1) Bone marrow after training was not extracted a singlevariety cells, bone marrow, mesenchymal stem cells account for only a smallpart, need human influence factors, make its augmentation, purification, andfinally form the cells with stem cells as community.(2) MSCs first slowgrowth after fast again slowly, is typical of the S curve, we found that in theexperiment of MSCs P4within the strong ability to form stable, metabolicexuberant, and can be used in the subsequent experiment.(3) The best time isthe first time for liquid12to24hours, depending on the concretecircumstances, in general liquid interval for3to5days, going down toposterity time according to the experimental requirements can be flexible.(4)The most suitable environment for the growth of MSCs containing10%tirebovine serum and90%DMEM medium (L), the most suitable for MSCs to muscle cells into the environment for10%90%serum, horse DMEM (L) and10μ mol/L5-Aza-CR constitute induction training system.(5) After inductiondesmin and myosin stem cells protein are positive, expression that5-Aza-CRMSCs induction into muscle cells to differentiate is successful.(6) Classicsappraisal, the experiment of stem cells extracted from the cell surface antigenCD29mark, CD44positive, CD34, CD45negative, the system of cultivatingthis experiment mainly for bone marrow cells mesenchymal stem cells, highpurity, active big, and can be used in the experiment.(7) After the examination,the SD rats mesenchymal stem cells in bone marrow transplantation into thedetrusor unable to model the rat, can continue to live in rats in the bladdermuscularis, transplantation continuous observation mice after3months, wasnot found to have adverse reaction, the experiment proves: SD ratsmesenchymal stem cells in the bone marrow allograft safe and effective. |
PDF Full Text Request