BACKGROUND: The functional gene fragments integrate into gene vector, which is then transfacted into target cells or joint cavity, through the transgenic target cells continue to secrete a large number of functional gene product, local therapeutic concentrations could be maintained within a long period of time, thus repairing articular cartilage injury. OBJECTIVE: To transfect rabbit articular chondrocytes using recombinant retroviral vector-mediated transforming growth factor-βl (TGFβ_1) in vitro, and to observe its expression and its effect on biological characters of chondrocytes. METHODS: Rabbit chondrocytas were isolated by use of trypsin digestion method. Vector was PLNCX_2 Hind Ⅲ/Not Ⅰ doubly digested and dephosphorylated, connected with some multiple cloning sites and RFP gene following pDsRed_2 double digestion, to build PLNCX_2-RFP. TGFβ_1 gene was amplified from the PGEMT-TGF and connected with PLNCX_2-RFP following double digestion, to build PLNCX_2-TGFβ_1-RFP. Subsequent to packaging retroviral vector, viral supernatant titer was detected. The cultured and transfected chondrocytes in rabbit knee joint were divided into 3 groups: control group (without any transfection), transfected PLNCX_2 group and transfected PLNCX_2-TGFβ_1-RFP group, continued screening 2 weeks to observe the cellular changes. Cell supematant transfected stably were collected for detecting the effect of gene transfection on the chondrocytes with NO detection kit, ELISA assay was applied to determine human TGFβ_1 expression in cell culture supernatant. RESULTS AND CONCLUSION: The recombinant gene PLNCX_2-TGFβ_1-RFP was identified correct sequence by the enzyme digestion sequencing TGFβ_1 and RFP, which showed that the eukaryotic expression vector PLNCX_2-TGFβ_1-RFP had been successfully built as expectation. They were then transfected into packaging calls and cultured, the virus titer was defined as 1×10~6 CFU. Following stable transfection of cartilage cells, red fluorescence can be observed, proving successful transfection. After continuous screening 2 weeks, the scattered adherent calls formed positive clones, and gradually diffusely integrated, cell clusters appeared with common dual cores, the calls proliferated actively. NO concentration in the transfected PLNCX_2-TGFβ_1-RFP group was higher than that of transfected PLNCX_2 group (P < 0.05), no difference was significant between control group and transfected PLNCX_2 group. The control group and the group transfected PLNCX_2 showed no TGFβ_1 expression, while TGFβ_1 concentration was (28.08±3.73) ng/L in the transfected PLNCX_2-TGFβ_1-RFP group. PLNCX_2 ratroviral vector-mediated human TGFβ_1 can be effectively transfected into rabbit knee joint cartilage cells and obtain stable expression, while the transfected cartilage calls proliferate actively.

ObjectiveTo study the expression of vascular endothelial growth factor (VEGF) and aquaporin 1 (AQP1) in the synovium of rheumatoid arthritis (RA) patients,and their correlation with angiogenesis.MethodsThe expression of VEGF and AQP1 in the synovial tissue was detected with immunohistoehemistry in 7 patients with RA,12 patients with osteoarthritis (OA).The expression of VEGF and AQP1 positive cells and vessels was scored,and the correlation of positive cells with vessels was analyzed.Rank-sum test and Spearman's correlation analysis were used for statistical analysis.ResultsThe expression of VEGF positive cells was significantly correlated with vessels in the synovium of RA group(rs=0.971,P＜0.01 ).No significant correlation was found between VEGF positive cells and vessels in synovium of the OA group (rs=0.235,P=0.462).There was no evident difference in the expression of VEGF positive cells between RA and OA group (Z=-0.390,P=0.711 ).The expressions of AQP1 positive cells were significantly correlated with vessels in the synovium of RA group (rs=0.828,P=0.022).No significant correlation was found between AQP1 positive cells and vessels in the synovium of OA group (rs=0.396,P=0.203).There was no notable difference in the expression of AQP1 positive cells between RA and OA group (Z=-0.302,P=0.773).The expressions of VEGF positive cells were significantly correlated with AQP1 positive cells in RA group (rs=0.891,P=0.007).No significant correlation was found between VEGF positive cells and AQP1 positive cells in OA group (rs=0.338,P=0.283).ConclusionThe expressions of VEGF and AQP1 are significantly correlated with angiogenesis in the synovium of RA,and they play an important role in the pathology of RA.Moreover,there may be synergistic effect between VEGF and AQP1 in RA.

