[Objective]To explore the distribution and expression changes of tight junctional protein JAM-1 in rat models after intracerebral hemorrhage (ICH) and their significance.[Methods]One hundred and twenty-eighty healthy male SD rats were randomly divided into normal control group (n=16) and ICH group (n=112),and the ICH models were induced by stereotactically injecting 75 uL autologous blood into the right caudate nucleus.Seven time points after ICH (6,12,24 and 48 h,and 3,7 and 14 d after ICH,16 rats for each time point) were chosen.BBB permeability was evaluated by Evans blue dye extravasation.The distribution and expression of JAM-1 were detected by immunofluorescence and real-time quantitative PCR.[Results] As compared with that in the normal control group,BBB permeability in the ICH group significantly increased at 24 and 48 h,and 3 and 7 d after ICH (P＜0.05).JAM-1 expression decreased at blood vessels at 12,24 and 48 h after ICH,and JAM-1 expressed at the circulatingleukocytes3 dafterlCH,and abundant JAM-1 positive cells around hematoma were noted in the ED-l-positve macrophages 7 d after ICH.JAM-I mRNA significantly decreased at 12,24 and 48 h after ICH,and significantly increased 7 d after ICH as compared with that in the normal control group (P＜0.05).[Conclusion] JAM-1 experssion changes not only participate in regulation of BBB permeability but also play roles in inflammatory insult after ICH.

Objective To investigate the proliferative differences of adipose-derived stem cells (ADSCs) from neonatal suckling SD rats (5-d-old) and adult ones under the same culture condition.Methods ADSCs were isolated from the subcutaneous adipose tissues of neonatal suckling SD rats and adult ones,and then,type Ⅰ collagenase digestion was employed to obtain the ADSCs; the morphology of these cells was detected.The expressions of such cell surface markers as CD45,CD29 and CD90 were observed. The number of ADSCs on the 4th d of culture under the same condition and with the same planted density was compared between the neonate and adult rats. In vitro culture of the second generation of ADSCs was performed in the 96-well plates, and CCK-8 and alamar blue kit were employed to compare and quantitate the proliferative differences; optical density was observed by microplate reader. Results The ADSCs from neonatal SD rats and adult ones expressed the stem cell biomarkers: the expression of CD45 was negative, and that of CD29 was 98.04％ and 93.17％,respectively,and that of CD90 was 94.92％ and 93.3％,respectively,for neonate SD rat and adult ones.The cell counting results indicated that the number of ADSCs from neonatal rats ([8.87±0.13]×105 cells) was larger than that of adult ones ([4.51±0.36]×105 cells) after being cultured under the same condition and at the same planted density. The optical density value of ADSCs in neonatal rats was significantly higher than that in adult ones on the 6th and 7th d of culturing detected by CCK-8 kit and on the 2nd-7th d of culturing by alamar blue assay. Conclusion The proliferative ability of ADSCs from neonatal rats is greater than that of adult ones.

Objective To investigate the effect of nimesulide (NIM), a selective cyclooxygenase-2 (COX-2) inhibitor, on angiopoietins (Ang) gene expression of human glioma xenografts in nude mice and its significance. Methods Human SHG44 glioma cells were inoculated subcutaneously in 16 nude mice to establish xenograft models, and then these mouse models were randomly divided into NIM treatment group and control group. NIM (6 mg/kg) and saline were poured into the stomachs of the mice in each group, respectively, once daily for 35 d. The mRNA expressions of Ang-1 gene and Ang-2 gene in the xenografts were determined by RT-PCR. Microvessel density (MVD) in the xenografts was assessed by immunohistochemical technique. The tumor growth curve was drawn and the inhibition ratio of tumor growth was calculated. Results NIM could significantly inhibit the glioma xenografts growth with its inhibition rate reaching 42.03%. The mRNA expression of Ang-2 gene in NIM treatment group (0.2032±0.0185) was significantly lower than that in control group (0.6024±0.0289, P＜0.05), but that of Ang-1 gene showed no significant changes; therefore, the mRNA ratio of Ang-2/Ang-1 genes was decreased (0.5825±0.0621 vs. 1.5847±0.1948, P＜0.05). MVD in the xenografts of the NIM treatment group was significantly lower than that in the control group (P＜0.05). Conclusion NIM, by down-regulating the mRNA expression ofA ng-2 gene and changing the mRNA ratio of Ang-2/Ang-1 genes, can inhibit the tumor growth

