Objective To explore the best serum-free cultural way in neural stem cells from the hippocampus of the newborn rats and observe the characteristics of growth,proliferation,and induced differentiation of neural stem cells.Methods The neonatal rats′ hippocampus tissues were dissociated mechanically.The cells were cultured in the serum-free cultural medium which were added separately basic fibroblast growth factor(bFGF),epithelium growth factor(EGF),bFGF+EGF by suspended cultural way.The neural stem cells were identified by nestin immunofluorescence.After induced differentiation of embryonic cow serum,the differentiated cells were identified.Results The serum-free medium added bFGF and EGF could perfectly induce neural stem cells to proliferate and the differentiated cells expressed the specific antigen of neurons,astrocytes and oligodendrocytes.Conclusion The serum-free medium with bFGF and EGF can be used to culture neural stem cells from hippocampus tissue of rats in vitro.

Blood Vessels ; anatomy & histology ; Facial Nerve ; blood supply ; Humans

Objective Insulin autoantibody (IAA) is known to exist in sera of type 1 diabetes mellitus (T1DM) patients and pre-T1DM individuals. The aim of this study was to establish a novel microtiter plate radioimmunoassay (RIA) for IAA and evaluate its clinical value. Methods Diluted 125Ⅰ-insulin was mixed with 5 ul serum samples in a 96-well microtiter plate and then incubated for 72 h on an orbital plate shaker (4℃). The immunocomplexes were transferred to another protein a coated Millipore plate, and then the plate was washed with Tri-Buffered Saline Tween-20 (TBT) buffer. Counts per minute (CPM) was measured with liquid scintillation and luminescence counter. The positive cut-off point of IAA index was defined as ≥0.06 based on the 99-percentile of the distribution in 317 healthy individuals. The specificity and sensitivity of the assay were calculated from the samples provided by the fourth Diabetes Autoantibodies Standardization Program (DASP 2005). The IAA levels were determined in 71 T1 DM and 551 newly diagnosed type 2 diabetes (T2DM) patients, and 317 healthy controls. The t test, non-parametric test, x2 test and linear correlation analysis were performed on the data using SPSS 11.5 software. The concordance rate was estimated with Kappa value. Results (1) The optimized testing condition was described as 2×104 CPM of 125Ⅰ-insulin, 5 ul serum sample and slowly horizontal shaking for 72 h. (2) The intra-assay CV was 4.8%-8.9% and inter-assay CV was 6.4%-10.5%. Based on DASP 2005 samples, the specificity and sensitivity of the assay were 97% (97/100) and 50% (25/50), respectively. Ninety-six serum samples with different IAA levels were selected and tested to compare between our new method and a domestic IAA RIA kit. The results showed that the IAA indices from the two methods were positively correlated (r= 0.678, P<0.001). The concordance rate was 72.9 %(Kappa value=0.402). There were 25 samples with discordant results, which were positive for IAA titer using the corresponding microtiter plate RIA but negative using the novel RIA kit. (3) In TIDM group the positive rate of IAA was 19.7% (16/71), higher than the healthy controls (0.9%, x2=54.36, P<0.001). The subgroup of T1DM children (with 0-9 years) showed the highest IAA positive rate (55.6% ,x2=4.85, P<0.05). In T2DM group the frequency of IAA was 1.5% (8/551), which had no significant difference comparing with that of healthy controls (x2= 0.95, P >0.05). Conclusions Our proposed microtiter plate RIA method for IAA is highly sensitive and specific, likely to be feasible for clinical application. The frequency of IAA is high in children with T1DM.

Cathepsin B from Helicoverpa armigera (HCB) belongs to the group of cysteine proteinases. HCB is proved being involved in the degradation of yolk proteins during embryonic development,which is an acidic preferring enzyme and is resistant to SDS. The expression of the proenzyme may offer a model for investigating the activation of the enzyme. The HCB gene was constructed into pPIC9K and expressed in Pichia pastoris KM71 strain . After induction by methanol, HCB was expressed and secreted into the medium. The molecular weight of the recombinant procathepsin B was determined as about 38 kDa. The expressed product was confirmed to be HCB by immunoblotting assay using specific rabbit anti-HCB polyclonal antibody. The activity of the product was assayed by in situ hydrolysis (gelatin-SDS-PAGE). These results showed that HCB with proteolytic activity was expressed in P. pastoris KM71. This proenzyme can be used for further research on the activation of the proenzyme or industrial production.

