Construction of a prokaryotic expression vector of human tau multi-epitope peptide and immunogenicity of the expressed product / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a prokaryotic expression vector of human tau multiepitope peptide for examining the immunogenicity of a TauP1/P2 DNA vaccine in mice using the expressed product.</p><p><b>METHODS</b>The coding sequence of Tau multiepitope peptide gene was amplified from the plasmid pVAX1-Tau by PCR and inserted into the prokaryotic expression vector pGEX-4T-2 to construct the recombinant plasmid pGEX-4T-2-TauP1/P2. The positive recombinants were transformed into E.coli BL21 cells, and the expression of fusion protein GST-TauP1/P2 was induced by IPTG and identified by SDS-PAGE. Mice was immunized with TauP1/P2 DNA vaccine and the production of the specific antibodies was detected by Dot-blot analysis using the purified fusion protein.</p><p><b>RESULTS</b>A gene fragment 300 bp in length was amplified. Enzyme digestion and DNA sequencing verified correct construction of the prokaryotic expression plasmid pGEX-4T-2-TauP1/P2. The expression of target fusion protein GST-TauP1/P2 was detected by SDS-PAGE. Specific antibodies against TauP1/P2 were detected in the serum of mice immunized with the DNA vaccine using GST-TauP1/P2 fusion protein.</p><p><b>CONCLUSION</b>The constructed prokaryotic expression plasmid of human Tau multiepitope peptide is capable of expressing the target fusion protein, which specifically recognizes the specific antibodies against TauP1/P2 in mice immunized with TauP1/P2 DNA vaccine.</p>

Feridex labeling of bone marrow stromal cells of crab-eating macaque / 中国组织工程研究

BACKGROUND: Studies regarding Feridex in vitro cell labeling are mainly in rodents, while little information is known on primate crab-eating macaque.OBJECTIVE: To explore the feasibility of protocols using Feridex and transfection agents for in vitro magnetic labeling of bone marrow stromal cells (BMSCs) in crab-eating macaque.METHODS: Under the sterile condition, the crab-eating macaque BMSCs were obtained by means of density gradient centrifugation following a bone puncture. Feridex-poly-l-lysine complexes were used to magnetically label BMSCs. The efficiency and cellular viability of Feridex-poly-l-lysine labeled BMSCs were evaluated by Prussian blue staining, electron microscopy, and trypan blue dye exclusion test. The proliferation and differentiation ability of Feridex-poly-l-lysine labeling BMSCs were also investigated by inverted phase contrast microscope and immunocytochemistry. RESULTS AND CONCLUSION: BMSCs could be effectively labeled by Feridex and labeling efficiency was around 99%. Tiny blue stained fine particles and numerous vesicles coated with the electron-dense magnetic iron particles could be found in the cytoplasm of Feridex-poly-l-lysine labeled BMSCs under optical microscopy and transmission electron microscopy respectively. Cell viability, proliferation and differentiation ability of labeled BMSCs were not affected by Feridex-poly-l-lysine labeling. Results indicated that Feridex might be used to label BMSCs of crab-eating macaque.

Transfection of pEGFP-C2 in brain mediated by targeting liposome P-MMA-DOSPER / 生物医学工程学杂志

This research tried improving the specificity and efficiency of gene transfection in gene therapy and tried making the liposome a better gene transfer vector to brain by use of the monoclonal antibody (anti-Lex/SSEA-1)-mediated targeting of liposome. The derivatized monoclonal antibody was conjugated to the liposome DOSPER to form the targeting liposome P-MMA-DOSPER. Then, the pEGFP-C2 encapsulated in P-MMA-DOSPER or DOSPER was injected into the lateral ventricle of SD rats respectively, and the brains were taken for frosted slice 1, 3, 7 or 14 days later. The expression of GFP was observed under fluorescent microscope. There was a lot of expression of GFP around the lateral ventricle of rats in each group. But the indirect fluorescence antibody test showed the ratio of GFP+/nestin+ cells to nestin+ cells of every marking time point in the group of P-MMA-DOSPER was higher than the one in the group of DOSPER; the difference was found to be statistically significant (P<0.01). The results proved that the P-MMA-DOSPER can permeat the ependyma and can transfer gene into the nerve stem cells in vivo safely and effectively.

Comparison of analgesic effect following different thalamotomy in rats / 中国神经精神疾病杂志

Background To provide clinical evidence for ablative application by comparison of the analgesic effect following different thalamotomy in rats.Methods Thirty rats were randomly assigned into sham and 4 thalamotomies groups: central medial thalamic nucleus ( CM), parafascicular thalamic nucleus ( PF), ventral posterolateral thalamic nucleus (VPL), and CM +Cigulum (cg). Two μL 10% phenol dissolved in glycerin were used for stereotactic thalamotomy. The thermal pain thresholds before and after procedures were evaluated with the tail stimulate test. The formalin test was carried out in an open field apparatus where the animal formalin-induced responses (licking duration, flexing duration, and flinching frequency of the injected paw) were recorded for 60 min.Results Changes of pain thresholds in all ablative groups were significantly higher than that in the sham group, especially it was higher in VPL group. Differences of the factor thalamotomy were found to be due to the shorter licking in the ablative groups than that in the sham group (P ＜0.01 ), whereas flexing duration and flinching frequency were only slightly affected by thalamotomy. Moreover, licking duration was lower in VPL group than in CM and CM + cg groups ( P ＜0.05), whereas nociceptive responses did not differ between the CM and CM+cg groups (P ＞0.05).Conclusions In acute period, CM, PF, VPL, CM + cg neurolysis all showed to elevate the thermal pain threshold and to reduce the pain-induced behavioral responses related to supraspinal neural circuits (licking of the injected paw). Among them, the damage of VPL might be the most active one. CM + cg damage did not get better antinociceptive effect than single CM ablation.

