Objective To observe the morphologic protection effect of intra-articular injection of osteoprotegerin (OPG)on articular cartilage in a rabbit model of osteoarthritis (OA).Methods Sixty male New Zealand rabbits were randomly divided into 3 groups:OPG group (n=20),sham-operated group (n=20) and PBS group (n=20).In OPG group and PBS group,each rabbit underwent anterior cruciate ligament transection (ACLT) in left knee joint,then 0.1 ml OPG solution or PBS were injected into the left knee for 8 weeks (5 times a week) in OPG group and PBS group,respectively.In sham-operated group,the anterior cruciate ligament was just exposed without transection,and then the incision was closed.All rabbits were sacrificed 12 weeks after operation,and the left knee joints were obtained.The Pelletier score and Mankin score were used to evaluate the macroscopic and microscopic cartilage morphology.Results The score of femoral condyle cartilage and tibial plateau cartilage was 1.80±0.89 and 1.80±0.77 in OPG group,respectively,and 3.10±0.97 and 3.20±0.77 in PBS group.However,there was no statistical difference in Pelletier score between the sham-operated group and the OPG group.Consistent with the macroscopic data,the OPG group has a significantly lower Mankin score involving cartilage structure (2.65±0.88),chondrocyte (1.35±0.71),Safranin O staining (1.83±0.83),tidemark integrity (0.30±0.47) and total score (6.13±1.97) compared with the PBS group (4.52±1.09,1.85±0.63,2.80±0.75,0.65±0.49,and 9.83±1.98,respectively).The OPG group has a significantly higher total Mankin score compared with the sham-operated group (4.80±1.25),however,there were no statistical differences in four subitem scores between the two groups.Moreover,the cartilage thickness in OPG group was 371.84±38.94 μm,255.09±74.82 μm in PBS group,and 404.68±15.97 μm in the sham-operated group,and the differences were statistically significant between three groups.Conclusion OPG has a protective effect on articular cartilage and can slow the progression of OA by reducing the grade of synovitis,decreasing the volume of osteophytes,and suppressing the loss of cartilage thickness.

Objective To study the effect of intra-articular injection of dehydroepiandrosterone (DHEA) on the balance of ADAMTS/TIMP-3 system in a rabbit osteoarthritis models.Methods Sixty rabbits were underwent bilateral anterior cruciate ligament transection (ACLT).Rabbits were randomizedn to the following treatment:one knee of each rabbit was treated with 100 μmol/L DHEA dissolved in dimethylsulphoxide (the experimental group) and the other knee was treated under the same schedule using dimethylsulphoxide (the control group) 4 weeks after transection,once a week for eight weeks.Twelve weeks after ACLT,all rabbits were killed after X-ray assessment and the knee joints were evaluated by gross morphology and histology.The concentration of hydroxyproline and glycosaminoglycan in the cartilage were analyzed.The mRNA expression of ADAMTS-4,ADAMTS-5,tissue inhibitor of metalloproteinases-3 (TIMP-3),transforming growth factor (TGF)-β1.Aggrecan and Collagen Ⅱ in the cartilage were analyzed using reverse transcription polymerase chain reaction (RT-PCR).The protein expression of aggrecan ARGxx and Collagen Ⅱ in the cartilage were analyzed by Western blot.Results By Mann-Whitney test,Gross morphologic scores on femoral condyle and tibial plateau in the control group were significantly higher than the experi-mental group (Z=-3.517,P＜0.01 ; Z=-2.518,P＜0.05).By unpaired Student's t test,histological evaluation showed that the grade of cartilage damage in the experimental group [(5.3±1.2) μg/ml] were less severe than that in the control group (10.1 ± 1.3,P＜0.01).The concentration of hydroxyproline [(5.7±0.3,23.6± 1.7) μg/ml] and glycosaminoglycan (30±4) in the experimental group increased significantly when compared with the control group [(4.6±0.5),(18.5±1.4),(24±4) μg/ml,P＜0.01].The mRNA expression of ADAMTS-4 (0.15±0.03)and ADAMTS-5 (0.10±0.04) in the experimental group decreased significantly compared with the control group (0.29±0.08,0.15±0.05; all P＜0.05).The mRNA expression of TIMP-3 (0.85±0.10),TGF-β1(1.2±0.4),Aggrecan (0.87±0.31) and Collagen Ⅱ (2.74±0.59) in the experimental group increased significantly when compared with the control group (0.70±0.13,0.8±0.4,0.49±0.16,2.2±0.5; all P＜0.05).The protein expression of Aggrecan ARGxx (0.53±0.10) in the experimental group decreased significantly when compared with the control group (0.81±0.12,P＜0.01).The protein expression of Collagen Ⅱ (2.3±0.7) in the experimental group increased significantly when compared with the control group (1.7±0.5,P＜0.05).Conclusion DHEA protects against cartilage degradation and inhibits the progression of OA,TGF-β1,Aggrecan and Collagen Ⅱ in cartilage may be the mechanism of the protective effect of DHEA on OA.

