Experimental Study Of TAT Mediate EGFP Into Mice Tissues And BDNF To Treat PD Model Rat

Parkinson 's disease (PD) is a common chronic degenerative disease of central nervous system (CNS) in the middle—aged and old people. It is also one of the main causes of disability for these people. The pathology of this disease is retrograde degeneration of dopaminergic neuron in the substantia nigra of midbrain and striatum which result in the functional disorder of transmitter system in striatum system. Clinically, cardinal symptoms are static tremor, movement retardation, hypokinesia, rigidity and posture balance disturbance and so on. Incidence and disability of PD are high, which puts a heavy burden on society,family and individual.At present, only symptomatic treatment can not prevent dopaminergic neuron from degenerating. Transplantation of embryonic stem cells is limited because of insufficient materials source and the low survival rate of transplanted cells. Gene therapy is also faced with the problems of safety and toxicity. So it is very important to find some new agents and medicines to prevent and treat PD.Brain derived neurotrophic factor(BDNF) belongs to the family of neurotrophin. It can promote many kinds of neurons to survive, especially the strong function of nutrition and protection to the dopaminergic neuron. BDNF can not only promote survival differentiation and growth of the dopaminergic neuron but also prevent dopaminergic neuron from degeneration caused by neurotoxicity substance.So BDNF could be a new kind of drug to treat Parkinson. But BDNF is macromolecular substance which can not permeate blood brain barrier(BBB), so it is difficult to apply clinically. TAT is trans—activator of HIV—1,and it can transmembrane into cell and transduct the covalent—bonding heterogenic protein into cell, At the same time, it also can permeate the BBB. Foreign scholars had fused TAT with protein whose molecular weight was (15～200)×10³ and found these fusion proteins could been transducted into cells. It was reported that TAT fused SOD—Cu,Zn could transmembrane the beta Cell of the islet to treat diabetes. Also TAT fused the p53 and p27 can lengthen the life span of the mice which suffered from the tumor. At present, the study of TAT fusion protein in the field of nerves system focuses on cerebral ischemia. There is no report that the TAT fusion protein treats the PD.We firstly utilized the enhanced green fluorescent protein(EGFP) as marker, constructed the plasmid TAT—EGFP and expressed the fusion protein, observing the protein distribution in different tissues after intravenous injecting the protein into mice, so that we can confirm the protein transduction ability, especially the ability of permeating BBB. Facts mentions aboveprovided the theory foundation of TAT mediates medicine to treat central nervous system disease. Then we constructed the plasmid TAT—BDNF and expressed the fusion protein, after detecting its neuroprotective activity by incubation with dopaminergic neuron in vitro. TAT—BDNF was intravenously injected into rat model of PD. Its neuroprotective effect was evaluated by observing the ethology and histomorphology of rats.in order to find a convenient and effective treatment for PD.The Study Includes Three Parts:Partâ… TAT Mediates EGFP to Transmembrane Transport into Mice TissuesMethods1. With the template pEGFP-N3, pEGFP was constructed by PCR and gene cloning technique.2. Double strands was obtained by annealing the two synthesized DNA fragment encoding TAT protein transduction domain, then it was ligated to pEGFP after digestion by restriction enzyme to construct the expression vector of pTAT—EGFP.3. pTAT—EGFP was transformed into E.coli BL21 (DE3), the fusion protein was expressed by IPTG. Molecular weight was detected by SDS—PAGE gel electrophoresis. Protein EGFP was obtained also in this step.4. The protein was purified by Ni²⁺ affinity column. 5. The fusion protein was intravenously injected into mice by vena caudalis. Four hours later, frozen section was made from brain, heart muscle, liver, spleen, kidney and so on. Distribution of the fusion protein in different tissues was observed in fluorescence microscope.At the mean time, EGFP protein was intravenously injected into the control group mice.Results1. 