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 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 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

OBJECTIVE: To prove the existence neurons in the rat corpus callosum, the potential function of these neurons and their connection. METHODS: Immunohistochemistry was used performed to examine the expressions of NeuN, a mature neuron marker,and N-type voltage-dependent valcium channel alpha1-subunit (Cav2.2)in the section of the rat corpus callosum. Horseradish peroxidase (HRP) normal sodium solution (30%), the retrograde tracer,was injected under the frontal forceps of corpus callousm and HRP absorbed by the process of neurons in the brain slices was stained with tetramethyl benzidine. RESULTS: There were some NeuN positive cells in the rat corpus callosum and Cav2.2 was detected in some of these NeuN positive cells.Neurons with positive HRP were found in the rat corpus callosum and some of these neurons connected to the cortex or corpus striatum. CONCLUSION There are a few neurons in the corpus callosum of adult rats and some of them express Cav2.2. Neurons in the corpus callosum have connections with the brain cortex or corpora striatum.
Animals ; Calcium Channels, N-Type ; biosynthesis ; Corpus Callosum ; cytology ; metabolism ; DNA-Binding Proteins ; Male ; Nerve Tissue Proteins ; biosynthesis ; Neural Pathways ; physiology ; Neurons ; cytology ; Nuclear Proteins ; biosynthesis ; Random Allocation ; Rats ; Rats, Sprague-Dawley

We transfected human neural stem cells using lentiviral vectors encoding glial cell line derived neurotrophic factor (GDNF) to study its expression level in vitro and to get a stable cell line expressing GDNF. First, GDNF gene was sub-cloned into the lentiviral transfer vectors. Then, the recombinant lentiviral supernatants were packaged by 293T cells through three plasmids transient co-transfection method using standard lipofectamine reagent. The viral titers were tested by the transfection efficiency of 293T cells. At the same time, human neural stem cells (hNSC) were transfected under different multiplicity of infection. GDNF gene expression level and protein secretion level of hNSC were tested by real-time PCR and ELISA methods after transfection. Lentiviral vectors encoding GDNF were constructed. Using lentiviral vectors encoding GDNF we successfully transfected human neural stem cells, and got a stable neural stem cell lines over-expressing GDNF. Furthermore, the results indicated that GDNF expression was influenced by the multiplicity of infection. Human neural stem cells could over-express GDNF through lentivial vectors tranfection. Its gene expression level and protein expression level correlate with the multiplicity of infection.
Embryonic Stem Cells ; metabolism ; Genetic Vectors ; genetics ; Glial Cell Line-Derived Neurotrophic Factor ; biosynthesis ; genetics ; Humans ; Lentivirus ; genetics ; Nerve Tissue ; cytology ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

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 examine the expression of nestin and neurogenin 3 (Ngn3), the markers of pancreatic stem cells, in the human fetal pancreas.

METHODSThe human fetal pancreas tissue of 12 and 14 weeks were examined for the expression of nestin and Ngn3 using the techniques of immunofluorescence dye and RT-PCR.

RESULTSBoth nestin and Ngn3 expressed widely in 12 and 14 weeks before in human fetal pancreatic tissue. In these positive cells there was no co-expressing insulin or glucagon. There were nestin and Ngn3 co-expressing cells in ducts but not in the islets. The results of RT-PCR also indicated the expression of nestin and Ngn3.

CONCLUSIONSThere was no expression of the markers of mature endocrine cells in the nestin and Ngn3 positive cells, and they were the marks of no-differentiation cells in the human fetal pancreatic tissue.

Basic Helix-Loop-Helix Transcription Factors ; biosynthesis ; genetics ; Fluoroimmunoassay ; Humans ; In Vitro Techniques ; Intermediate Filament Proteins ; biosynthesis ; genetics ; Microscopy, Fluorescence ; Nerve Tissue Proteins ; biosynthesis ; genetics ; Nestin ; Pancreas ; cytology ; embryology ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo determine cell proliferation and nestin expression in the ependyma of adult rat spinal cord after injury.