Objective To explore the effect of chondroitin sulfate enzyme ABC (chABC) on glial scar in rat models of brain traumatic injury (TBI). Methods Thirty-eight Wistar rats were randomly divided into 5 groups, including normal control group (n=2), model group (rat models of TBI,n=9), 1.0 U/mL chABC treatment group (n=9), 2.5 U/ml chABC treatment group (n=9) and 5.0 U/ml chABC treatment group (n=9). After performing TBI by free falling in the later 4 groups, rats of the model group were given no treatment, while those of the other 3 groups were administrated with different concentrations of chABC by local injection respectively. One, 2 and 4 w after TBI, HE staining was performed on the brain tissues of these rat models;and immunohistochemical assay and Western blotting were employed to evaluate the secreting of chondroitin sulfate proteoglycans (CSPGs) and the therapeutic effect of chABC on glial scar. Data were statistically analyzed using t-test. Results Pathological test revealed the scars in the treatment groups were significantly fewer than those in the model group 2 w after TBI, with 5.0 U/mL chABC treatment group enjoying the fewest level (P<0.05). Immunohistochemical assay showed that the secreting of CSPGs in the treatment groups and model group was significantly increased than that in normal control group 2 w after TBI (P<0.05);the 5.0 U/ml chABC treatment group showed an obvious reduction of CSPGs secreting as compared with the model group (P<0.05). Western blotting indicated that the treatment groups showed an obvious reduction of CSPGs secreting as compared with the model group 1, 2 and 4 w after TBI (P<0.05);an obvious gradual reduction of CSPGs secreting in the model group, 2.5 and 5.0 U/ml chABC treatment groups was noted 1, 2 and 4 w after TBI (P<0.05). Conclusion ChABC could degrade the glial scar by degrading the CSPGs molecules and improve the microenvironment of local axonal regeneration after TBI;In this experiment, the highest concentration of chABC (5U/ml) shows the best effect on removing the glial scar.

Objective To investigate the effects of transient axonal glycoprotein-1 (TAG-1) on activity of U251 cells and expressions of AICD, p53 and EGFR genes in the cells. Methods The viability of U251 cells was tested by MTT assay at 48 h following the addition of various concentrations of TAG-1 (0, 5, 10 and 20 μg/mL). The expression ofamyloid precursor protein (ALP) was detected by immunofluorescent staining. The apoptotic cells were examined by TUNEL. Real-time PCR was employed to detect the influence of TAG-1 on the expressions of AICD, p53 and EGFR genes in U251 cells. Results TAG-1 did not play an inhibitory effect on the proliferation of the U251 cells. APP was abundantly expressed on membrane of the U251 cells. U251 cells did not show apoptotic cells but increased expressions of AICD, p53 and EGFR genes were noted when U251 cells were exposed at 10 μg/mL of TAG-1. Conclusion TAG-1 plays an important role in regulating the proliferation of glioma and may not induce the apoptosis of U251 cells through the signal pathway of TAG-1/APP/AICD/p53 or TAG- 1/APP/AICD/EGFR.

Objective To detect the immunogenicity of the recombinant DNA vaccine that encoded for neurite growth inhibitors: Nogo-A, oligodendrocyte myelin glycoprotein (OMgp), tenascin-R (TN-R) and myelin-associated glycoprotein (MAG) after the nerve injury under the help of pAdEasy, a kind of adenovirus plasmid being the vector of the DNA. Methods Sixteen 5-w-old Lewis rats were randomized into DNA vaccination group (vaccine group) and pAdEasy group. Rats in the vaccine group were immunized once weekly for a consecutive 8 w by bilateral injection of the recombinant plasmid into the musculus tibialis. The immunized animals in the 2 groups were exsanguinated each time before the vaccination for sera collection, and the qualitation and quantitation of the antibodies in the serum were detected by Dot-blot analysis and ELISA. Results The vaccine group could produce fusion-protein antibodies against Nogo-A, MAG, OMgp and TN-R at the 6th w of vaccine injection, while pAdEasy group could not. The valency of antiserum was shown by ELISA as 1:1 000 000 at the 6th w of vaccine injection and kept this level stably. Conclusion The DNA vaccine exclusively induces the generation of the fusion-protein antibodies against Nogo-A, MAG, OMgp and TN-R in vivo, which controls the favorable immunogenicity.