720bp PCR product was confirmed by agarose gel electrophoresis, which was in line with the expectancy of EGFP.2. The enzyme digestion products of recombinant plasmid pEGFPand pTAT—EGFP are 720bp and 5000bp, it is consistent with the expectancy of EGFP and pET28a. After gene sequencing, the recombinant plasmid sequence is 100% consistent with the sequence in Genebank. It showed that the EGFP gene was inserted the plasmid correctly, the recombinant plasmid were obtained successfully.3. induced by IPTG, the expressed fusion protein TAT—EGFP was maximum in 0.5 mmol/L IPTG,30℃and 3h, in the same way, the expressed protein EGFP was maximum in 0.5 mmol/L IPTG, 30℃and 4h. Molecular weight of TAT—EGFP and EGFP are 28KD and 27KD by SDS-PAGE gel electrophoresis detection.4. The purity coefficient of protein exceeds 85% after purification by Ni²⁺ affinity column.5. Four hours later, in experimental group mice, green fluorescence was detected in different tissues, including brain, heart muscle, liver, spleen, kidney and so on. Among them, the fluorescence in liver and kidney were the most obvious, while in heart muscle and brain were moderate, and so did in skeletal muscle. In control group mice no fluorescence were detected in all tissues.Partâ…¡Experiment Study of Construction and Expression of pTAT—BDNF and the Neuroprotective Effect of TAT—BDNF to Aminergic NeuronMethods1. RNA was extracted from human brain tissue, and human BDNF gene was clonedby RT—PCT.2. BDNF gene was ligated to prokaryotic expression vector pTAT/HA afterdigestion by restriction enzyme,then the recombinant expression vector of pTAT/HA—BDNF was constructed.The recombinant vector was identified by double restriction enzyme digestion and gene sequencing.3. pTAT/HA-BDNF was transformed into E.coli BL21 (DE3), the fusion protein TAT—BDNF was expressed by IPTG. Molecular weight was detected by SDS—PAGE gel electrophoresis.4. Inclusion was purified by Ni²⁺ affinity column after washing and denaturation.5. Inclusion body of the fusion protein TAT—BDNF was further detected by Western—blot.6. Embryos were taken out from the pregnant mice, the midbrain neurons were isolated and cultivated. Dopaminergic neuron was identificated by TH and MAP2 double immunofluorescence stain.7. Dopaminergic neurons were primary cultivated, and nutrient medium was exchanged twice a week. The growth state was observed by microscope.8. Cells were divided into 5 groups when cultivated in the sixth day, and different concentration TAT—BDNF was added into the third, the forth and the fifth group. In the tenth day, except for the first group, 100μg 6—OHDA was added into all groups, then cells continue to been cultivated for 24 hours. Protection to dopaminergic neuron was observed by detecting cell vigor using MTT method.9. Apoptosis was detected by hoechst 33258 and PI double immunofluorescence stain after 6—OHDA was added.Results1. Human BDNF gene of 763bp was obtained by nest PCR from the human brain,it was in line with the expectancy of BDNF.2. The product of pTAT/HA—BDNF digestedby restriction enzyme was about 720bp and 3.0kb which is in line with the BDNF gene and the pTAT/HA vector, so it showed that human BDNF had already inserted the vector correctly. Gene sequencing confirmed that pTAT/HA—BDNF according with the sequence in Gene bank. The recombinant vector was obtained successfully.3. pTAT/HA—BDNF was transformed into E.coli BL21 (DE3), and SDS—PAGE gel electrophoresis showed the protein of 35KD was produced by IPTG By changing the expression condition, maximum fusion protein was expressed.4. Most of expressed protein was reside in the sediment in the style of occlusion body, occlusion body was purified by Ni²⁺ affinity column after washing and denaturation.5. TAT—BDNF had the immunogenicity of both poly—histidine and BDNF by Western—blot, which showed that the fusion protein was integrity.6. It was identified that dopaminergic neuron were above 80% in the midbrain neurons by TH and MAP2 double immunofluorescence stain. This satisfied us that it is enough for the next step.7. The dopaminergic neuron was observed in different time, cell adherence,nervous process eruption and extension into reticulation can be observed by microscope.8. Cell growth index was detected by MTT chromatometry, the result showed that cell survival rate in the group of pre—disposal treatment using different concentration TAT—BDNF increased significantly than that of 6—OHDA group.Futhermore, the cell survival rate of 50, 20μg BDNF is higher that of 5μg BDNF.9.Slight apoptosis can be observed in normally cultivated dopaminergic neurons by Hoechst 33258 and PI double immunofluorescence stain,a lot of cell apoptosis in 6—OHDA group, apoptosis in the group of pre—disposal TAT—BDNF treatment decreased obviously than that of 6—OHDA group. Furthermore, neuroprotective effect of 50μg/ml and 20μg/ml is more significant than that of 5μg/ml treatment.Partâ…¢The Therapeutical Effect of Fusion TAT—BDNF on Rat Model of PDMethods1 Using stereotaxic apparatus, PD models were made by injecting the 6—OHDA into unilateral Striatum of the rats.2 Distribution detection of the TAT—BDNF: Rats were randomly divided into two groups—study group and control group, and there are 3 rats in each group. Study group rats were intravenously injected 0.1ml(500μg) TAT—BDNF by vena caudalis when rats were made into the PD models, rats of control group were injected 0.1ml physiological saline. Four hours later, rats were killed and midbrain tissues were taken out, whether or not TAT—BDNF got into brain tissue was observed by HA immunofluorescence histochemical stain. 