Construction Of The Vector Of Human β-NGF Gene And Gene Expression In Rabbit's Bone Marrow Mesenchymal Stem Cells

Nerve growth factor(NGF) was found by Levi- Montalcini et al in 1952 , for which they were rewarded the Nobel Prize of Physiology in 1986.NGF is one of the most important neurotrophic factors in immune system, hematogenious system, genitourinary system, endocrine system and particularly the nervous system. NGF exerts great effect on development, growth, differentiation of nerve cells and their surviving and rehabilitating. NGF has also been proven effectively in the clinic treatment of nervous system disease, such as Alzheimer's disease and nerve injury .The molecule of NGF is composed of three sub-units ( α ,β and Y ).For the β sub-units represents almost entirely its biology function, NGF is also called β -NGF. At present, it is still one of the important task in the field of neuroscience that the expression of the NGF gene and the biology function of its protein, as well as its clinic application. NGF can be abstracted directly from animal tissue, but this method provided only small amount farless than enough. Now the manual recombining NGF protein can solves this problem. However, the natural NGF protein is of high molecular weight, it could not permeate through the blood-brain barrier(BBB).So it is key to find a proper method in reason for remedy . The gene therapy is one of the most promising ways.People have made lots of researches on the donate cell for transplant and genetherapy. Bone marrow mesenchymal stem cells(BMSCs) is a group of multi-potential stem cells which can differentiate to bone cell, fat cell, cardiac muscle cell and nerve cell. BMSCs is dominate in such aspects as (1)it is easily obtained and culted ;(2)it permeates through BBB and survives for a long time in brain;(3)it express transfected gene;(4)it seldom causes immune reaction. So BMSCs has better foreground as the seed cell applied to cell transplant and gene therapy. The aim of the current study is to construct the eukaryotic expression vector of human β -NGF gene and make it express in rabbit's BMSCs. Methods1. The human genomic DNA was extracted from the leucocyte. A pair of specific primers was designed. The prep- P -NGF gene fragment was amplified by polymerase chain reaction(PCR) and inserted into the pMD18 vector. The plasmid of pMD18- P -NGF was analyzed by restriction enzyme digestion and DNA sequencing.2. The human prep- P -NGF gene was obtained from the plasmid of pMD18- P -NGF by restriction enzyme digestion of BamHI and EcoKV and inserted into the eukaryotic expression plasmid of pcDNA3.0. Then the plasmid of pcDNA3.0- P -NGF was identified by the restriction enzyme digestion.3. Collected and planted the rabbit's BMSCs. The mononuclear cells from the rabbit of marrow were isolated by centrifugation over Lymphoprep and cultivated with density of 1 × 10⁶ cells/ml in T-50 tissue culture flasks in humidified atmosphere with 5%CO₂ and 37℃.4.Through the technique of gene transfection by the cationic liposome, the plasmid of pcDNA3.0- P -NGF was transfected into the rabbit's BMSCs. Screened the cells with the G418, the postive cell clones were selected and cultured.5. To identify whether the postive cell can express human β -NGF, its mRNA was tested by RT-PCR and protein by ELISA. Results1. Proven by restriction enzyme digestion and DNA sequencing, the fragmentamplified is the same to human prep- P -NGF gene(726bp).2. The eukaryotic expression plasmid of pcDNA3.0- P -NGF was successfullyconstructed.3. The rabbit's BMSCs could establish steady , culture invitro.4. After transfected and acreened by G418, these positive cell clones were obtained and proliferated.5. By the techniques of RT-PCR and ELISA, these postive cells were tested toexpress the mRNA and protein of human P -NGF. Conclusions1. The human prep- P -NGF gene has been succeesfully cloned and the eukaryotic expression plasmid of pcDNA3.0- P -NGF can be constructed.2. The human P -NGF can be expressed from the transfected rabbit's BMSCs.