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

Objective To offered some prophase works by preparing Neurocan protein, antiserum, and assaying their characteristics, in order to construct the isopropy-β-D-thiogalactoside (IPTG)-participated DNA vaccine, which can neutralize the inhibitors in the injured CNS following the immune administration and then promote the nerve regeneration. Methods Neurocan gene was syntbetized with HisTag label in beginning and enzyme-cut sites at amphi- of the sequence. The prokaryotic expression plasmid, PET30a-Neurocan, was constructed as usual, converted into the Escherichia coli, and induced by IPTG to express positively. The interest protein was identified by SDS-PAGE and Western blot respectively. The preimmune serum was as the negative control during the ELISA assay of antiserum valency. The immune serum was as the first antibody, and the goat-anti-rabbit labeling with alkaline phosphatase (AP) was employed as the second one. Coloration was with NBT/BCIP method. Results The correct sequence of the synthetic Neurocan gene was clearly showed by identification with enzyme-cut, PCR and sequencing. The Neurocan protein expressed by prokaryotic showed its molecular weigh as 55 000 following the SDS-PAGE identification, and it could specifically bind with anti-HisTag, which implied the interesting protein just as the expression product of Neurocan gene. The valency of antiserum was shown by ELISA as 1:1 000 000, the purpose strap of which was confirmed by Western blot. Conclusions Neurocan protein could be successfully expressed in prokaryotic, the antibody of which could be specifically obtained by immune administration to the rabbit. The Neuroncan antibody could bind with the Neurocan protein specifically.

Objective To analyze the feasibility of local LINGO-1 polyclonal antibody administration for treatment of spinal cord injury in adult rats. Methods Twenty-four adult female SD rats were randomized into sham-operated group, rabbit IgG group and LINGO-1 antibody group. In the latter two groups, partial transaction of the T9 segment of the spinal cord was performed to completely sever the dorsal eorticospinal tract, followed immediately by administration of rabbit IgG and anti-LINGO polyclonal antibody via a mini-osmotic pump, respectively. At 3 and 28 days after the operation, the T8～10 segments of the spinal cord were harvested to prepare cryosections, and immunofluorescence staining was used to analyze the penetration of LINGO-1 polyclonal antibody into the spinal cord tissue and its specific binding to LINGO-1 molecules. Results In LINGO-1 antibody group, the presence of rabbit antibodies was detected at the injured sites of the spinal cord at 3 and 28 days after the operation. The mean immunofluorescence density was significantly lower in L1NGO-1 antibody group than in rabbit IgG group at 3 days after the operation (P<0.05). In rabbit IgG group, the mean immunofluorescence density for LINGO-1 in the crysections pre-treated with LINGO-1 polyclonal antibody was significantly lower than that in sections pre-treated with rabbit IgG(P<0.05). Conclusion Locally administered LINGO-1 polyclonal antibody can penetrate into the injured sites in the spinal cord in a wide time window and recognizes LINGO-1 molecule specifically, suggesting the feasibility of passive immunotherapy for spinal cord injury.

Objective To observe the time course of changes in synaptophysin (P38) expression in the cortex and hippocampus of rats with posttraumatic epilepsy (PTE), and explore the role of synaptic plasticity in the epileptogenesis of PTE. Methods Thirty-seven male Sprague-Dawley rats were randomized into normal control group (n=5), sham-operated group (n=12) with intracortical saline injection, and PTE model group (n=20) with stereotactic FeCl<,2> injection (0.1 mol/L, 10 μ1) into the motor cortex. The expression of P38 in the brain cortex and hippocampus of the rats was detected immunohistochemically at 1 h and 7, 14 and 30 days after the injections. Results Most of the rats with FeCl<,2> injection developed isolated epileptiform discharges soon alter the injection. Compared with the sham-operated groups, the rats in PTE group showed significantly decreased P38 expression in the right frontal cortex at all the time points of measurement (P<0.05). At 1 h after FeCl<,2> injection, P38 expression in the polymorphic layer, stratum lacunosum and stratum radiatum of the right hippocampai CA3 area and DG molecular layer underwent no significant changes (P>05), but at 7 days, the expression increased significantly in all the stratum regions of the right hippocampal CA3 area, and this high expression level was maintained till 30 days after the injection. Conclusion Synaptic plasticity alterations in relation to P38 expression changes in the cortex and hippocampus may play an important role in the epileptogenesis of PTE in this rat model.