3 Midbrain tissues of rats were extracted and TAT—BDNF was identified by Western—blot so that we can confirm that TAT—BDNF distributed into brain tissues.4 Neuroprotective effect of TAT—BDNF rats model of PD: Rats were randomly divided into four groups—normal group,model group, study group, control group. There were 16 rats in each group. Study group rats were intravenously injected 0.1ml(500μg) TAT—BDNF by vena caudalis when they were made into the PD models, then every 7day to 28 day, same dosage TAT—BDNF was injected into Study group rats. Control group rats were injected 0.1ml physiological saline. In different time, rats of every group were examined in ethology, and rotation circle numbers were record and compared.5 Dopaminergic neurons that TH immunization was positive were counted by TH immunohistofluorescence in 28th day. Number of dopaminergic neurons in different group was compared.6 Substantia nigra of midbrain was detected by TUNEL, and TUNNEI positive cells were counted and compared in the different groups.Results1 Using the HA immunofluorescence histochemical stain method, we saw the fusion protein homogeneous distribution in mid—brain of rat by fluorescence microscope, However, no fluorescence was detected in the rats of control group, which confirmed that TAT can mediate BDNF to permeate the BBB in CNS of rats.2 Western-blot analysis showed that there was fusion protein in the midbrain of rats which was injected the TAT—BDNF,however, there was no fusion protein in the midbrain of control rats. It indicated that fusion protein had already permeated the BBB into central nervous system of rats.3 The rotational behavior of study group rats was distinctly different from model group threats. The rotation speed of study group rats was decrease,and the extent of rotation was lighten. Rats of normal group did not rotate. Statistical results showed that the rotational behavior in four group rats was not discrepancy in first two weeks. However, in the third week, the rotational behavior of the study group rats was significantly different from the control group rats,and in the forth week, the difference was extreme in two group rats. 4 In normal rats, there were many large cells of TH—immunoreactive(IR) positive located mainly in the reticular compacta of substantia nigra, arranging along the long axis of substantia nigra region. These cells were pyramid or ellipse and their fibers were slender. TH positive cells were very few in rats of model group, cells distributed sparseness and their outline and neurite were fuzziness. TH positive cells and fibers were increased significantly in rats of study group, and the outline and neurite of neuros were distinct.5 A great quantity of positive cells in substantia nigra of midbrain were observed in the rats of model group and the control group by TUNEL, these cell nucleuses were buffy and pyknosis, and brown apoptotic bodies were lytic. Analysis results showed that the positive cells in study group rats were less than that of control group significantly. There were not positive cells in normal group rats. It illustrated that the degeneration of DA neurons was related with apoptosis after 6—OHDA was injected into striatum of rats, and TAT—BDNF can keep DA neurons from apoptosis.Conclusions:1 Green fluorescence was detected in different tissues in study rats,such as brain, heart muscle, liver, spleen, kidney and so on, After injecting TAT—EGFP by vena caudalis while no fluorescence was detected in all tissues of control group mice, which indicated that TAT can mediated EGFP to permeat into tissues of mice, especially into CNS. These results provide us a new way to treat CNS disease using large molecular substance.2 Fusion protein TAT—BDNF that we expressed could protect the dopaminergic neurons from the neurotoxicity of 6—OHDA, which indicated that TAT—BDNF has the neuroprotective activity.3 We made the rats model of PD by two point injecting 6—OHDA into unilateral striatum of the rats successfully. The damage of 6—OHDA to dopaminergic neurons is gradually, so this model was more similar to the process of Parkinson disease than the models with other methods. Which indicated that this model is available for observing the therapeutic effect of medicine at the stage of progressive degeneration of neurons.4 When fusion protein TAT—BDNF was used to treat the rats model of PD, we found that the ethology of study group rats was improved more obviously than the control group rats. There were more TH positive neurons and less TUNEL positive neurons in study group rats than the control group rats. These illustrated that TAT—BDNF could permeat BBB into the central nervous system, and TAT—BDNF had the neuroprotetive effect to dopaminergic neuron.