Objective To investigate the treatment of spinal cord injury (SCI) by recombinant adeno-associated virus (AAV) transducing the hNGF gene, and construct and produce the vector of hNGF recombinant AAV. Methods The resulting gene of hNGF was inserted into the KpnⅠ-BamHⅠ site of vector plasmid pSSHG-Neo to construct the vector of hNGF recombinant AAV. The recombinant AAV viral stock was packaged. Renal embryo 293 cell was co-transfected with the rAAV vector of plasmid pSSHG/hNGF, packaging plasmid pAAV/Ad and helper adenovirus pasmid pFG140 instead of adenovirus by calcium phosphate precipitation. Results The recombinant viral stock vector of plasmid pSSHG/hNGF was constructed successfully. The results of dot blot showed that we had obtained the rAAV stocks of high titre 1.46?10 12 PFU?mL -1. Conclusion We prepared the viral stock of rAAV-hNGF that can serve as the experimental study of gene therapy of SCI.

Objective To express the hNGF? gene segment encoding mature peptide (hNGF? ) in E.coli and determine its bioactivity. Methods The resulting gene of hNGF? was subclonedinto the hNGF? site of the expression vector plasmid pBV220. The ligation products were used to transform the competent E.coli DH 5?. The proteins of hNGF? were expressed by temperature induction. The expression products were dealed with solubilizing inclusion bodies and refolding protein. It was introduced into the expressed hNGF? tests of neurite growth of dorsal root knot of chicken embryo and tests of Brdu incorporation into PC12 cells was a biologically active protein. Results The recombinant plasmid pBV220/NGF? was successfully constructed. The NGF? was inserted to pBV220 plasma, a prokaryotic expression vector. Expression of NGF? in E.coli was induced by raising temperature to 42℃. SDS-PAGE electrophoresis showed that NGF? protein existed in inclusion. The solubility protein of NGF? was obtained through purification of inclusion by centrifugation and technique of protein repatriation. Recombinant NGF? protein was purified by affinity chromatography of heparin SepharoseCL-6B. The purity of NGF? was higher than 90% and yield of NGF? was 1.8～2.0mg/L expressing bacteria. The bioactivity of NGF? expressing prokaryotic cell was 1?10 5BU/g according to rule concerning examination of biological products in China. Conclusion The hNGF?gene with bioactivity can be expressed in E.coli.

Objective: To study the effect of Interleukin(IL)-17 on neutrophil apoptosis and try to explain the possible mechanism involved. Methods: Neutrophils isolated from healthy donors were incubated in enriched RPMI 1640 cell culture medium at 37 ℃ in 5% carbon dioxide. Subgroups were incubated with IL-17, heat-denatured IL-17 (X-IL-17), dexamethasone (DEX), or buffer alone. Apoptosis was assessed by morphologic changes, by detecting DNA strand breaks. Production of proapoptotic protein Bax by neutrophils was evaluated by immunocytochemistry. Results: At the time of neutrophil incubation, neutrophils in the control subsets exhibited morphologic evidence of apoptosis. A steady rise in apoptosis index (AI) was noted, with (1.54?0.08)% for 0 h, (11.48?1.80)% (compared with 0 h, P0.05) for 0 h, (20.47?6.22)% ( compared with control 12 h, P0.05).Neutrophils apoptosis was accompanied by DNA fragmentation. In all groups, the increasing of Bax immunoreactivity was strongly related with more apoptotic neutrophils (r=0.932, P

ObjectiveTo study the cloning and sequencing of mature fragment of human bone morphogenetic protein-4 gene. Methods The template DNA was obtained from the human osteosarcoma cell line U2OS. By using RT- PCR method, the cDNA coding for the mature fragment of BMP-4 was amplified, cloned into the vector pUC19, and sequenced by Sanger Dideoxy-mediated Chain Termination method. Results The mature fragment of BMP4 cDNA was obtained by RT-PCR and determined by sequencing. Through the computer search on Genebank, the analysis showed that the homology of nucleotides and amino acids between cDNA of rhBMP4 mature fragment of this study and the published sequence was 99％. Sequence analysis showed that there were two differences, one was at base 1154 (201): G→C, which had no influence on the corresponding amino acids (Val). Another was at basel222 (269):C→T, the mutation at the base 1222 had the change of Ala to Val. Conclusion The mature fragment of BMP4 gene has been cloned. The results will be of great significance in treatment of skeletal injuries and diseases.

