Experimental Study Of TAT-BDNF, A Neotype Neurotrophin, Therapy For Acute Spinal Cord Injury In Rats

BackgroundRepair of spinal cord injury(SCI) is always a challenging problem in medical science. Recently, while the development of building industry and transportation, Morbidity of SCI increases year by year. Traumatic SCI causes disability in 11,000 Americans yearly. Most SCI usually cause different degrees of deformity, and lay an often overwhelming financially burden on the patients and their families, so do the society. This signals an urgent need for new treatments that will improve quality of life and lessen suffering after SCI. It becomes an major subject for basic and clinic research in the spine surgery and neuroscience. Encouraging progress has been made on the mechanisms of SCI. It has been recognized that much pathological factors involved in the spinal cord following injury after primary injury process, which cause more serious injury. To attenuate the secondary degeneration has been considered critical to the regeneration of spinal cord and outcome of the patients of SCI. During last two decades, or so. Investigator developed varies kinds of pharmacological strategies are developed to reduce injury and enhance the recovery, till today, only high-dose intravenous administration of methylprednisolone in 8h is proved clinicallyuseful for post traumatic human spinal cord, but the therapy isn't content. Therefore, to develop more effective strategies for reducing secondary degeneration is urgently needed.Many studies shown that insufficient neurotrophin is one of the reasons of limited regeneration ,also can not prevent second injury. The investigation on BDNF should be focus among the neurotrophin. The biological effect of BDNF has been extensively studied after being cloned since 1989. It not only plays an important role in regulating neuronal survival, differentiation, proliferation, maturation ,outgrowth and retaining the survival and normal physiologic function during the development of the central nervous system(CNS), but also can protect neurons from injury and prompt regeneration. In 2004, investigator found that BDNF is necessary and sufficient for spinal respiratory plasticity following SCI. It has the potential benefits for patient suffered upper cervical cord to recover his respiratory plasticity.The size and cationic nature of the protein preclude its passage across the blood brain barrier(BBB). using exogenous BDNF to treat CNS injury have encountered difficulty in effectively delivering therapeutic dose of neurotrophin to the intended neuronal targets, gene therapy may deliver proteins to the site of SCI,but is not suited for acute SCI therapy because the delivery of genes into spinal cord tissues and expression of therapeutic proteins are time-consuming. Moreover, spinal cord protein synthesis is severely compromised in injured areas after SCI. A new and efficient delivery technology termed protein transduction has been indentified to cargo exogenous macromolecules or charged compounds into living cells. In fact, it is a small sequences in some protein termed protein transduction domain (PTD). When exogenous proteins are linked to PTD, which can delivery them into celld or cross the blood brain barrier. The protein transduction domain (PTD) of the HIV-1 TAT protein is an 11-amino-acid peptide with the ability to rapidly translocate into cellsboth in vivo and in vitro. Fusion of this PTD with proteins has been shown to facilitate an effective transduction of the fused cargos into cultured cells or across BBB while preserving their biological activity. Therefore, for the studies described herein, we constructed TAT-BDNF fusion protein and administered BDNF intravenously in vivo to evaluated whether administration of a TAT-GDNF fusion protein may prevent spinal cord injury after SCI. so that, we can establish a technologic line of protein therapy for SCIObjective1. To harvest human BDNF gene and evaluate the biological effects.2. To construct and produce TAT-BDNF by prokaryotic expression and identify its biological effect in vitro. Also it can provide a neotype therapeutic protein for SCI.3. To investigate the contribution of TAT-BDNF fusion protein on embryonic stem cells differentiation. And to be an evidence for combine with embryonic stem cells transplantation treatment for SCI.4. To evaluate the transduction efficiency and beneficial effects of TAT -BDNF fusion protein in vitro for acute spinal cord injury in rats. So to establish a technologic strategy of protein therapy for SCI.Methods1. we harvested human BDNF gene from human embryonic brain cDNA lib by reversetranscriptase polymerase chain reaction (RT-PCR). BDNF was cloned into multi-clone-sites in pIRES2-EGFP vector with restriction enzymes. Furthermore, we subclone BDNF-IRES2-EGFP fragment into EcoRI/Hpa I sites of MSCV vector to construct recombinant virus vector MSCV-BDNF-IRES2-EGFP (MBIE) which can express both BDNF and ehanced green fluorescent protein(EGFP). we obtained transfected PT67 cells which can stable packaging MBIE virus constantly after G418 selecting, and then using the virus infected neural stem cell line C17.2. The stable transfected C17.2 that express BDNF and EGFP was selected with clone cylinder. The primary cultured DRG from E16d kunming mouse were co-culture with BDNF transfeted C17.2 for lOd. C17.2 was identified by x-gal stain. The expression of BDNF was assessed by immunohistochemistry enzyme linked immunosorbent assay(ELISA) and RT-PCR .and