To observe the clinical effect of Yisui decoction plus western medicine in treating multiple system atrophy patients, totally 65 patients from China-Japan Friendship hospital during 2008-2012 with complete clinical data and received consecutive traditional Chinese medicine and western medicine treatment for more than 3 months were observed changes of traditional Chinese medicine symptom score, part 1 of unified multiple system atrophy rating scale, orthostatic hypotension before treatment and after 3 months treatment. After 3 months treatment, total effective rate of traditional Chinese medicine symptom was 70.8%. Compared with before treatment, score of part 1 of unified multiple system atrophy rating scale was obviously reduced after 3 month treatment (P < 0.001). Ex- cept swallow function without significant improvement, the remaining projects of unified multiple system atrophy rating scale were im- proved obviously (P < 0.05), of which the most obvious differences were orthostatic symptoms, falls and intestinal function (P < 0.001). Orthostatic hypotension after 1 month treatment and 3 month treatment was obviously better than before treatment (P < 0.001). There was no significant difference in orthostatic hypotension between 1 month treatment and 3 month treatment. The research results show that Yisui decoction plus western medicine has a certain effect on improving clinical symptoms of multiple system atrophy patients, especially has a significant effect on orthostatic hypotension, and can maintain a stable clinical effect in a certain period of time.
Adult ; Aged ; Humans ; Hypotension, Orthostatic ; drug therapy ; Male ; Medicine, Chinese Traditional ; adverse effects ; methods ; Middle Aged ; Multiple System Atrophy ; drug therapy ; Retrospective Studies ; Treatment Outcome

Objective To evaluate the role of c-Jun N-terminal kinase (JNK) signaling pathway in paclitaxel-induced apoptosis in hippocampal neurons of rats, and the relationship with nuclear factor kappa B (NF-κB) pathway.Methods The primarily cultured hippocampal neurons were seeded in 96-well plate at a density of 1×106 cells/ml (200 μl/hole) , and were randomly divided into 4 groups (n=8 each) using a random number table: control group (C group), paclitaxel group (P group), JNK inhibitor SP600125 group (S group), and SP600125 + paclitaxel group (S+P group).Paclitaxel 2 ml (1 μmol/L) was added to group P.SP600125 2 ml (10 μmol/L) was added to group S.In group S+P, SP600125 2 ml (10 μmol/L) was added, the cell were then incubated for 1 h, and then paclitaxel 2 ml (1 μmol/L) was added.The cells were then incubated for 24 h.At 24 h of incubation, the apoptosis in hippocampal neurons was detected by flow cytometry, and the expression of NF-κB p65 was measured by Western blot.The apoptosis rate was calculated.Results Compared with group C, the apoptosis rate was significantly increased, and the expression of NF-κB p65 was up-regulated in P and S+P groups, and the apoptosis rate was significantly decreased, and the expression of NF-κB p65 was down-regulated in group S (P＜0.05).Compared with group P, the apoptosis rate was significantly decreased, and the expression of NF-κB p65 was down-regulated in group S+P (P＜0.05).Conclusion JNK signaling pathway mediates paclitaxel-induced apoptosis in hippocampal neurons of rats, and the mechanism is likely related to inhibition of NF-κB pathway activation.

To study the toxicokinetics of bakuchiol, hepatic and renal toxicity in rats after single oral administration of Psoraleae Fructus and combined with Glycyrrhizae Radix et Rhizoma, in order to provide scientific evidences for clinical safe medication use. A total of 35 SD rats were randomly divided into seven groups: vehicle (distilled water) control group, Glycyrrhizae Radix et Rhizoma group, positive control (aristolochic acid A) group, Psoraleae Fructus (40 g x kg(-1)) group( both male and female rats), Psoraleae Fructus and Glycyrrhizae Radix et Rhizoma (40 +20) g x kg(-1) group (both male and female rats). HPLC-UV method was used to determine the concentration of bakuchiol in rat plasma at different time points after single oral administration. Plasma alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), plasma creatinine (Cr), N-acetyl-β-D-glucosaminidase (NAG) and kidney injury molecule 1 (Kim-1) were measured after administration for 24 h. The main toxicokinetics parameters of bakuchiol in rats exert significantly gender difference. When Psoraleae Fructus combination with Glycyrrhizae Radix et Rhizoma, the total area under the plasma concentration-time curve( AUC), C(max), and plasma clearance (CL) of bakuchiol were increased, respectively; CL, half-life (t½) were decreased, and T(max) were prolonged. The biochemical indicators (including ALT, AST, BUN, Cr and KIM-1 level) in different dose of Psoraleae Fructus groups, were found no statistically significant difference when compared with vehicle control group. The level of NAG in both Psoraleae Fructus and compatibility with Glycyrrhizae Radix et Rhizoma groups were significant increased (P < 0.05). There are obvious effects on toxicokinetics of bakuchiol in rats when Psoraleae Fructus combined with Glycyrrhizae Radix et Rhizoma. Renal toxicity induced by Psoraleae Fructus at high dose was observed after single oral administration and no liver damage in rats was found.
Administration, Oral ; Animals ; Female ; Glycyrrhiza ; toxicity ; Kidney ; drug effects ; Liver ; drug effects ; Male ; Phenols ; pharmacokinetics ; toxicity ; Psoralea ; toxicity ; Rats ; Rats, Sprague-Dawley ; Rhizome ; toxicity ; Toxicokinetics

