Isolation And Culture Of The Mesenchymal Stem Cells Derived From Bone Marrow Of Goat

Bone mesenchymal stem cells (BMSC) is another kind of multipotential tissue stem cells besides of hematopoietic stem cell within bone marrow. These cells can be expanded and induced ,either in vivo or in vitro, to terminally differentiate into osteoblasts,chondnocytes,adipocytes,tenocytes,myotubes and neural cells. The multipotential of these cells, their easy isolation and culture, as well as their high expansive potential make these cells an attractive cell and gene therapeutic tool. Human bone morphogenetic protein 2(hBMP-2) is a member of TGF-β superfamily, which mostly exsit bone marrow . Their function that can induce original BMSC to differentiate to bone cells is crucial during bone formation, as well as bone revision, and of all members it is the most powerful in inducition formation of bone . In order to use hBMP-2 gene treat, the experiment have done the following things, and explored hBMP-2 gene transfer to goats BMSC by lipofection method. Thus layed a ideal foundation for the following animal models derived from the technology of transfer gene.1. BMSC were obtained from aspirates from goat marrow blood, and layed over the Percoll, after gradient centrifugation at 1 500 rmp for 30 min ,all of the mononuclear cells were plated in culture dish with DMEM+10%FBS and incubated at 37℃ with 5% CO2 in a fully humidified atmosphere. After 2 hours ,cells began to adhere to the bottom and gradually increasedly afterwards . The next day nonadherent cells were discarded thoroughly though washing with PBS. Fresh complete medium was added and replaced every 3 or 4 days, many adherent cellular configurations displayed mostly fibroblast-like cell with shuttle shape or flat shape. The growth curves of primary and passaged cells have the same shape "S", The sub-culturing demonstrated that BMSC begin to die after 9 passages. 2. According to the freezing methods of epithelioid cell, different density freezing media with serum and DMSO were used to freezed the cells, so as to select rational freezing medium. The results indicated that DMEM+15%FBS+10%DMSO and DMEM+10%FBS+10%DMSO have the same efficiency , taking into account of practicing economy, of the two the latter is far better. 3. The first,the fifth and the ninth generation of BMSC were inoculated on the 24-plates covered by microscopic coverglass, β-mercaptoethanol and Chinese angelica were added respectively into the plates to induce, and observed morphology changes through optics microscope. Immunohistochemical method was used to evaluate the induced cells. The experiment result showed thatβ- mercaptoethanol and Chinese angelica have the same induction efficiency, more than 70% cells induced by the two reagents expressed neurofilament(NF) and neuron-specific enolase(NSE). It suggested that goat BMSC also can differentiate into neuroncytes.4. The total RNA was harvested from human placenta tissue with TRIZOL, and hBMP-2 gene was gained by RT-PCR, then integrated into pMD18-T vector . After being transformed into E.coli.JM109, tested by sequencing, which indicated that the homology both cloned fragment and the sequence of GeneBank is 99.9%. So hBMP-2 gene was cloned successfully.5. The target fragment was digested by restriction endonuclease from reconstructed vector, and inserted into the multiple cloning site of pEGFP-C1. The conclusion can be made that hBMP-2 gene expression vector of green fluorescence was reconstructed successfully. At the time, feasibility is discussed that hBMP-2 gene express in goat BMSC. It layed solid foundation for hBMP-2 gene in vivo directly, and gave many ideal animal models for curing bone diseases of human.