Construction And Indentification Of Astrocytes Lines Expressing BDNF Steadily

Neurotrophin(NT) is a kind of little protein which product by nerve tissues or their target tissues. Since NT has firstly been found in 1950s, there are six family members including nerve growth factor( NGF), brain-derived neurotrophic factor( BDNF), neurotrophin-3( NT-3) ,neurotrophin-4/5 (NT-4/5), neurotrophin-6(NT-6), neurotrophin-7 (NT-7).For NT take a important effect on neuronal generate,differentiation survival,against neuronal apoptosis ,protecting and repairing pathological changes or impaired neuron .Therefore, in many neutron degenerate diseases with the character of neuron death or analosis ,such as Alzheimer's disease(AD),Parkinson's disease(PD),Amyotrophic lateral sclerosis(ALS),NT has a extensive therapia perspective. Consequently, studying on NT is always a very important field in neutronscience and it's a hot spot of study and research topic in neutrobiology . Nowaday, we cured central nervous system diseases with NT ,it is the most difficult that NT cannot pass blood-brain barrier (BBB) in adult brain .On the other hand ,by peripheral administration ,because NT have positive charge ,it soon enter liver for metabolish. It is unstability in blood circulation. So there is no other way in which NT can enter inner brain ,except the method of injecting directly to cerebral ventricle. In order to obtain easily transfection,highly express and harmless target cell, we choose astrocyte as target cell . Accordingly , we establish BDNF eukaryote expression vectors, and then stably transfect astrocyte. As a result, we gain stable transfection astrocyte and it can effectually express and secrete biological activative BDNF . Because the development of engineered , it makes possible to extraorgan synthesis BDNF. In this study , we gain the RNA of rat brain . According to the gene of BDNF which take from Genbank designed and synthesized primer. We amplified BDNF by reverse transcript-polymerase chain reaction (RT-PCR )and recombined it with pRK plasmid . And then this plasmid transferred into Bacterium coli DH5α. It was culture on the nutrient medium including X-gal , IPTG and Chanamyn ,and white coenobiums were recombined bacterium In order to gain a bulk of BDNF recombined bacterium,we cultured dilatedly it .Next step is to enzyme cut the colon production, and a great deal of copy BDNF fragment connect with pCI-neo mammalian expression vector. The result of BDNF sequencing show the sequence of inserting part seem to literature report .That prove PCI-neo/BDNF recombined plasmid construct successfully. Because unneurocyte such as fibroblast,immortal nerve cell line and neurocyte have many personal defect. But the astrocyte have the advantages of harvesting easily ,culturing in vitro easily ,transduce gene easily and can express and secrete gene production and have a long life .So we choose astrocyte with fissionability as target cell in our research. Using the recombinative vectors to transfect astrocyte, it is sifted by G418.Consequentely, the transfected astrocyte can grow on G418 and untransfected astrocyte are all death. Then we cultured dilatedly positive cloned cell and gain the astrocyte of stablely expressing BDNF. Whether the target cell can express objective gene after transfection or...