Through genetic recombination technique, the rat glial cell line-derived neurotrophic factor (rGDNF) cDNA was in-serted into polylinker site of retroviral vector pLXSN, to generate a recombinant plasmid pLXSN-GDNF as transfer vector. Therecombinant plasmid was verified with restriction analysis, PCR, dot blot hybridization and Southern blot hybridization. The re-sults showed that GDNF cDNA was cloned correctly into retroviral vector pLXSN, recombinant retroviral vector was construct-ed. It is concluded that the eukaryotic cell expression vector was constructed successfully for gene therapy of Parkinson's,Alzheimer's and other central nervous system diseases.

In order to investigate effect of basic fibroblast growth factor(bFGF) on the prolifiration and differentiation of theneural progenitor of embryonic hippocampus of rats in vitro, 25 ng/ml bFGF was employed into the serum-free medium of cultureof hippocampal neural cells of embryonic day 18 rats in the present study. The effect of bFGF on the viability of cells culturedwas detected by MTT colorimetric method and the effects of bFGF on proliferation and differentiation of hippocampal neural pro-genitors were analyzed qualitatively and quantitatively by means of immunochemistry, for the nestin, neurofilament, galactocere-broside and glial acidic fibrillary protein. The results showed that the OD value of experimental group was higher than that ofcontrol group by 1.5 and 1.8 times at 4 d and 8 d respectively. The quantitative analysis of each kinds of cells indicated that thenumber of neural progenitors, neurons and oligodendrocytes in experimental group were increased about 2 times as many as thatin control group, but no differences of astrocytes between the two groups at 4 d. However, the number of four kinds of cells aug-mented about 1.7 times at 8 d. The results of this study suggest that bFGF can not only promote the survival, proliferation, butalso facilitate the differentiation of neural progenitor of hippocampus to neurons and glial cells. To obtain more many purifiedneural progenitors in vitro, the embryonic day 18 is not an appropriate age. It is better to get younger embryo brain to culture and to add enough bFGF.

Objective To explore whether the neural stem cells(NSCs) can act directly as a gene target cell which can be infected by the recombinant retrovirus and express the products of exogenous genes after infection. Methods The NSCs were cultured with supernatant containing the recombinant retroviruses with the genes of NGF or GDNF for two days.After screened with G418,the infected NSC were expanded at the present of bFGF in culture.The PC12 cells and the neurons of ventral midbrain of rat were cultured by the medium from the infected NSC,which were called as GDNF\|containing conditioned medium NGF or GDNF\|containing conditioned medium the morphological changes of the dopamine neurons of the ventral midbrain and expression of exogenous genes of the infected NSCs were detected by immunohistochemistry staining. Results It was estimated that about fifty percent of NSCs via retrovirus\|mediated NGF or GDNF gene transduction were G418\|resistant.These infected NSCs began to differentiate.Long and radical processes reached out from the sphere of proliferation and the cells migrated towards outside along the processes.The NSC infected with gene of NGF showed an astroid\|shape with larger body and processes.The NSC infected with gene of GDNF showed a shuttle\|shape with a smaller body and long processes.The PC12 cells increased in the NGF\|containing conditioned medium and stretched out long neurites.The dopamine neuron of the ventral midbrain which were immunoreactive for TH also showed a larger body and longer processes in the GDNF\|containing conditioned medium.Most of G418\|resistant NSCs were immunoreactive for NGF or GDNF. Conclusion NSC can act directly as a gene target cell which not only be infected by the recombinant retrovirus,but also express and secrete the products of exogenous genes.

Objective To investigate the survival,differentiation and gene expression of the neural stem cells(NSCs) modified by the gene of NGF or GDNF in the brain of AD rat model after transplantation. Methods The NGF or GDNF genetically modified NSCs labled with BrdU were implanted into the lateral cerebral ventricle of the AD rat model.The rats were killed three weeks after transplantation.The brains were cut and the sections were processed for single or double immunochemistry staining with antibodies against BrdU,Nestin,GFAP,NF,NGF and GDNF. Results BrdU\|positive cells were found in the lateral cerebral ventricle.Some of them migrated into the parenchyma and located in the wound,fibria\|fonix,hippocampus,corpus callosum,septum,subventricle zone and beside the blood vessels.Cells of doubled labeling with anti\|BrdU and GFAP were more often seen in the cortex,whereas more cells with anti\|BrdU and NF in the hippocampus,and both of them in the ventricle.Doubled labeling cells against BrdU and NGF,and against BrdU and GDNF were found in the ventricle and parenchyma.Conclusion\ The NGF or GDNF genetically modified NSCs can not only survive well,but also differentiate into neuron and astrocyte,and express the exogenous genes of NGF and GDNF in the host brain