Experimental study on the catecholaminergic effects of paraventricular nucleus projected from medullary visceral zone in "immune-to-brain communication" access / 中国组织工程研究

BACKGROUND: Lipopolysaccharide(LPS), as a polyclonal immune exciter, can simulate immune excitation status, which is useful in the observation of whether the catecholaminergic neurons in paraventricular nucleus of hypothalamus(PVN) projected from medullary visceral zone(MVZ) react towards LPS stimulation that is to provide a theoretical gist for the researches on the protection of brain function.OBJECTIVE: To observe whether PVN catecholaminergic neurons projected from MVZ react towards LPS stimulation for the exploration of the impacts of MVZ-PVN catecholaminergic access in "immune-to-brain communication".DESIGN: A randomized controlled study using experimental animals as subjects.SETTING: An Institute of Neurosurgery and Neurology of one Military University of Chinese PLA.MATERIALS: The study was conducted in the Department of Neurosurgery of Zhujiang Hospital affiliated to Southern Medical University and the Institute of Neurology of the Fourth Military Medical University of Chinese PLA from January to December in 2002. Ten healthy adult SD rats in cleanness grade were obtained from the experimental animal center of the Fourth Military Medical University of Chinese PLA.METHODS: WGA-HRP was injected into PVN of one side of the rat, and the immune exciter LPS was injected into the abdominal cavity after 48 hours of survival to induce immune response. Samples were stained by triple labels of WGA-HRP method and double immunohistochemical staining of anti-Fos and anti-TH antibodies.MAIN OUTCOME MEASURES: To observe the distributions and expressions of WGA-HRP labeled cells, Fos protein, and catecholaminergic neuron(labeled by TH) in MVZ.RESULTS: Seven immune-reactive(IR) positive neurons were found in MVZ, i. e., HRP, Fos or TH single labeled cells, Fos/HRP, Fos/TH or HRP/TH double labeled cells, and Fos/HRP/TH triple labeled cells. Fos/HRP double labeled neurons and Fos/HRP/TH triple labeled neurons accounted for 12. 5% and 39.6% of HRP labeled cells respectively.CONCLUSION: MVZ reacts to LPS immune stimulation, which could upload the immune message to PVN through Catecholaminergic neurons.MVZ might be a relay station in "immune-to-brain communication", which exerts immune modulatory impact through "MVZ→PVN" access.

Distribution character of functional unit of reaction neuron and astrocyte in medullary visceral zone 1 hour after epileptic attack: a laser confocal microscopic research / 中国组织工程研究

BACKGROUND: It is considered traditionally that epilepsy is a kind of complicated nervous conduct disorder caused by abnormally excited neuron in different area in brain. While the research on the function of astrocyte in epileptic attack is very rare.OBJECTIVE: To study the reaction of neuron and astrocyte in medullary visceral zone after epilepsy induced by pentetrazole in rats.DESIGN: A randomized controlled experimental research.SETTING and MATERIALS: The experiment was done in the Neurosurgery Laboratory of Zhujiang Hospital Affiliated to Southern Medical University and Neuroscience Institute of Fourth Military Medical University of Chinese PLA. Fourteen healthy adult SD rats, weighing 180 - 220 g, clean grade, were provided by the Experimental Animal Center of Fourth Military Medical University of Chinese PLA.INTERVENTIONS: Distribution of neuron and astrocyte in MVZ 1 hour after epileptic attack was shown by laserconfocal microscopic technique combined with triplication immunofluorescence histochemistry of anti-Fos protein, anti-tyrosine hydroxylase(TH) and anti-glial fibrillary acidic protein(GFAP).MAIN OUTCOME MEASURES: Observation of distribution of positive cells of Fos, GFAP and TH in MVZ and relationship between GFAP positive astrocyte and neuron.RESULTS: Fos positive neurons and GFAP positive astrocytes in MVZ increased significantly. Triplication immunofluorescence histochemistry showed reaction neuron(Fos positive) closely related with reaction astrocyte(GFAP positive) . Three kinds of N-ASC compounds with different labels were found, which were TH +/Fos +/GFAP + three labeled compound, TH + /GFAP +/Fos- and Fos+/GFAP +/TH- two labeled compound.CONCLUSION: Neuron and astrocyte in MVZ reacted strongly when epilepsy attacks. N-ASC as a functional unit may regulate onset of epilepsy.