- [Show abstract] [Hide abstract] ABSTRACT: In vivo electroporation works as an effective method to transfer exogenous genes into postnatal rodent forebrain. Nevertheless, two deficiencies were found in the reported methods. First, surgical operation brings unnecessary trauma to newborn pups. Second, the procedure was complicated and the transfection efficiency was relatively low. Here we improved the previous electroporation method and make it more simple and efficient. The pulse voltage was decreased to 90 v. DNA injection into one pup's forebrain could be completed within 30 s without any surgical operation. More than 94% of injected neonates survived. Almost 100% of the survivors expressed the introduced gene and the expression persists as long as 20 days after injection. Thus, this method offers a powerful new way for gene function study in postnatal neurogenesis and neural development.
- [Show abstract] [Hide abstract] ABSTRACT: Human DIXDC1 is a member of Dishevelled-Axin (DIX) domain containing gene family which plays important roles in Wnt signaling and neural development. In this report, we first confirmed that expression of Ccd1, a mouse homologous gene of DIXDC1, was up-regulated in embryonic developing nervous system. Further studies showed that Ccd1 was expressed specifically in neurons and colocalized with early neuronal marker Tuj1. During the aggregation induced by RA and neuronal differentiation of embryonic carcinoma P19 cells, expressions of Ccd1 as well as Wnt-1 and N-cadherin were dramatically increased. Stable overexpression of DIXDC1 in P19 cells promoted the neuronal differentiation. P19 cells overexpressing DIXDC1 but not the control P19 cells could differentiate into Tuj1 positive cells with RA induction for only 2 days. Meanwhile, we also found that overexpression of DIXDC1 facilitated the expression of Wnt1 and bHLHs during aggregation and differentiation, respectively, while inhibited gliogenesis by down-regulating the expression of GFAP in P19 cells. Thus, our finding suggested that DIXDC1 might play an important role during neurogenesis, overexpression of DIXDC1 in embryonic carcinoma P19 cells promoted neuronal differentiation, and inhibited gliogenesis induced by retinoic acid.
- [Show abstract] [Hide abstract] ABSTRACT: By using of Escherichia coli DH5alpha to express GST-Ccd1 fusion protein and which was purified by affinity chromatographic separation. The purified target protein was used to immunize rabbits to prepare polyclonal antibody. The previously constructed recombinant prokaryotic expression vector pGEX-5X-1-Ccd1 was transformed into Escherichia coli DH5alpha and was induced to expression by IPTG. The recombinant target protein was expressed with soluble state in Escherichia coli expression system which was separated and purified by chromatographic column stuffed with glutathione Sepharose 4B. The prepared antigen was used to immunize rabbits to get anti-Ccd1 specific rabbit original polyclonal antibody. ELISA data demonstrated that the antibody titer of the serum was up to 1:40 000. Immunohistochemistry analysis indicated that the "home-made" antibody had a specific interaction with Ccd1 protein and which could be used for extended experimental research. The anti-Ccd1 polyclonal antibody we had prepared had a high quality of potency and specificity. The antibody was demonstrated by experiments that which could totally fulfill the requirement of immunoblotting and immunohistochemistry study of Ccd1. The antibody provided an useful experimental tool to profoundly research the tissue expression profile, intercellular location and biological function of Ccd1.
- [Show abstract] [Hide abstract] ABSTRACT: To identify the genes differentially expressed in development of human glioma, and to study the expression of some genes in different grade gliomas. Oligonucleotide microarray (including 218 genes related to neural system development) was adopted and hybridized with probes which were prepared from the total RNAs of glioma specimens and normal brain tissues. Differentially expressed genes between the normal tissues and glioma tissues were assayed after scanning oligonuceltide microarray with ScanArray 4000, and some of these genes such as smad1, Hmp19 and TRIP3 were verified by real-time quantitative PCR(real-time-Q-PCR) method. In comparison with the genes in the normal brain tissue, 5 down-regulated and 5 up-regulated genes in glioma specimens were revealed by means of microarrays, and the expression of smad1, Hmp19 and TRIP3 were verified by real-time-Q-PCR assay. Multiple genes play important roles in development of glioma. cDNA microarray technology is a powerful technique in screening for differentially expressed genes between glioma tissues and normal brain tissues. This study is helpful for judgement of invasion and prognosis of gliomas, and provides more target genes for targeted therapy.
- [Show abstract] [Hide abstract] ABSTRACT: Intermittent hypoxia has been found to prevent brain injury and to have a protective role in the CNS. To address the possible causes of this phenomenon, we made investigative effort to find out whether intermittent hypoxia affects neurogenesis in the adult rat brain by examining the newly divided cells in the subventricular zone (SVZ) and dentate gyrus (DG). The adult rats were treated with 3000 and 5000 m high altitude 4 h per day for 2 weeks consecutively. 5-Bromo-2-deoxyuridine-5-monophosphate (BrdU) immunocytochemistry demonstrated that the BrdU-labeled cells in the SVZ and DG increased after 3000 and 5000 m intermittent hypoxia. The number of BrdU-labeled cells in the SVZ returned to normal level 4 weeks following intermittent hypoxia. However, the BrdU-labeled cells in the DG had a twofold increase 4 weeks subsequent to intermittent hypoxia. From these data, we conclude that intermittent hypoxia facilitates the proliferation of neural stem cells in situ, and that the newly divided cells in the SVZ and DG react differently to hypoxia. We are convinced by these findings that the proliferation of neural stem cells in SVZ and DG may contribute to adaptive changes following intermittent hypoxia.
- [Show abstract] [Hide abstract] ABSTRACT: To investigate the effect of hyperthermia on apoptosis of cultivated striatum neurons in the rat. After 30 min hyperthermia in 43 degrees, the Ca2+ concentration, the mitochondria membrane potential of the neuron were detected by Laser Scanning Confocal Microscopy (LSCM). The apoptosis of striatum neurons was detected by TUNEL staining. Heat stress at 43 degrees C for 40 min caused an increase in the Ca2+ concentration of striatum neurons and a decrease in the mitochondria membrane potential of the striatum neurons. The striatum shows more apoptosis neurons after heat stress.