| Objective: Bone marrow stromal stem cells（BMSCs）are the most commonlyused seed cells in bone tissue engineering.It has a strong proliferation ability,Under certain conditions, it can be induced to the tissue that differentiate intomesodermal origin. such as bone, cartilage, fat, etc. It can repair and reconstruc-tion tissue defect due to various reasons such as trauma, infection, tumor tissueetc,and provide an ideal treatments for them, It is a technology that have a goodapplication prospect. Bone marrow stromal stem cells （BMSCs）is a goodcarrier for bone tissue engineering, with its purpose as the carrier to carrygenetic model NGF(Nerve gorwth factor) and BMP2(Bone morphogeneticprotein) transplantation into the fracture model of rats in order to promote thehealing of fracture is this topic (BMP2and NGF genes transfection marrow stromal stem cell transplantation to treat fracture experiments, the item numberis81160223) of the main research content. Bone marrow stromal stem cells（BMSCs）in the bone marrow is extremely low content, only accounted for0.001%-0.1%, bone marrow nucleated cells in vitro is difficult to get. It is veryimportant for the cells of tissue engineering and vivo or vitro experiments tocultivation BMSCs what is high purity, strong vitality and have the samebiological characteristics in vitro.To establish a simple and effective way tocultivation, purification, identification bone marrow stromal stem cells, and toidentify the cell morphological observation, surface markers andmultidirectional differentiation capacity test. In order to further understand thebiological characteristics of bone marrow stromal stem cells and provide reliabletheoretical basis for its application in clinical.Methods:.To take off the neck of4weeks healthy SD rats to death. Soakthem in75%alcohol about10minutes. Remove the femur and tibia underaseptic conditions.clean soft tissue dissection and muscle on the surface of thefemur and tibia. after rinse with PBS, cut open on both sides of the extremities,use1ml syringe with Low Glucose DMEM, from the long bone end insertsyringe rinse twice, after washing liquid put in15ml sterile tubes, with3mlstraw blowing appropriate marrow cells.Put the suspension in70mm cell culturedish, adding suitable amount of complete medium (Low Glucose DMEM+15% fetal bovine serum,+1%Penicillin and streptomycin).Placed at37℃and5%CO2thermostatic cultivation in the box. By sticking cultivation method tofurther extend and purification cells, observe its growth characteristics.Observation and analysis of the relevant characteristics of the cells, Draw thegrowth curve. the growth state good P7generation cells in flow cytometryinstrument to detect cell surface markers CD29, CD45, CD90and type withcontrast, growth in good condition of P5generation of cell differentiation in thedirection of osteogenesis, adipogenic and separately alizarin red and oil red Ostaining, observed induced results.Results: By the method of SD rat BMSCs cultured in vitro colony was likeadherent growth, cell morphology mainly fibroblast-like cells, cell clustersunder the microscope visible growth, similar to the swirling, fish-shaped ordaisy petal.P1-P5of BMSCs are fast generation proliferation and cellmorphology stability and better growth status.Then slow down gradually, P10generations obvious aging cells form, the cell edge is rough, body becomes largeand irregular shape.Growth curve showed that bone marrow stromal stem cellsin normal cell growth characteristics and active growth.Using flow cytometryinstrument to identify the cell phenotype: BMSCs expressed CD29and CD90,without expressing CD45, It explains the cells cultured by this experimentaccord with rat BMSCs phenotypic characteristics. Osteogenesis induced liquidinduced cultured BMSCs can be obviously calcium nodules after alizarin red staining, and the control group did not see the calcium nodules. Adipogenicinduced liquid induced cultured BMSCs can be obviously lipid drops after oilred O staining, and the control group is not obvious. SD rat BMSCs culture bythis experiment has the ability of osteogenesis and Adipogenic differentiation.Conclusions: This experiment is a relatively simple method to isolation andpurification BMSCs.It can obtained the bone marrow stromal cell of high purity,good activity and traits homogeneous.Cells to grow strong and can berepresented more than10generation continuous stability.The operation is simple,but it can do lots of separation, purification and amplification BMSCs, thosecells have general biological characteristics of bone marrow stromal stem cells, after induction training has a multi-directional differentiation potential. Canprovide abundant source of seed cells for tissue engineering, have importantpractical significance. |
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