The human brain-derived neurotrophic factor (hBDNF) gene was cloned by polymerase chain reaction and the recombinant adeno-associated viral vector inserted with hBDNF gene (AAV-hBDNF) was constructed. Cultured rat hippocampal neurons were treated with Abeta(25-35) and serued as the experimental Abeta-induced neuronal damage model (AD model), and the AD model was infected with AAV-hBDNF to explore neuroprotective effects of expression of BDNF. Cell viability was assayed by MTT. The expression of bcl-2 anti-apoptosis protein was detected by immunocytochemical staining. The change of intracellular free Ca ion ([Ca2+]i) was measured by laser scanning confocal microscopy. The results showed that BDNF had protective effects against A-induced neuronal damage. The expression of the bcl-2 anti-apoptosis protein was raised significantly and the balance of [Ca2+]i was maintained in the AAv-hBDNF treatment group as compared with AD model group. These data suggested that recombinant AAV mediated a stable expression of hBDNF in cultured hippocampal neurons and resulted in significant neuron protective effects in AD model. The BDNF may reduce neuron apoptosis through increasing the expression of the bcl-2 anti-apoptosis protein and inhibiting intracellular calcium overload. The viral vector-mediated gene expression of BDNF may pave the way of a novel therapeutic strategy for the treatment of neurodegenerative diseases such as Alzheimer's disease.

Objective To study the prokaryotic expression of fusion gene A?-HBcAg and analyze the immunoreactivity and immunogenicity of expression protein. Methods Recombinant plasmid pBV220/A?-HBcAg was transformed into E.coli DH5?, and expressed by temperature inducing. The bacteria were split by ultrasonic wave. The expression of the fusion protein was studied by SDS-PAGE and Coomassie brilliant blue staining. The immunoreactivity of the fusion protein was determined using ELISA. After immunized intraperitoneally with the fusion protein, 5 Balb/c mice's sera titers of anti- A? and anti-HBc were evaluated by ELISA. Results Fusion protein was in sediment of the split bacteria as inclusion bodies and its expression level was 5% of the total sediment protein. The fusion protein had both immunoreactivity of A? and HBcAg. The titers of anti-A? and anti-HBc were very low after 3 times of immunization. After immunization for 5 times, the titers reached 1∶800 and 1∶3 200 for anti-A? and anti-HBc, respectively. Conclusion Recombinant gene A?-HBcAg can be expressed in E.coli DH5? and the expression protein has certain immunoreactivity and immunogenicity. It indicates that further work should be done to enhance the expression level of fusion gene A?-HBcAg and improve the immunogenicity of the fusion protein.

Objective To prepare a hybridoma secreting stab le monoclonal antibodies against ?-amyloid peptide (A? 1-42) with high titer. Methods By genetic engineering technology, A ? gene was recombined with the MIR of HBcAg to get the A? and HBcAg fusi on protein. Spleen cells from BALB/c mice immunized with A? and HBcAg f usion protein were fused with mouse myeloma cells SP2/0. Results Two strains of hybridomas (1H 7 and 1F 3) secreting stable monoclonal antibodies raised against A? 1-42 were ob tained. The subtypes of A? 1-42 antibodies were IgG 3. C onclusion The A? 1-42 monoclonal antibodies obtained have high titers and specificity.

Objective To obtain highly purified astrocytes and identify the cells in each stage to support further studies.Methods The cerebral cortex of a neonatal SD rat was isolated and prepared into single cell suspension.The obtained cells were purified by differential adherence and shook at a constant temperature.By inverted phase contrast microscopy and HE staining,cell morphology was observed.The immunofluorescence staining with anti-mouse GFAP was used to identify the cells.Results The primary cortical cells developed rapidly at 3 d after culture and covered the flasks at 9-12 d.At this time,the cells showed stratification and the astrocytes lay at the lower layer.GFAP positive rate was only about (67.2 ±7.1)%.After the first passage,GFAP positive rate increased obviously (84.0±6.0)%. However, oligodendrocytes and microglias could not be removed completely,and the cells also showed stratification.Through 3 times of passages,we obtained many single species of astrocytes showing satellite shape with 2 or 3 processes,big cell body and round or oval-shaped nuclei leaned to one side.Immunofluorescence staining showed that nearly all of the cells were strong positive and the positive rate reached as high as (97.6 ± 2.4 )%.Conclusion Through differential adherence and shaking at a constant temperature,more astrocytes of high purity and in good state can be obtained.