application of Immunocytochemical stain to evaluate the biologic effects of stable gene-modified neural stem cells on embryonic mouse DRG neurons in a dual-chambered cocultivation system.2. With designed primers ,the signal peptide sequence in BDNF gene was kick out by RT-PCR. Purified polymerase chain reaction fragments were cloned into the Xho I/EcoR I sites of the pTAT-HA vector (gifts from Dr Steven F. Dowdy,USA). TAT-GDNF was expressed in Escherichia coli strain BL21 (DE3) pLysS and lysed by sonication. Ecoli lysates were denatured in 8 mol/L urea and GuHcl. Bacterial debris was pelleted and supernatant was subjected to metal- affinity chromatography using an Ni-NTA matrix . The purity of the eluted protein was assessed using SDS-PAGE, renaturated using GSH/GSSG system and quantitated by BCA. In order to evaluate the protein transduction effect of TAT-BDNF, it was added into cerebellar granule cells culture media on 3Days. lOmin later, Immunofluorescence analysis of transduction of TAT-BDNF were performed. lOOuM glutamate and lOOng/ml TAT-BDNF were added in the cultured media for cortical neurons of rats. After 12 hours, Immunofluorescence assays were performed using conjugated FITCantibodies to study TAT-BDNF protection of cortical neurons from excitotoxic insult.3. Embryonic stem cells were cultured in the absence of leukemia inhibitory factor and MEF for 4d and formed embryonic bodies(EBs). EBs were exposed to RA (106 M) for 4 days. Cells were then dissociated, plated onto glass coverslips and allowed to differentiate in DF12/N2 medium for 7, 14 or 21 day. In treatment group, BDNF and TAT-BDNF were added to induce differentiation. The production of specific cells from differentiating ES cells was evaluated by following the NF200 which is a marker of mature neuron, and the expressions of tyrosine hydroxylase (TH) and glutamic acid decarboxylase (GAD), which are markers of dopaminergic and GABAergic neurons, which are the major source of synaptic inhibition in the nervous system. Immunofluorescence assays was used to evaluate the effect of TAT-BDNF on ES cells differenitiation.4. Immunocytochemical and western blot analysis of central nervous system tissue TAT-BDNF content were executed after intravenous TAT—BDNF fusion protein. Additionally, TAT-BDNF or vehicle was intravenously applied immediately after acute compressed spinal cord injury . TUNEL stain and Nissle stain were used to analyze neuroprotective effect of TAT-BDNF at 3Days and 7 Days while apoptosis cell ratio is at peak. The function of hindlimb movements were evaluated in BBB scales and as a reference to evaluate the decreasing effect of the function loss .Results1. The fragment and sequences of BDNF were confirmed by essay of DNA sequences and electrophoresis. The gene transfected Packaging cells PT67which express EGFP were selcected. The gene-modified neural stem cells C17.2 were obtained after being infected by recombinant virus supernatant and selected by clone cycline. RT-PCR analysis demonstrated expression of human-BDNF. ELISA confirmed the presence of secreted BDNF and showed that The level of BDNF production from NSC-BDNF transfected was at a rate of 14.6+0.8 ng /24h/106cells even after 3 months.With immunohistochemical analysis, Compared with the control,the longer neurite outgrowth of cultured DRG cells and the more survival neurons were showed in BDNF transfected neural stem cells group.2. The BDNF fragment without signal peptide sequences was confirmed by essay of DNA sequences and electrophoresis. The pTAT-HA-BDNF plasmid is then transformed into a BL21(DE3) and Optimization of culture conditions were determined that was induced 4h under 26 °C, 0.5mmol/L ITPG The identities of the overexpressed proteins were confirmed by SDS-PAGE and Western blot analysis with monoclonal antibody. Immunofluorescence outcome shew that TAT-BDNF can rapidly delivery into cerebellum granular cell cultured in vitro. During the study of excitotoxic cortical neurons induced by glutamate. TAT-BDNF can decrease the apoptosis ratio and enhance neurons survival.3. Embryonic stem cells cultured on the MEF were proliferating like a clone clump, and express SSEA-1. when withdraw LIF and MEF ,embryonic stem cells aggregate termed embryonic bodies. After induced by RA, the addition of BDNF during the differentiation stages promotes neurons survival and neurite longer and thiner, and increase the proportion of mature neuron. The study also show that embryonic stem cells can differentiated into dopaminergic neurons and GABA neurons in vitro.4. Immunocytochemical and western blot analysis of central nervous system tissue confirmed that intravenous TAT-BDNF can cross blood-brain barrier throughout CNS. Additionally, TAT-BDNF significantly decreased the apoptosis ratio and enhance neurons survival in vivo after acute spinal cord injury in rats. According to the time curves of BBB scale of hindlimb movements, TAT-BDNF group relic function was more than control group.Conclusions1. Not only the structure of human BDNF gene that harvested in this study is correct, but also the protein encoded by this gene have biological effect.2. TAT-BDNF fusion protein produced by prokaryotic expression system can effectively enter neurons cultured in vitro and protect neurons from excitotoxic insult.3. TAT-BDNF fusion protein can enhance embryonic stem cells differentiation of neuron and prompt neuron mature.4. Intravenous TAT-BDNF fusion protein can reduce second injury across blood brain barrier and improve function of spinal cord.