10.3969/j.issn.1007-3969.2013.04.008

BACKGROUND:Apoptosis of chondrocytes eventualy leads to osteoarthritis. New studies find many mechanisms of chondrocyte apoptosis, but the mechanism of endoplasmic reticulum stress-induced chondrocyte apoptosis is stil in the first stage. OBJECTIVE: To review how the endoplasmic reticulum stress causes the chondrocyte apoptosis and to explore a new method of treatment for osteoarthritis. METHODS: A computer-based online search of PubMed database, CNKI database and Wangfang database between January 2000 and January 2015 was performed to search related articles with the key words of “endoplasmic reticulum stress, chondrocyte, apoptosis” in English or in Chinese, respectively. The word “AND” was used for the connection between the word retrieval. Literatures related to osteoarthritis were selected; the articles published lately in authoritative journals were preferred. RESULTS AND CONCLUSION: A total of 62 literatures were primarily selected, and 57 documents were involved in result analysis according to inclusion criteria. PERK, IRE1 and ATF6 play an important role in the chondrocytes. Endoplasmic reticulum stress-mediated apoptosis mechanisms include two kinds, UPR and Ca2+start signal, but the specific mechanism and the interactions between apoptosis pathway are unclear. Inhibitory molecules for chondrocyte apoptosis in these signaling pathways as treatment targets for osteoarthritis may provide new methods for the treatment of osteoarthritis.

The 1H-NMR fingerprints of three different species tibetan medicine sea buckthorn were established by 1H-HMR metabolomics to find out different motablism which could provide a new method for the quality evaluation of sea buckthorn. The obtained free induction decay (FID) signal will be imported into MestReNova software and into divide segments. The data will be normalized and processed by principal component analysis and.partial least squares discriminant analysis to perform pattern recognition. The results showed that 25 metabolites belonging to different chemical types were detected from sea buckthorn,including flavonoids, triterpenoids, amino acids, carbohydrates, fatty acids, etc. PCA and PLS-DA analysis showed three different varietiest of sea buckthorn that can be clearly separated by the content of L-quebrachitol, malic acid and some unidentified sugars, which can be used as the differences metabolites of three species of sea buckthorn. 1H-NMR-based metabonomies method had a holistic characteristic with sample preparation and handling. The results of this study can offer an important reference for the species identification and quality control of sea buckthorn.
Hippophae ; metabolism ; Magnetic Resonance Spectroscopy ; methods ; Medicine, Tibetan Traditional ; Metabolomics

The effects of presynaptic nicotinic acetylcholine receptors (nAChRs) on excitatory synaptic transmission in CA1 pyramidal neurons of the rat hippocampus were examined by blind whole-cell patch clamp recording from hippocampal slice preparations. Local application of the nAChRs agonist dimethylphenyl-piperazinium iodide (DMPP) did not induce a postsynaptic current response in CA1 pyramidal cells. However, DMPP enhanced the frequency and amplitude of spontaneous excitatory postsynaptic current (sEPSC) in these cells in a dose-dependent manner. This enhancement was blocked by the selective nicotinic alpha-7 receptor antagonist alpha-bungarotoxin, but not by the antagonist mecamylamine, hexamethonium or dihydro-beta-erythroidine. The frequency of miniature excitatory postsynaptic current (mEPSC) in CA1 pyramidal neurons was also increased by application of DMPP, indicating a presynaptic site of action of the agonist. Taken together, these results suggest that activation of presynaptic nAChRs in CA1 pyramidal neurons, which contain alpha-7 subunits, potentiates presynaptic glutamate release and consequently modulate excitatory synaptic transmission in the hippocampus.
Animals ; Bungarotoxins ; physiology ; Dimethylphenylpiperazinium Iodide ; pharmacology ; Glutamic Acid ; pharmacology ; Hippocampus ; physiology ; Male ; Neurons ; physiology ; Nicotinic Agonists ; pharmacology ; Pacemaker, Artificial ; Patch-Clamp Techniques ; Rats ; Rats, Wistar ; Receptors, Nicotinic ; physiology ; Receptors, Presynaptic ; physiology ; Synapses ; physiology ; Synaptic Transmission ; alpha7 Nicotinic Acetylcholine Receptor

AIMTo investigate the changes of neurotransmitter concentration in spinal cord after exercise-induced central fatigue and study the mechanism of central fatigue at spinal level.

