The Study On Bone Marrow Stromal Cells Transfected With Noggin Gene Differentiating Into Neuron-like Cells In Vitro

The study on bone marrow stromal cells transfected with Noggin gene differentiating into neuron-like cells in vitroObjectiveCentral nervous system disease such as stroke and degeneration can lead the damage even death to the nerve cells .Of course the functions of these cells are lost. How to repair the neural pathway. The key is what can take the place of the lost nerve cells. Because of the non-reproducibility of the adult nerve cells , we put our attention on the stem cells .The cells that can differentiate to nerve cells include embryonic stem cells, nerve stem cells, bone marrow stem cells .The factors of source and ethics constraint the development of embryonic stem cells and nerve stem cells. Then , bone marrow stem cells have relative wide prospect.bone marrow stromal cells(BMSC), have wide potential of differentiation. It belongs to multipotent stem cells. With the definite cultural condition in vitro, BMSC can differentiate into sclerotomal cell, cartilage cell, adipose cell, sarcoblast, and so on..It even can differentiate into horizontal cell, oligodendroglial cell and neurone. Because of the wide potential of differentiation , BMSC is the ideal tool of cell therapy and gene therapy. At present , three ways can differentiate BMSCs into nerve cells--antioxidant agent, growth factor, and transgenic way. We lead Noggin gene -an important neural inducer in embryos into the way of transgenic induction.Noggin was originally isolated from Xenopus embryos by Harland R.M. and Smith W.C. in 1992 . Subsequently , people found that noggin plays an important role in normal development , especially of inducing dorsal development. As a endogenous neural inducing signal , noggin not only can be found in early vertebrate embryogenesis but also in the central neural system (CNS) and peripheral neural system (PNS) of adult mammalian . Noggin has been identified to be the neural inducer in vivo and in vitro , which effect neural induction through antagonizing bone morphogenetic protein-4(BMP4).Respecting above theory support, we construction the carrier with Noggin gene, transfect it into BMSCs. In our anticipation, this way can induce BMSCs into nerve cells, then , we detect the cells after differentiation.Methods1. After isolated by density gradient contrifugation with Percoll, the rats' bone marrow stromal stem cells were cultured withα--MEM containing 20% fetal bovine serum in the plastic flastic. When the cells covered the flask, they were passaged with pancreatin. Their morphology were observed by microscope. Detect the growth curve and vitality of BMSCs .2. We have the plasmid with the purposal gene-Noggin transformed into the E.coli strain DH5α. Then transformation colonies were picked at random, and the plasmid were extracted . Having been verified via restrictive endonucleases digest, PCR.3. The fourth and sixth generation of BMSCs were transfected the expression vector with Noggin by lipofectin. After culturation, observe the shape of the cells, detect the differentiated cells if they express specific marks of nerve cells, ex. NSE,MAP-2 and horizontal cells' specific marker ex. GFAP by Immunocytochemical stain, detect the expression of the Genes participating nerve cells' physiological functions ex. NCAM,GAP43,SYN1 and the marker gene of horizontal cells-GFAP.Results1. The cells isolated by density gradient contrifugation with Percoll are globular mononuclearcell , The vitality of the isolated cells was 97. 41%±1. 01.After cultured in the plastic flask for 10-14 days, the cells covered the bottom , the shape was radiat, paliform, or swirl. Then the cells can be passaged. After the cells were passaged for 3-4 times, they had the typical morphology .The growth curve was just like the shape of "S", the first and second day , the number of the cells was no marked change, even a little reduced. The third day, the number began to increase, the forth day , the cells entered logarithmic growth phase , the ninth day, the cells' number is stable. From the tenth day , the cells began old, and the number of the cells began reducing.2. After restrictive endonucleases digest and PCR , we can verify that the plasmid include the part about 760bp , just the length of Noggin gene.3. The BMSC transfected the vector displayed the neuron morphologies.It was confirmed that the differetiated cells expressed NSE, MAP-2 but no GFAP by immunocytochemistry; expressed the Genes participating nerve cells' physiological functions ex. NCAM,GAP43,SYN1 but no the marker gene of horizontal cells-GFAP.Conclusions Transfecting Noggin Gene can induce BMSCs into nerve cells, and after transfection the cells have a part of functions of nerve cells .