BackgroundOur previous study determined that tetrandrine (Tet) has an inhibitory effect on the proliferation of human Tenon capsule fibroblasts ( TCFs ) in vitro,but its mechanism is poorly understood.ObjectiveThis study was to investigate the effect mechanism of Tet on human TCFs.MethodsHuman TCFs were isolated and cultured from scleral tissue of donor using explant technique.The cells were identified by vimentin antibody staining and morphology.The third generation of cells were seeded in the culture plate at the density of 1 × 105 cells/ml.Twenty-four hours after inoculation,the Tet of 1 × 10-5 mol/L was added in the well of culture plate,and the cells cultured only in 1640 medium served as the control group.The apoptosis of the cells was assessed by TUNEL,and the expressions of bax,bel-2,transforming growth factor-β2 (TGF-β2 ) in TCFs were detected using immunochemistry.Results The cultured cells showed the features of the fibroblasts in shape with the positive response for vimentin.A number of TUNEL positive cells were seen in Tet group and no TUNEL positive response was found in control group.The expression levels (A value) of bax,bcl-2 and TGF-~ protein in TCFs were 0.577 ± 0.009,0.430±0.012 and 0.341 ±0.017 in Tet group,and those in control group were 0.320±0.015,0.819±0.021 and 0.624±0.014 respectively,showing statistically significant differences between two groups( t =33.277,-35.356,-28.093,P＜0.01 ).Conclusions Tet suppresses the proliferation of human TCFs through up-regulating the expression of bax and down-regulating the expressions of bcl-2 and TGF-β2 in vitro.

Adjuvants, Immunologic ; therapeutic use ; Adult ; Aged ; B-Lymphocytes ; immunology ; CD4-CD8 Ratio ; Carcinoma, Hepatocellular ; drug therapy ; immunology ; surgery ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Killer Cells, Natural ; immunology ; Liver Neoplasms ; drug therapy ; immunology ; surgery ; Male ; Middle Aged ; Perioperative Care ; Phytotherapy ; T-Lymphocyte Subsets ; immunology

Objective To investigate the expression and significance of heme oxygenase-1(HO-1) in gastric adenocarcinoma and its peritoneal metastatic tissues, as well as drug-resistant cell strains. Methods The expression of HO-1 in patients with (n=68) or without (n=46) peritoneal metastasis of gastric adenocarinoma was examined. The expression of HO-1 in cancerous tissue, peritoneal metastatic foci, and normal peritoneum was detected by immunohistochemistry. The expression of HO-1 protein in metastatic foci and drug-resistant cell strains was measured by Western blotting. Results The positive expression of HO-1 was 39.7% (27/68) in gastric adenocarcinoma tissues with metastasis and 41.2% (28/68) in peritoneal metastatic tissues, which was significantly higher than that in normal peritoneum (0%,0/68,P<0.01) and gastric adenocarcinoma tissues without metastasis (21.7%, 10/46, P<0.05). The Western blot study showed that the HO-1 expression in metastatic tissues was higher than that in normal peritoneum (P<0.05). The expression of HO-1 protein was markedly increased in GC9811-P drug-resistant cell strains compared with its parental cell strains (P<0.05). Conclusions The increased expression of HO-1 may be involved in the peritoneal metastasis of gastric adenocarcinoma, and related to the malignant potential. The underlying signal pathways in neopastic epithelium may also be related to the multi-drug resistance.

