To construct the recombinant adenovirus vector expressing specific siRNA for rat retinoic acid receptor-beta (RARbeta) gene, and to detect its effect on RARbeta expression and neuronal differentiation of all-trans retinoic acid (ATRA) treated mesenchymal stem cells (MSCs). First, we designed four pairs of siRNA sequence for rat RARbeta gene and annealed complementary oligonucleotides in vitro, then cloned double-stranded DNA in pSES-HUS vector containing U6/H1 double-promoter and recombinated with the backbone vector to construct pAd-siRARbeta plasmid. We infected MSCs by using adenovirus Ad-siRARbeta which was packaged in HEK293 cell line, then performed Real-time, Western blotting and immunoflourencence to detect the expression of RARbeta. We used combination of ATRA and MNM to induce MSCs into neural-like cells, then performed Real-time PCR and immunoflourencence to detect neuronal specific markers of induced neural cells. By using PCR, endonuclease cutting and gene sequencing, we confirmed that the target genes were correctly cloned in adenovirus vector. We could observe more than 60% RFP-positive MSCs at 24 h after adenovirus infection. The expression of RARbeta was significantly increased to 16.5 +/- 2.34 fold in ATRA treated MSCs (P < 0.05) and located in nucleus. Three of four pairs siRNA could effectively inhibit the expression of RARbeta with inhibition efficacy of (66.26 +/- 9.12)%, (48.70 +/- 5.78)%, (64.09 +/- 0.53)% (P < 0.05), especially siRNA-pool group with inhibition efficacy of (78.09 +/- 4.24)% (P < 0.01). Combination of ATRA and MNM induced MSCs into neural-like cells which expressed neuronal specific markers, Nestin, NSE, MAP-2, and Tau. Immunoflourencence result showed that about 50-88 present of cells were positive for Nestin, NSE, Tjul, however, adenovirus medicated expression of siRARbeta could effectively inhibit the expression level of neural specific proteins and the ratio of positive stained cells (P < 0.05). Therefore, we successfully constructed the recombinant adenovirus vector containing siRNA for rat RARP gene, adenovirus could effectively infect MSCs and inhibit the expression of induced RARbeta in ATRA treated MSCs, then inhibit neuronal differentiation of MSCs.
Adenoviridae ; genetics ; metabolism ; Animals ; Cell Differentiation ; genetics ; Down-Regulation ; HEK293 Cells ; Humans ; Intermediate Filament Proteins ; metabolism ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Nerve Tissue Proteins ; metabolism ; Nestin ; Neurons ; cytology ; RNA, Small Interfering ; genetics ; Rats ; Receptors, Retinoic Acid ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection ; Tretinoin ; pharmacology

OBJECTIVETo investigate the relationship between GLI3 gene and pathogenesis of idiopathic congenital talipes equinovarus (ICTEV).

METHODPotential mutations in the coding region of GLI3 were detected among 84 patients with ICTEV by denaturing gradient electrophoresis. Expression of GLI3 in the ICTEV patients' disease tissues was assessed by reverse transcription PCR. Following generation of rat model for ICTEV, mRNA and protein levels of GLI3 were evaluated by real-time PCR and immunohistochemistry and Western blotting.

RESULTSNo mutation was found in exons 1 - 8 and 13 of GLI3 gene among the 84 ICTEV patients. No expression of GLI3 gene was detected in the flexor hallucis longus of ICTEV patients or normal controls. Expression of Gli3, in terms of both mRNA and protein, was stronger in the hindlimb of ICTEV rat embryos compared with normal controls.

CONCLUSIONMutation in the coding region of GLI3 may not be responsible for the occurrence of ICTEV. However, there may still be connection between abnormal expression of the gene and pathogenesis of ICTEV.

Animals ; Clubfoot ; genetics ; metabolism ; pathology ; Gene Expression ; Genetic Predisposition to Disease ; Humans ; Kruppel-Like Transcription Factors ; biosynthesis ; genetics ; Mutation ; Nerve Tissue Proteins ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Zinc Finger Protein Gli3

OBJECTIVETo construct the recombinant plasmid pcDNA3.0-RGC32 and evaluate the effect of the response gene to complement-32 (RGC32) on cell cytoskeleton in vitro.