Objective To observe the effect of fleabane injection on serum level of D-dimer, fibrinogen and hypersensitive C-reactive protein in patients with IgA nephropathy and to explore the mechanism of fleabane injection for treating IgA nephropathy. Methods 29 patients with IgA nephropathy were given fleabane injection together with routine treatment. Another group of 28 patients with IgA nephropathy were only treated with routine treatment as con-trol. Determinations of 24 hours urine protein output(24HPQ), serum level of D-dimer(D-D), Fibrinogen(Fib) and hypersensitive C-reactive protein (hsCRP) were carried out pre- and post-study. Results Significant decrease in 24HPQ , serum level of D-D, Fib and hsCRP were observed in both treatment group and control group(P <0. 01 ～0. 05), but more significant in treatment group as compared with control group ( P < 0. 05). Conclusion Flea-bane Injection plus routine treatment could significantly decrease 24HPQ in patients with IgA nephropathy, and this maybe contribute to regulation of D-D, Fib and hsCRP by fleabane injection.

Objective Cloning and sequencing of the human neurotrophin-4(hNT-4) gene.Methods With the chromosomal DNA of human blood lymphocytes as template,hNT-4 coding genes were amplified by polymerase chain reaction(PCR) and recombinated into phage vector pGEM-T Easy,which were sequenced by using Sanger's single stranded DNA terminal termination method.Results The sequence of the cloned gene is completely the same as that reported in the literature(GenBank data base,M86528).Conclusion This study successfully cloning and sequenced the gene of mhNT-4,and it would be convenient for us to study the expression of mhNT-4 in eukaryote,and to continue the research on the gene therapy of Alzheimer's disease intensively.This study indicate that the hNT-4 is conservative in different races and individuals.

Objective To investigate integration and expression of adeno-associated virus (AAV) vectors in neuronal PC12 cells,the result of which can be applied in further gene therapy of diseases of the central nervous system. Methods Human neurotrophin-3(hNT3)genes were inserted into AAV vectors. Then the recombinat AAV plasmids were encapsidated as recombinant virions. PC12 cells were transfected with the virions and the positive cells were selected by G418. The transfection positive (hNT3 modified)PC12 cells were cultured for several generations and the cellular genomic DNA and total RNA were extracted. We investigated the integration locus of AAV vectors by Southern blot and transcript situation of foreign genes by dot blot. Results The hybridization tests showed that AAV introduced foreign genes were stably integrated, but at random locus, and robustly transcribed in hNT3 modified PC12cells. Conclusion AAV vectors can serve as high efficiency vectors of target genes in neuronal PC12 cells.

Objective Expressing the human matured brain-derived neurotrophic factor (mBDNF) gene in E.Coli and determining its bioactivity. Methods The resulting gene of mBDNF was subcloned into the EcoRI-BamHI site of the expression vector plasmid pBV220. The ligation products were used to transform the competent E. Coli DH5α. The proteins of mBDNF were experessed by temperature inducing. The expression products were dealed with solubilizing inclusion bodies and refolding protein. It was introduced into the embryonic chicken DRG to test whether the expressed mBDNF is a biologically active protein. Results The recombinant plasmid pBV/mBDNF was successfully constructed. By temperature inducing,under the control of the bacteriophage λ PL promoter, the experessed mBDNF protein was a 14Kd non-fusion protein,which existed in E. Coli as inclusion bodies. The size of expressed mBDNF is identical to the prediction. Bioactivity of the products was proved that it could support the cell survival and neurite growth in the primary cultures of embryonic 8-day-old chicken DRG neurons as compared to control.Conclusion The mBDNF gene can be expressed bioactively in E. Coli.

In the present study, we have cloned the gene of human neurotrophin-3 (hNT-3) from the genomic DNA of white blood cells (WBC) by polymerase chain reaction (PCR). The amplification products were cloned into pUC19 and sequenced. Genomic sequence comparison of the cloned fragment and the reported hNT-3 (GenBank M61180) reveals 7 base differences: 1 in the signal peptide, 3 in the prepro peptide, and 3 in the mature hNT-3. Except the 2 varied bases (16th, T to G; 285th, A to C) in the signal peptide and pro-sequence resulted in the change of their encoded amino-acids (Tyr→Asp; Gln→His), the other varied bases have no influence on their respective encoded amino-acids, and all the changes have no influence on the open reading frame (ORF) of the hNT-3.