Construction Of Recombinant Adeno-Associated Virus Vector Encoding BDNF And Its Protective Effects On Rat Cerebral Ischemic Injury

Cerebral infarction is a major public health problem greatly endangering human health, rehabilitation of which remains to be a medical challenge. As there is no practically effective way to rescue the dead nerve cells, patients are often subjected to irreversible neural dysfunction. When cerebral ischemic injury extends to the maximum, there is almost no way to recover the neural function. At present, studies on the treatment of cerebral infarction have progressed from improving blood supply of the ischemic area to protecting the brain or promoting regeneration of the injured brain tissue. In the research of repairing the injured brain tissue, the protective effect of BDNF has gradually aroused people's interest.Brain-derived neurotrophic factor (BDNF), which is the second member of neurotrophin family and widely distributed throughout the central nervous system, activates various signal transduction pathways in cells and exerts its biological function by binding with the high-affinity TrkB receptor to activate protein tyrosine kinase. BDNF not only plays an important role in regulating neuronal survival, differentiation, proliferation, maturation and outgrowth during the development of the central nervous system (CNS), but also is essential in retaining the survival and normal physiologic function of mature neurons.These concepts are being tested in clinical trials. Therapeutic use of Neurotrophic proteins seems practical for diseases of the neuron injury, where they can be given by systemic administration. Administration of exogenous neurotrophic factors after nerve injury has been shown to mimic the effect of target organ-derived trophic factors on neuronal cells. But the therapeutic use of these factors in human disease has been limited by the short serum half-life and dose-limiting side effects of these potent peptides. Effective gene transfer into injured brain by viral vectors is the potentials for gene therapy of CNS nerve injury.Gene introduction system is the core technique of gene therapy. The key point of this research is how to introduce the interested gene into organism cell in order to express effective and safely. Adeno-associated virus (AAV) offers many desirable features of other vectors without some of their potential drawbacks for application in gene therapy. The recombinant AAV vectors can transducer both dividing and non-dividing cells in vitro and in vivo. Efficient and long-term transduction in vivo and the lack of both cytotoxicity and cellular immune responses are the hallmark features of AAV mediated gene transfer. AAV was regarded as one of the extreme useful vectors in gene therapy in future. Although AAV vectors have been extensively used in gene therapy for genetic and metabolic diseases, few studies have used this vector system for ischemic brain injury therapy.In view of the above mentioned problems, our experiments were designed to study the protective effects of brain-derived neurotrophic factor (BDNF) gene mediated by recombinant adenovirus in vitro, on repair of nerve injury in the cultured hippocampal neurons and the MACO rat models. Concrete works are as follows.1. First of all, we constructed adeno-associated virus vector encoding the SD rat brain derived neurotrophic factor (BDNF) gene and examine its ability to express the BDNF gene in vitro. The specific BDNF sequence was cloned into the plasmid of pAAV-MCS in AAV helper-Free system to construct the BDNF expression plasmid pAAV-MCS-BDNF. The recombinant plasmids were identified by DNA sequencing and restriction digestion. Then the packaging cell lines (HEK293 cell) were cotransfected with the pAAV-MCS-BDNF together with the control plasmid pAAV-RV and pHelper by phosphate-calcium deposit method. The recombinant adeno-associated virus vector infected the hela cell and cultured hippocampal neurons, the expression of BDNF was detected by Western blot, RT-PCR and Elisa method. The results showed the recombinant adeno-associated virus vector carrying the BDNF sequence was constructed successfully, the viral titer was 1.29×108 and it could up-regulated the expression of the BDNF gene following with the time. The recombinant adeno-associated virus vector rAAV-BDNF can enhance the expression of BDNF gene significantly, which lays the basis for its application in the treatment of the neurodamaged disease.2. In the work above, we have adopted a universally accepted method to establish a stable system in culturing hippocampal neurons. On the basis of this cell model, we have carried on the AAV infection and checked the toxic effect. We finally controlled this kind of toxic effect within experimental error range permission. We further found that AAV-BDNF enhanced the expression level of endogenous BDNF in the hippocampal neurons. We detected AAV-BDNF upregulated the dendritic growth by the MAP2 stain (P<0.001). AAV-BDNF activated the BDNF-induced activation of downstream signaling pathways, especially the MAPK/ERK1/2 pathways. AAV-BDNF was required for serum free-induced neuronal protection, which is dependent on the activation of MAPK/ERK1/2 pathways.3. The virus was shot into the brain ventricle of MCAO rat. We checked if it can protect cell death .The control group is AAV-GFP. Brain infarction area was differentiated by TTC dyeing. The result showed infarction area near the brain ventricle of AAV-BDNF group is smaller than that of control group in certain degree. This explains that in rat MACO models, AAV-BDNF can block the injury effect of ischemia in the brain.Taken together, our results established an essential role of AAV-BDNF for neuronal protective effect, thus promoting neuronal survival and dendritic morphogenesis. Identifying the molecular mechanisms that control AAV-BDNF efficiency and its subsequent signal regulation should facilitate our efforts in understanding how to cure the nerve injury in the nervous system.