METHODSThe full-length cDNA of RGC32 was obtained by RT-PCR and inserted into the eukaryotic expression vector pcDNA3.0 to generate the recombinant plasmid pcDNA3.0-RGC32. After transfection of the recombinant plasmid into SW480 cells, the expression of RGC32 in the cells was detected by Western blotting. The cytoskeleton of SW480 cells was visualized before and after the transfection, and the changes in the cell migration ability was assessed by wound-healing assay.

RESULTSThe recombinant plasmid pcDNA3.0-RGC32 was successfully constructed. The expression of RGC32 was significantly increased in SW480 cells after transfection with pcDNA3.0-RGC32. Before the transfection, the microfilaments of SW480 cells were few and short without obvious polarity, but after the transfection, the microfilaments were increased and elongated with also an obvious polarity, and the invasive structures of lamellae and lamellipodia occurred. The migration ability of the cells was enhanced after transfection with pcDNA3.0-RGC32.

CONCLUSIONOverexpression of RGC32 can cause the reorganization of cytoskeleton and promotes the cell migration, which can be an important mechanism of RGC32 in promoting cancer metastasis.

Cell Cycle Proteins ; biosynthesis ; genetics ; Cell Line, Tumor ; Cell Movement ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; Cytoskeleton ; chemistry ; metabolism ; Genetic Vectors ; Humans ; Muscle Proteins ; biosynthesis ; genetics ; Neoplasm Metastasis ; genetics ; Nerve Tissue Proteins ; biosynthesis ; genetics ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo establish transgenic mice with GFP expression in the vascular endothelium during neovascularization.

METHODSThe vector nestin-hsp68-gfp containing nestin second intron was introduced into U251 cells and the expression level of GFP was detected by fluorescence microscopy. Transgenic mice were produced by microinjection. The genome of the offspring mice was screened by PCR, and GFP expression in the vascular endothelium was detected using immunohistochemistry.

RESULTSThirteen offspring mice were obtained and 2 of them were positive for GFP in the vascular endothelium as detected by PCR. GFP was detected in the offspring mice both at the embryonic stage and after birth.

CONCLUSIONSThe transgenic mice with GFP expression in the vascular endothelium during neovascularization have been successfully established.

Animals ; Animals, Newborn ; Base Sequence ; Endothelium, Vascular ; metabolism ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Intermediate Filament Proteins ; biosynthesis ; genetics ; Mice ; Mice, Transgenic ; Molecular Sequence Data ; Neovascularization, Pathologic ; genetics ; Neovascularization, Physiologic ; genetics ; Nerve Tissue Proteins ; biosynthesis ; genetics ; Nestin

In order to construct the recombinant retrovirus vector of human ngn3 gene and its packaging cell line, we successfully amplified the open reading frame (ORF) of ngn3 gene from human fetal pancreatic tissue by RT-PCR. The PCR products of human ngn3 gene was subcloned into pMD18-T vectors and sequenced. Results showed that its sequence was fully consistent with the ngn3 gene published in GenBank(GenBank Accession No. BC126468). The correct fragment was digested by EcoR I and Hpa I from recombinant pMD18-T vector and inserted into the same restriction enzyme sites of retroviral vector pMSCV-neo. We got recombinant retrovirus vector pMSCV-ngn3, which was identified by double restriction enzyme digestion and then transfected into PT67 cells by lipofectamine 2000. We established the PT67-ngn3 packaging cell line by G418 selection, which was detected by RT-PCR and immunohistochemistry staining. The detection results showed that the Ngn3 expressed at the mRNA and protein level in the packaging cell line. RT-PCR detection and electronic microscope analysis showed that the recombinant retroviral vector pMSCV-ngn3 was packaged into infectious virus particles and released into the supernatant of the cells. These results demonstrated that a PT67-ngn3 packaging cell line was successfully established, and this could facilitate the study of differentiation of the human fetal pancreatic progenitor cells into insulin-producing cells by using the ngn3 gene.
Basic Helix-Loop-Helix Transcription Factors ; biosynthesis ; genetics ; Cell Differentiation ; drug effects ; Cell Line ; Cloning, Molecular ; Fetus ; Genetic Vectors ; genetics ; Humans ; Insulin-Secreting Cells ; cytology ; Molecular Sequence Data ; Nerve Tissue Proteins ; biosynthesis ; genetics ; Open Reading Frames ; genetics ; Pancreas ; cytology ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Retroviridae ; genetics ; metabolism ; Stem Cells ; cytology ; Transfection