Animals ; Cell Differentiation ; genetics ; physiology ; Humans ; MicroRNAs ; genetics ; Ossification, Heterotopic ; genetics ; Signal Transduction ; genetics ; physiology

Objective To explore the effects of the autoantibody against the second extracellular loop of ?3-adrenoceptor(?3AR)from children with heart failure on isolated cardiomyocytes of adult rats.Methods The synthetic peptide corresponding to the sequence of the se-cond extracellular loop(176-202 amino acid residues)of the human ?3AR was used as the antigen to screen the anti-?3AR autoantibodies from the sera of the 50 children with heart failure and 50 healthy children by enzyme linked immunosorbent assay(ELISA);IgG in the children with heart failure of positive autoantibodies sera was purified.The effects of purified IgG per each group on contractile response of isolated cardiomyocytes of adult rats were observed using motion-edge detection system and double-provocation fluorescence photomultiplier system.Results Among the 50 children with heart failure,the anti-?3AR autoantibodies positive rate was 30%(15/50 cases),and ob-viously higher than that of healthy children[12%(6/50)cases,P

Nicotinic acetylcholine receptors (nAChRs) belong to ligand-gated ion channel receptors, of which α7 nAChR subtype is widely distributed in the cerebral cortex, thalamus, hippocampus, and also identified in microglia, macrophages, bone marrow cells, etc. Previous studies revealed that α7 nAChR is closely related to the function of the cholinergic anti-inflammatory pathway, and is a vital target for drug development of Alzheimer's disease and schizophrenia. The establishment of a stable α7 nAChR in vitro drug screening system is crucial for the efficient screening of novel drugs targeting this target. Recombinant expression of different subtypes of nAChRs on Xenopus laevis oocyte membranes and current detected by two-electrode voltage clamp (TEVC) is an advanced and complex model for novel drug screening. Molecular chaperones can assist the assembly of some nAChR subunits to form functional receptors, providing a stable expression model for the screening of compounds targeting this receptor. In this study, a molecular chaperone gene of α7 nAChR, transmembrane protein 35A (Tmem35a), was isolated and cloned from rats. We constructed the recombinant expression vector and obtained the cRNA of Tmem35a by in vitro transcription technique. Two cRNAs (Tmem35a and α7) were mixed and injected into X. laevis oocytes for expression. Then, the effects of this molecular chaperone on the current expression and pharmacological properties of α7 nAChR were evaluated by the TEVC. The results revealed that TMEM35A, also known as novel acetylcholine receptor chaperone (NACHO) could effectively increase the expression of α7 nAChR protein on oocyte membranes, and the amount of α7 nAChR protein was increased about 1-fold. The peak current induced by agonist acetylcholine (ACh) was increased about 10-fold. After injection of Tmem35a cRNA, the median effect concentration (EC₅₀) value of α7 nAChR to agonist ACh is 228.5 μmol·L^-1, which shows almost no difference from native α7 nAChR (EC₅₀: 223.3 μmol·L^-1), indicating the preservation of the normal properties of α7 nAChR. The results of this investigation indicate that the molecular chaperone NACHO effectively assists the heterologous expression of α7 nAChR in X. laevis oocytes, which provides a model for screening the potency of lead compounds targeting α7 nAChR. All animal experiments in this study were reviewed and approved by the Ethics Committee of Guangxi University (approval number: GXU-2023-0249).