Objective To investigate the expression of neuroprotective peptide [Gly14]-Humanin (HNG) in eukaryotic cells by gene engineering technique and analyze its biological activity. Methods By means of asymmetrical primer/template, double stranded cDNA of HNG with FLAG in its C-terminal was obtained, which was cloned into the plasmid pcDNA3.1(-), and the resultant recombinant vector pcDNA3.1(-)/HNG-FLAG was transfected into PC12 cells. At the same time, the recombinant vector pcDNA3.1(-)/EGFP was transfected to control the efficiency of transfection. The expression of HNG in the cells was determined by immunocytochemistry. In order to analyze the biological activity of the expressed HNG, 25μM Aβ25-35 peptide was added to the culture medium of the transfected cells for 24h, then cell morphology, MTT assay and Hoechst 33258 staining were observed. Results The eukaryotic expression vector of pcDNA3.1(-)/HNG-FLAG was identified by enzyme digestion and sequencing. HNG was highly expressed in PC12 cells. After exposure of PC12 cells to 25μM Aβ25-35 for 24h, cell viability decreased to (65.8±5.3)%, and the dystrophic changes of neuritis and nuclei condensation were obvious. When cells were pre-transfected with pcDNA3.1(-)/HNG-FLAG, Aβ25-35-induced cell death and morphological changes of cells and nuclei were suppressed. In contrast, pre-transfected with empty vector did not protect cells from Aβ25-35-induced toxicity. Conclusion The eukaryotic expression vector for FLAG-tagged HNG was successfully constructed and expressed in PC12 cells. Expressed HNG has biological activity.

The human brain-derived neurotrophic factor (hBDNF) gene was cloned by polymerase chain reaction and the recombinant adeno-associated viral vector inserted with hBDNF gene (AAV-hBDNF) was constructed. Cultured rat hippocampal neurons were treated with Aβ25-35 and serued as the experimental Aβ-induced neuronal damage model (AD model), and the AD model was infected with AAV-hBDNF to explore neuroprotective effects of expression of BDNF. Cell viability was assayed by MTT. The expression of bcl-2 anti-apoptosis protein was detected by immunocytochemical staining. The change of intracellular free Ca ion ([Ca2+]i) was measured by laser scanning confocal microscopy. The results showed that BDNF had protective effects against Aβ-induced neuronal damage. The expression of the bcl-2 anti-apoptosis protein was raised significantly and the balance of [Ca2+]i was maintained in the AAV-hBDNF treatment group as compared with AD model group. These data suggested that recombinant AAV mediated a stable expression of hBDNF in cultured hippocampal neurons and resulted in significant neuron protective effects in AD model. The BDNF may reduce neuron apoptosis through increasing the expression of the bcl-2 anti-apoptosis protein and inhibiting intracellular calcium overload. The viral vector-mediated gene expression of BDNF may pave the way of a novel therapeutic strategy for the treatment of neurodegenerative diseases such as Alzheimer's disease.

Objective To explore the effect on traditional experiment and case teaching method in regional anatomy study. Methods 80 students from 2014 medical students were randomly selected as the teaching subjects and divided into traditional group and case teaching group. The traditional group con-tained 40 students, using the traditional teaching method, while case teaching group had also 40 students with case teaching method. In the process of teaching, three clinical cases were introduced, including thesubtotal thyroidectomy thoracic outlet syndrome andpancreatic cancer. After the end of the course, the students conducted a unified questionnaire and examination. SPSS 18.0 was used for data line t test or chi square test between the two groups. Results The scores of the students in the case group in the selection questions, blanks and essay questions in the final exam were higher than those of the traditional group; The average total score of the case group was (85.69 ±11.61), while the traditional group was (73.19 ±18.66), and the difference was statistically significant (t=3.597, P=0.002). The results of the questionnaire showed that the students in the case group were higher than the traditional group, and the difference was statistically significant ( χ2=14.753, P=0.001). Conclusion The effect on regional anatomy study with case teaching method is better than the traditional teaching method, and it is a promising teaching reform for the med-ical students.

OBJECTIVE: To construct a recombinant prokaryoticexpression plasmid pET/ c-Aβ(15)-c, and evaluate the immunogenicity of its encoded fusion protein as expressed in E.coli. METHODS: The gene fragment HBc88-144 was amplified by PCR and subcloned to pUC19. The synthetic, double-strand Aβ(1-15) gene was inserted downstream of HBc1-71 in pGEMEX/c1-71. After restriction enzyme digestion, c1-71- Aβ(15) was spliced to HBc88-144, yielding the recombinant gene c-Aβ(15)-c; that gene was subcloned into pET-28a(+). The fusion protein (CA15C) expressed in the transformed E.coli BL21 was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) and analyzed by SDS-PAGE. The virus-like particle (VLP) formed by fusion protein CA15C was observed with transmission electric microscope (TEM). Four Kunming (KM) mice were given intraperitoneal injections of CA15C, and the anti-Aβ antibody elicited was detected by indirect ELISA. RESULTS: The sequence of the recombinant gene was confirmed by restriction enzyme digestion and DNA sequencing. After IPTG induction, the fusion protein was expressed, mainly in the sediment from the bacterial lysate. The expression level was 40% of total protein in the sediment. The CA15C could form VLP. After 5 rounds of immunization, the titer of anti-Aβ antibody in the sera of KM mice reached 1:10000, while the anti-HBc antibody was undetectable. CONCLUSION Recombinant c-Aβ(15)-c gene can be expressed in E.coli. The expressed protein can form VLPs and has a strong immunogenicity.
Alzheimer Disease ; prevention & control ; Amyloid beta-Peptides ; genetics ; Animals ; Base Sequence ; Genetic Vectors ; genetics ; Hepatitis B Core Antigens ; genetics ; immunology ; Humans ; Mice ; Molecular Sequence Data ; Peptide Fragments ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Vaccines, Virus-Like Particle ; genetics ; immunology ; metabolism