A chimeric protein called Wallerian degeneration slow (Wld(S)) was first discovered in a spontaneous mutant strain of mice that exhibited delayed Wallerian degeneration. This provides a useful tool in elucidating the mechanisms of axon degeneration. Over-expression of Wld(S) attenuates the axon degeneration that is associated with several neurodegenerative disease models, suggesting a new logic for developing a potential protective strategy. At molecular level, although Wld(S) is a fusion protein, the nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1) is required and sufficient for the protective effects of Wld(S), indicating a critical role of NAD biosynthesis and perhaps energy metabolism in axon degeneration. These findings challenge the proposed model in which axon degeneration is operated by an active programmed process and thus may have important implication in understanding the mechanisms of neurodegeneration. In this review, we will summarize these recent findings and discuss their relevance to the mechanisms of axon degeneration.
Animals ; Axons ; physiology ; Humans ; Mice ; Mice, Mutant Strains ; Models, Neurological ; Mutant Proteins ; genetics ; physiology ; Mutation ; NAD ; biosynthesis ; Nerve Degeneration ; etiology ; genetics ; physiopathology ; Nerve Tissue Proteins ; genetics ; physiology ; Nicotinamide-Nucleotide Adenylyltransferase ; genetics ; physiology

OBJECTIVETo express and purify the fusion protein of extracellular domain of human Ig domain-containing, neurite outgrowth inhibitor (Nogo) receptor-interacting protein-1 (LINGO-1(aa76-319)) in prokaryotic cells and prepare the rabbit anti-LINGO-1 polyclonal antibody (pAb).

METHODSThe 732 bp DNA sequence of hLINGO-1(aa76-319) was obtained from pCMV-SPORT6 by PCR and inserted into pET30a(+) plasmid to construct the prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319), which was subsequently transformed into E.coli. The target fusion protein was expressed with IPTG induction and purified by Ni(2+)-NTA affinity chromatography column. The antiserum against hLINGO-1(aa76-319) was obtained from the rabbits immunized with hLINGO-1(aa76-319), and the titer of the pAb was determined using enzyme linked immunosorbent assay (ELISA) and its specificity identified using Western blotting.

RESULTSThe prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319) was constructed successfully. Efficient expression of the target fusion protein was achieved with IPTG induction at the optimal concentration of 0.4 mmol/L and culture temperature at 37 degrees celsius; for 2.5 h. The hLINGO-1(aa76-319) fusion protein was effectively expressed in E.coli as inclusion bodies, and the soluble protein was obtained through denaturation and refolding procedures, and the purified fusion protein showed a purity above 90%. The titer of the anti-hLINGO-1(aa76-319) pAb obtained by immunizing the rabbits with the purified protein reached 1:1.6x10(6), and Western blotting confirmed its good specificity.

CONCLUSIONThe fusion protein hLINGO-1(aa76-319) with high purity has been obtained and the anti-hLINGO-1(aa76-319) pAb obtained shows a high titer and good specificity, which provide important experimental basis for further functional investigation of LINGO-1.

Animals ; Antibodies ; immunology ; isolation & purification ; Antibody Specificity ; Escherichia coli ; genetics ; metabolism ; Humans ; Immune Sera ; immunology ; Membrane Proteins ; biosynthesis ; genetics ; immunology ; Nerve Tissue Proteins ; biosynthesis ; genetics ; immunology ; Plasmids ; genetics ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

OBJECTIVETo observe the expressions of nestin and glial fibrillary acidic protein (GFAP) and their association with reactive astrocytes following spinal cord injury in adult rats.

METHODSAdult rats with compression injury of the spinal cord were divided into 7 groups (n=6) and examined at 1, 3, and 5 days and at 1, 2, 4 and 8 weeks after the injury. The recovery of the locomotor function after the injury was evaluated with Basso, Beattie and Bresnahan (BBB) scale, and the degree and scope of the spinal injury were assessed using toluidine blue staining. Immunohistochemistry, double immunofluorescent labeling and an image analysis system were employed to observe nestin and GFAP expression and cell proliferation in different regions of the spinal cord.

RESULTSThe bilateral hind limb locomotor function of the rats declined severely 24 h after the spinal cord injury and underwent substantial recovery in 1 or 2 weeks after the injury, but followed by rather slow recovery afterwards. Toluidine blue staining of the spinal cord 24 h after the injury showed significant pathological changes in the neurons. The extension of the tissue injury increased with time till 1 week after the spinal cord injury. The site of injury and the adjacent tissues presented with markedly increased nestin and GFAP expressions 24 h after the injury, and nestin+/GFAP(-) cells dominated in the ependymal region around the central canal, whereas nestin+/GFAP+ dominated in the in other regions, showing significant difference from the control group. Nestin and GFAP expression reached the peak level 3 to 7 days after the injury and declined gradually till reaching nearly the control level at 2 weeks.