Objective To investigate the effect of a single or combined transplantation of the neural stem cells(NSCs) modified with gene of NGF or GDNF on the cholinergic neurons of basal forebrain of AD model rat. Methods The NSCs modified with gene of NGF or GDNF were implanted in single or combined into the lateral cerebral ventricle of the rats after fibria\|fornix transection.The rats were killed three weeks after transplantation and the brain sections concluding basal forebrain were cut coronally on a freezing microtome and were processed by immunohistochemistry staining with antibodies against ChAT.The numbers of ChAT positive neurons of medial septum(MS) and vertical diagonal band(VDB) were analyzied statistically with one way of Student\|Newman Kaels. Results In MS,the percentages of ChAT positive neurons at the lesion side to the intact side in NGF group was 81% which was significantly higher than that in the lesion group(34%),NSC group(36%) and GDNF group(50%), P 0\^05). Conclusion\ The injury cholinergic neurons can be protected in different extent after a single or combined transplantation of the neural stem cells modified with gene of NGF or GDNF.Among these three groups,greater protection was found in NGF group and NGF+GDNF group,and lesser protection in GDNF group.\;[

Objective To investigate the memory amelioration of the AD model rat after a single or combined transplantation of the neural stem cells(NSCs) modified with gene of NGF or GDNF. Methods The AD model rat was made by cutting unilaterally the fibria\|fornix of male SD rat.Eight to ten days after surgery,the genetically modified and unmodified NSCs were implanted into the lateral cerebral ventricle of the rats.Two weeks after transplantation,the amelioration of memory impairment of the rats were detected by Morris water maze. Results The average escape latencies of the last three blocks in NGF and NGF+GDNF groups were lesser,and the percentages of swim distance in the platform quadrant were greater than that in NSC group( P

OBJECTIVETo investigate in vivo survival of retinal ganglion cells (RGCs) after partial blockage of optic nerve (ON) axoplasmic flow by sub-retinal space or vitreous cavity injection of brain-derived neural factor (BDNF) produced by genetically modified neural progenitor cells (NPCs).

METHODSAdult Sprague-Dawley (SD) rat RGCs were labeled with granular blue (GB) applied to their main targets in the brain. Seven days later, the left ON was intra-obitally crushed with a 40 g power forceps to partially block ON axoplasmic flow. Animals were randomized to three groups. The left eye of each rat received a sham injection, NPCs injection or an injection of genetically modified neural progenitors producing BDNF (BDNF-NPCs). Seven, 15 and 30 days after ON crush, retinas were examined under a fluorescence microscope. By calculating and comparing the average RGCs densities and RGC apoptosis density, RGC survival was estimated and the neuro-protective effect of transplanted cells was evaluated.

RESULTSSeven, 15 and 30 days after crush, in the intra-vitreous injection group, mean RGC densities had decreased to 1885 +/- 68, 1562 +/- 20, 1380 +/- 7 and 1837 +/- 46, 1561 +/- 58, 1370 +/- 16, respectively with sham injection or neural progenitors injection. However, RGCs density in the groups treated with intra-vitreous injection of BDNF-NPC was 2101 +/- 15, 1809 +/- 19 and 1625 +/- 34. Similar results were found in groups after sub-retinal injection. Higher densities were observed in groups treated with BDNF-NPCs. There were statistically significant differences among groups through nonparametric tests followed by the Mann-Whitely test. RGC apoptosis density in BDNF-NPC at each follow-up time was less than in other groups.

CONCLUSIONSA continuous supply of neurotrophic factors by the injection of genetically modified neural progenitors presents a highly effective approach to counteract optic neuropathy and RGC degeneration after partial ON axoplasmic flow blockage.

Animals ; Apoptosis ; Axonal Transport ; Brain-Derived Neurotrophic Factor ; genetics ; Cell Survival ; Gene Transfer Techniques ; Genetic Therapy ; Glaucoma ; therapy ; Male ; Rats ; Rats, Sprague-Dawley ; Retinal Ganglion Cells ; cytology ; Stem Cells ; physiology ; Vitreous Body ; metabolism