The Effect Of Gene Clone And Gene Engineering Cells Of Brain-derived Neurotrophic Factor (BDNF) On Brain Slice In Vitro

ObjectiveParkinson's disease(PD) is a chronic degenerative disease of the extrapy-ramidal system. Primary pathological change is degeneration of dopamine neurons in the substance nigra of midbrain resulting in denaturalization and vanishing of dopamine nerve fibers in the striatum. The damage of the substance nigra- stiatum pathway causes decreasing of the content of dopamine in the substance nigra. This is the cause typical symptoms of PD in clinic. At present remedial study of PD in clinic include two aspects: one is drug administration, such as L- dopa, in order to supply enough dopamine to improve clinical symptoms. But this type of medication can not cure PD but improve clinical symptoms. The other is nerve nutrition factorial therapy so as to save the surviving neurons and renew the function of the denaturalized neurons and nerve fibers.Brain-derived neurotrophic factor (BDNF) belongs to nerve growth factors. The relation between BDNF and development of the nerve system is very osculating. BDNF act on neuron's growth, multiplication, deposit and repair-ment. A large number of members of nerve growth factor family are cloned and used to cure nerve system disease recently.According to the investigation trend above, we constructed the substance nigra - striatum pathway in vitro by brain slice triple culture of neocortex - striatum - substantia nigra. BDNF was cloned by RT - PCR and BDNF eukaryotic expression plasmid pEGFP - N1 - BDNF was constructed. The recombined pEGFP - Nl - BDNF plasmid was transfected into bone marrow stromal cells( BMSCs) with lipofectamine. We checked up constructed gene engineering cell by fluo - microscope and immunocytochemitry and collected the gene engineering cell' s medium or put the cells to act as breeding layer of brain slice triple culture. We observed the effect of gene clone and gene engineering cells of BD-NF on brain slice in vitro.MethodsA. The isolation, culture of BMSCs and the induced differentiation of rat bone marrow stromal stem cells towards neural cells1. The isolation and attachment culture of rat bone marrow stromal stem cells2. The expansion and purification of rat bone marrow stromal stem cells to provide higher purification seed cells for experiment.3. The identification of BMSCs: the surface markers CD71 and CD34 of the cultured cells were identified by immunocytochemitry.4. The preparation induce medium;gather brain slices culture fluid.5. The identification of induced cells by immunocytochemitry: the expression of the differentiated cells Nissl bodies, NSE, GFAP and TrkB was identifiedB. The construction of pEGFP - Nl - BDNF plamid and establishment of BMSCs cell with stable expression of BDNF.1. To distill overall RNA of rat embryo brain.2. To expand BDNF gene by RT -PCR3. To construct and checkup carrier of PMD -18 - T - BDNF4. To construct and checkup carrier of eukaryotic expression plasmid pEG-FP-N1 -BDNF5. The recombined pEGFP - Nl - BDNF plasmid was transfected into BMSCs with lipofectin6. To observe transfected BMSCs and identify BDNF by immunofluores-cence.C. The effect study of transfected BMSCs with BDNF on the and the stria-turn brain slice in vitro.1. Organotypic brain slice triple culture of neocortex - striatum - substia -nigra of neonatal rat.2. The ability that the slices absorb EB was tested with LCM5103. To identify TH neurons and axons by immunohistochemitry.4. To mensurate sensity and content TH - positive cells and fiber.ResultsA. The isolation, culture of BMSCs and the induced differentiation of rat bone marrow stromal stem cells towards neural cells1. Twelve hour after inoculation, marrow single nucleus fully attached to the bottom of the culture bottle. The cells mainly took the shapes of shuttle and circle. Fifteen day cell dyed by Spectrophotometric increased swirl.2. The immunocytochemitry of the surface markers CD71 and CD34 of P6 cells. The results were that CD71 was weakly expressed in all cultured cells. CD34 was not observed. This proved that cultured cell is an offset of BMSCs.3. Slightness cell appeared Nissl reaction result, which were induced by brain slices medium.4. 60% cell of cultured BMSCs expressed NSE and TrkB, GFAP was not observed in this cells.B. The construction of pEGFP - Nl - BDNF plamid and establishment of BMSCs cell with stable expression of BDNF1. Eighteen hour after transfected plasmid into BMSCs, we could see green fluorescence in pEGFP - Nl - BDNF groups and pEGFP - Nl empty carrier. In reverse fluomicroscope, GFP positive BMSCs were about 60 percent.2. Transfected BMSCs is green fluorescence by immunofluorescence. positive BMSCs were about 50 percent . BDNF was expressed intransfected cell.C. The effect study of transfected BMSCs with BDNF on the and the striatum brain slice in vitro.1. ExDeriment croups brain slices crowed rapidlv and had wider halo thancontroll group. The fibres of neuron from the stratum growth to neocortex with hasten direction. Neocortex spreaded tuber without direction.2. The fibres of TH - positive neuron from the substa - nigra growth to stri-atum. At 20 day and 30day. The density of positive TH - fiber density was that Experiment groups fl and I were markedly denser than controll group (p < 0. 05). but at 30 day Experiment groups II were markedly denser than controll group(p<0.01).3. At lOd and 20d, there was not red fluores - cell in experiment groups II and I . But at 20d, about lOpercent cell died and were dyed red by EB in controll groups. A Larger area red fluores - cell appeared at 30d in controll groups brain slices.Conclusion1. Expression of the neuron - specific marker NSE and formation of Nissl body were observed in BMSGs after induction with brain slices medium, which may attribute to the activation of Erk signaling pathway by nerve nutrition factors.2. The eukaryotic expression vector pEGFP - Nl - BDNF were constructed and confirmed with sequencing. The recombined pEGFP - Nl - BDNF plasmid was transfected into BMSCs by using lipofectamine. We proved that transfected BMSCs can expressed BDNF protein by immunofluorescence. So gene engineering cells of BDNF with fluorescence marker were obtained.3. The brain slice combining triple culture of neocortex - striatum - substa - nigra on Millicell - CM membrane is a simple and effective method in vitro.This method may be used for PD investigation .4. Transfected BMSCs is induced to differentiate toward neuron by brain slices medium and BDNF, which may promote the growth of brain slice and neocortex - striatum.