CONCLUSIONCompression injury of the spinal cord induces up-regulated expressions of nestin and GFAP, and nestin expression is positively correlated to the reactive astrocytes, which, along with the neural stem cells, respond to spinal nerve injury and possibly play a role in repair of the central nervous system injury.

Animals ; Astrocytes ; pathology ; Glial Fibrillary Acidic Protein ; biosynthesis ; genetics ; Intermediate Filament Proteins ; biosynthesis ; genetics ; Male ; Nerve Tissue Proteins ; biosynthesis ; genetics ; Nestin ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; metabolism ; pathology ; Stem Cells ; cytology ; metabolism ; Up-Regulation

OBJECTIVETo study the effects of heat-killed Penicillium marneffei (PM) on the expressions of toll-like receptor-4 (TLR-4), toll-like receptor-2 (TLR-2) and dendritic cell associated C-type lectin-1 (Dectin-1)and the production of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). in mouse peritoneal macrophages.

METHODSMouse peritoneal macrophages were cultured in the presence of heat-killed yeast-phase PM for 24 h, and the average fluorescence intensity of TLR-2, TLR-4, and Dectin-1 in the macrophages was detected using flow cytometry. Fluorescent staining of the macrophages was performed to observe the fluorescence of TLR-2, TLR-4, and Dectin-1 with confocal microscopy. TNF-alpha mRNA in the cell culture supernatant was measured with real-time PCR, and TNF-alpha protein detected using enzyme-linked immunosorbent assay (ELISA).

RESULTSThe average fluorescence intensity of TLR-2, TLR-4 and Dectin-1 in the macrophages was increased in response to a 24-h PM stimulation, and the stimulated macrophages produced large amounts of TNF-alpha.

CONCLUSIONPM up-regulates the expression of TLR-2, TLR-4 and Dectin-1 in mouse peritoneal macrophages, and their expressions are directly associated with macrophage activation.

Animals ; Cells, Cultured ; Lectins, C-Type ; Macrophages, Peritoneal ; cytology ; immunology ; metabolism ; Male ; Membrane Proteins ; biosynthesis ; Mice ; Mice, Inbred BALB C ; Nerve Tissue Proteins ; biosynthesis ; Penicillium ; immunology ; Toll-Like Receptor 2 ; biosynthesis ; Toll-Like Receptor 4 ; biosynthesis ; Tumor Necrosis Factor-alpha ; biosynthesis

Even though there is no direct evidence to prove the cellular and molecular changes induced by radiofrequency (RF) radiation itself, we cannot completely exclude the possibility of any biological effect of mobile phone frequency radiation. We established a carousel-type exposure chamber for 849 MHz or 1763 MHz of mobile phone RF radiation to expose RF to the heads of C57BL mice. In this chamber, animals were irradiated intermittently at 7.8 W/kg for a maximum of 12 months. During this period, the body weights of 3 groups-sham, 849 MHz RF, and 1763 MHz RF-did not show any differences between groups. The brain tissues were obtained from 3 groups at 6 months and 12 months to examine the differences in histology and cell proliferation between control and RF exposure groups, but we could not find any change upon RF radiation. Likewise, we could not find changes in the expression and distribution of NeuN and GFAP in hippocampus and cerebellum, or in cell death by TUNEL assay in RF exposure groups. From these data, we conclude that the chronic exposure to 849 MHz and 1763 MHz RF radiation at a 7.8 W/kg specific absorption rate (SAR) could not induce cellular alterations such as proliferation, death, and reactive gliosis.
Animals ; Apoptosis/*radiation effects ; Body Weight/radiation effects ; Brain/pathology/*radiation effects ; Cell Proliferation/*radiation effects ; *Cellular Phone ; Dose-Response Relationship, Radiation ; Gliosis/etiology/pathology ; In Situ Nick-End Labeling ; Mice ; Mice, Inbred C57BL ; Nerve Tissue Proteins/biosynthesis/genetics ; Proliferating Cell Nuclear Antigen/biosynthesis/genetics ; Radio Waves/*adverse effects