An Experimental Study Of The Effect Of A New Type Neurotrophic Factor On The Glucose Deprivation, Hypoxia/Reoxygenation Injured Cell Model

The cerebral ischemia/reperfusion injury is believed to be a key factor in inducing the cerebral ischemic stroke, unfavorable prognoses of the thrombolysis therapy and the resuscitation of the heart, lung, and brain. Its pathophysiological process is very complicated. Studies have found that the organism is capable of producing endogenous neurotrophic factors participating in repairing injured nerves after cerebral ischemic injury. Thus we presume if huge amount of exogenous brain-derived neurotrophic factors (BDNFs) participate in the process of cerebral ischemia/reperfusion injury, the pathological process of cerebral ischemia/reperfusion injury may possibly be effectively attenuated or reversed. However, exogenous BDNFs, as macromolecular protein, are difficult to be medicated through the peripheral path. Neither can they pass through the blood brain barrier (BBB). Therefore, how to make exogenous BDNFs effectively pass through the BBB has become the high light of studies.In this study, the carrier pET-PTD-BDNF able to fuse and express in prokaryotic organisms was constructed by taking the BDNF gene with optimized nucleotide sequence as the model through modification of pET-32a carrier by gene engineering subclone technologies. The carrier was introduced into the engineering bacterial strain BL21. IPTG was added to induce its expression. Results showed the constructed expression vector pET-PTD-BDNF was completely correct. After the BL21 containing recombinant plasmid pET-PTD-BDNF was induced by IPTG, SDS-PAGE analysis showed that the pET-PTD-BDNF plasmid BL expressed PTD-BDNF fusion protein. Besides, it was soluble expression. Further analysis of Western blot showed the obtained protein was the interest protein.Secondly, the optimal conditions for MTT colorimetry was determined using the orthogonal test by taking SH-SY5Y cells as the subject as follows: (0.2×10⁸/L, 100μl) as the cell number in each well, 6 hours as the staining time, and 5 g/L as the dose in the MTT. The biological activities of BDNF were detected under these conditions. When the concentration of BDNF was 50μg·L^-1, the cellular absorbance was not significantly increased along with increased concentrations of BDNF. It was able to get favorable dose-effect relation.Finally, the glucose deprivation, hypoxia/reoxygenation injured SH-SY5Y neural cell model was established. The morphology of cells was observed under microscope. The cell survival rate was determined using MTT colorimetry. The cell vitality was detected using trypan blue exclusion test. The LDH release rate of SH-SY5Y cells was determined. The cell apoptosis was observed using Annexin V-FITC and Propidium Iodide double staining methods under a fluorescent microscope. The cell death and apoptosis were detected using flow cytometry. The protective effects of the new type neurotrophic factor PTD-BDNF, the exogenous BDNF (the referential product), pcDNA3.1/BDNF (the gene fragment expressed by gene transfection method) on glucose deprivation, hypoxia/reoxygenation injured SH-SY5Y neural cells were compared.Results under microscope showed huge amount of died cells in the injury model group, while cells survived more in the pre-protective group and the protective group with BDNF and PTD-BDNF, better in the pre-protective group than in the protective group. After ATRA induction, compared with those of the control group, the numbers of died and apoptotic SH-SY5Y cells with transfection of expressed BDNF gene were obviously reduced when they received the glucose deprivation, hypoxia/reoxygenation. The numbers of died and apoptotic cells were insignificantly increased when compared with those of the BDNF and PTD-BDNF pre-protective groups. In the tests of the survival rate, death rate, and LDH release rate of SH-SY5Y cells, the cell survival rate of the injury model group was obviously lower than that of the normal control group, while its cell death rate and LDH release rate were obviously raised. The cell survival rate was improved, the cell death rate and the LDH release rate lowered in the pre-protective group and the protective group with BDNF and PTD-BDNF, with better effects obtained in the pre-protective group than in the protective group. In the experimental groups of gene transfection, compared with the transfection injury model group, the cell survival rate was significantly improved, the cell death rate and the LDH release rate lowered in the transfection injury model+ATRA group. The same conclusion was obtained in the experiment of detecting SH-SY5Y cell death and apoptosis by the flow cytometry.Conclusion: The new type neurotrophic factor, PTD-BDNF can entry the injured cells, showing protective effects on glucose deprivation, hypoxia/reoxygenation injured cells. Besides, better effects were obtained pre-medication than post-injury medication. The BDNF gene fragment introduced by gene transfection method and the administration of exogenous BDNF (the referential product) show the same protective effects on glucose deprivation, hypoxia/reoxygenation injured cells.