METHODSRat spinal cord injury models were established by aneurysm clip compression, and nestin expression and proliferation of ependymal cells at different times were shown with pathological and immuno-histochemical staining.

RESULTSEpendymal cells adjacent to the injured site demonstrated a dramatic increase in nestin expression 24 hours after compression. Proliferating cell nuclear antigen was positive, and significant proliferation was observed after 7 days. Nestin expression was down regulated as time went by.

CONCLUSIONNormally quiescent mature ependymal cells appear to revert to an embryonic state in response to spinal cord injury.

Animals ; Cell Division ; Ependyma ; cytology ; metabolism ; Immunohistochemistry ; Intermediate Filament Proteins ; biosynthesis ; Male ; Nerve Tissue Proteins ; Nestin ; Rats ; Rats, Wistar ; Spinal Cord Injuries ; metabolism ; pathology

OBJECTIVEFurther studies have been conducted to evaluate the roles of Ngn3 in adult islet maintenance and renewal.

METHODSIslets were isolated from 6 - 8 week old male C57BL/6 mice. After common bile duct cannulation, the pancreas was resected and digested in collagenase V (2.5 mg/ml). Islets were then handpicked and 10 - 12 islets were plated in 60 mm culture dish and cultivated with RPMI-1640, which contained 12.5 mmol/L HEPES, 5.2 mmol/L glucose and 2% fetal bovine serum (FBS). Islet cells were analyzed by immunocytochemistry methods for A6, insulin, glucagon, nestin, Ngn3 and 5-bromo-2'-deoxy-uridine (BrdU).

RESULTSThe results of these studies indicated that less than 15 percent of proliferated islet cells were Ngn3 expressing cells, in which about one third of the Ngn3 positive cells co-expressed A6. The existence of Ngn3 in cultured islet cells is consistent with the results from other's findings both in embryogenesis and adult islet studies. A significant finding of our study is that the existence of A6 and Ngn3 co-expressing cells in the cultured islet. A6 is a marker for identifying bile duct epithelial cell oriented hepatic progenitor cells. Islet-derived A6 cells are possibly born in the adult pancreatic duct and migrate into islets. A6 cells co-express Ngn3 when these cells commit to endocrine lineage within the islets. More interestingly, islet-derived A6 positive cells have the potential to transdifferentiate into hepatic cells.

CONCLUSIONThe presence of Ngn3(+) and A6(+) cells in the cultured islets suggests that the four established islet cell types arise from a common endocrine lineage residing within the adult islets. A6 and Ngn3 are useful markers for understanding intra-islet adult stem cell lineages in our future studies. This approach may allow for significant advances in understanding the IPC proliferation and differentiation, and open the possibility of using intra-islet adult stem cells for diabetes treatment.

Animals ; Basic Helix-Loop-Helix Transcription Factors ; Cell Differentiation ; Cell Lineage ; Cells, Cultured ; Islets of Langerhans ; cytology ; Male ; Mice ; Mice, Inbred C57BL ; Microfilament Proteins ; Nerve Tissue Proteins ; biosynthesis ; Protein-Tyrosine Kinases ; biosynthesis ; Stem Cells ; cytology ; metabolism

OBJECTIVETo observe the position and quantity of nestin expression in SD rat eyes in different stages of postnatal development.

METHODSImmunocytochemical method was used to identify nestin expression in the eyes of SD rats of 1 to 30 days old.

RESULTSNestin expression was detected in the retina and extraocular muscles of SD rats. The expression varied with the time of postnatal development, distributing in the entire retina layers in earlier stages and confined in the nerve fiber layer in later stages. The quantities of nestin expression in the extraocular muscles decreased gradually with growth.

CONCLUSIONNestin expression in the retinas and extraocular muscles of SD rats decreases during the postnatal development.

Animals ; Animals, Newborn ; Eye ; growth & development ; metabolism ; Immunohistochemistry ; Intermediate Filament Proteins ; biosynthesis ; Nerve Tissue Proteins ; biosynthesis ; Nestin ; Oculomotor Muscles ; growth & development ; metabolism ; Rats ; Rats, Sprague-Dawley ; Retina ; growth & development ; metabolism ; Time Factors