METHODSEstablish exercise-induced central fatigue model according to Bedford incremental loading training. The rats were divided into three groups, control group (C), immediately after training group (IT), rest 3 hours after training group (RT). Then using high performance liquid chromatography (HPLC) to detect the concentration of neurotransmitter in spinal cord.

RESULTSAmino acid neurotransmitters in spinal cord increased in IT group: Glu, GABA increased significantly (P < 0.05), Gly also increased but have no statistic difference; while in RT group, amino acid neurotransmitters got back to normal. However monoamine neurotransmitters NE, 5-HT tended to decrease in IT group. In RT group 5-HT decreased dramatically (P < 0.05).

CONCLUSIONExercise-induced fatigue changed the concentration of neurotransmitter in spinal cord.The results suggested that neurotransmitter in spinal cord might have relationship with exercise-induced fatigue, especially 5-HT might have more important effect on recovery of fatigue.

Animals ; Fatigue ; metabolism ; Male ; Motor Neurons ; metabolism ; Neurotransmitter Agents ; metabolism ; Physical Conditioning, Animal ; Rats ; Rats, Wistar ; Spinal Cord ; metabolism ; Synaptic Transmission

Objective To screen the optimized Buyang Huanwu Decoction (BYHWD);To verify it. Methods H2O2 was used to induce PC12 cell oxidative stress models. MTT method was used to determine the prevention effects of BYHWD at different concentrations (0.1, 0.2, 0.5, 1.0, 2.0, 3.5 mg/mL) on in vitro oxidative stress cell models to define the optimized concentration. Orthogonal design was used to divide BYHWD single medicine into decomposed BYHWD groups, control group (only with DMEM), normal group (without H2O2 and medicine processing), and model group, to investigate the protective effects on PC12 cells. Optimized BYHWD was screened to decide the compatibility ratio of each medicine. MTT was used to detect the cell survival rate in each group. Middle cerebral artery occlusion was used to replicate MACO rat models. SD rats were randomly divided into sham-operation group, model group, BYHWD group and optimized BYHWD high-, medium-and low-dose groups. Each medication group was given relevant medicine for gavage. The screened results were verified. Results Compared with other decomposed BYHWD groups, the protective effects of the compatibility of Astragali Radix+Chuanxiong Rhizoma+Pheretima on PC12 cells was the best (P<0.05), which was nearly equaled to BYHWD. Compared with the model group, BYHWD and the optimized one could evidently reduce cerebral cortex infarction area and improve the impaired brain edema (P<0.05), and the medium-dose group was the best. Conclusion The optimized BYHWD ratio is:Astragali Radix:Chuanxiong Rhizoma:Pheretima=10:3:1.

OBJECTIVE: To investigate the numbers, sizes and types distribution of diatoms in drowned and postmortem immersed rabbits' lungs. METHODS: Sixty-two rabbits were randomly divided into drowning group (n = 30), postmortem immersion group (n = 30) and land death group (n=2), and the diatoms in each lung lobe were analyzed quantitatively and qualitatively by microwave digestion and scanning electron microscopy. RESULTS: In the drowning group, the diatoms were detected in each lung lobe with Cyclotella and Melosira in the majority. In the postmortem immersion group, Cyclotella was in the majority. And the diatoms weren't detected in some lung lobes in postmortem immersion. There were significant differences in the detection rates of upper lobe of left lung, middle lobe and cardiac lobe of right lung in two groups (P < 0.05). CONCLUSION Based on the microwave digestion and scanning electron microscopy, the numbers, sizes and types distribution of diatoms in drowned and postmortem immersed rabbits' lungs can be analyzed and used as references for testing theory.
Animals ; Autopsy ; Diatoms/isolation & purification* ; Drowning ; Lung/microbiology* ; Microscopy, Electron, Scanning ; Microwaves ; Rabbits

The main species and research status were introduced for the haemostatic dressing of the prehospital first aid in foreign countries and China.The advantages and disadvantages,problems and futural development were analyzed for kinds of haemostatic dressing.It's pointed out that the haemostatic dressing tended to be multi-functional and complicated in the future.Dressing and haemostatic system had to be designed based on traumatic conditions in case multi function could not be realized.