Objective To understand the situation of downward dislocation of intrauterine device (IUD) and the impact related to the effectiveness of IUD use, in China. Methods An epidemiological survey with cross-sectional, retrospective and prospective study designs was conducted to investigate 18 922 IUD users who were selected by a multi-phase stratified cluster sampling method. Results IUD's downward dislocation had been an important unsuccessful issue related to the IUD insertion that accounted for 20% of total the cases of failure. The top three failure outcomes would include extrusion,removal due to downward dislocation and unintended pregnancy. Respectively, the cumulative rates and the ranking due to IUD failure (per 100 women) in the first, third, sixth and twelfth month were shown as follows: extrusion appeared as 0.33%, 1.13%, 2.21% and 4.30%; removal as 0.10%, 0.37%, 0.80% and 2.34% ; while unintended pregnancy were 0.03%, 0.14%, 0.41% and 1.14%. Conclusion IUD' s downward dislocation made great impact on the effectiveness of IUD use that should call for attention from relative governmental sectors and researchers in the areas of prevention, diagnosis and treatment.

AIMTo establish quality control requirements and methods for recombinant adeno-associated virus(rAAV) type 2/human blood coagulation factor IX (rAAV-2/hFIX).

METHODSIdentification of rAAV genome fragments, potential contaminants including wild type AAV(wtAAV) and helper virus, were detected by PCR. Purity of rAAV-2/hFIX was analyzed by cation-exchange HPLC and SDS-PAGE. Virus partical numbers were performed by dot blot assay. hFIX expression was demonstrated by ELISA and potency of hFIX was verified by APTT.

RESULTSIdentity of rAAV-2/hFIX was proved. Residues of wtAAV and helper virus were conformed to requirements. Purity of rAAV-2/hFIX were more than 98%. Partical numbers of rAAV-2/hFIX were more than 1.0 x 10(15) VG.L-1. hFIX expression was more than 20.0 micrograms.L-1. hFIX potency was verified by APTT following rAAV-2/hFIX injected to FIX gene knockout mice, potency results conformed to requirements.

CONCLUSIONThe methods and requirements had been established for quality control of rAAV-2/hFIX.

Animals ; Dependovirus ; genetics ; Factor IX ; biosynthesis ; genetics ; Female ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; Genome, Viral ; Humans ; Male ; Mice ; Mice, Knockout ; Quality Control ; Random Allocation ; Recombinant Proteins ; biosynthesis ; genetics

Animals ; Bone Marrow Cells ; cytology ; Bone Marrow Transplantation ; Cell Differentiation ; Cell Proliferation ; Humans ; Liver Cirrhosis ; pathology ; surgery ; Liver Function Tests ; Mesenchymal Stem Cell Transplantation ; Rats ; Stem Cells ; cytology

BACKGROUNDNeurone atrophy and loss are major causes of chronic neurodegenerative disorders such as Alzheimer's disease. Despite many pharmacotherapies for neurodegeneration, there are no accepted treatments. We investigated the feasibility of human nerve growth factor beta (hNGFbeta) gene expression mediated by recombinant adeno-associated viruses type-2 (rAAV-2) vector in the central nervous system (CNS) after blood brain barrier (BBB) disruption.

METHODSrAAV-2 containing hNGFbeta gene was constructed. The ability of hNGFbeta gene mediated by rAAV-2 vector (rAAV-2/hNGFbeta) to transfect cells in vitro was confirmed by both ELISA and bioassay of hNGFbeta in the culture supernatant of BHK-21 cells infected by rAAV-2/hNGFbeta. rAAV-2/hNGFbeta and rAAV-2/green fluorescence protein (GFP) were administrated separately to rat brains through internal carotid intubation after BBB disruption with hypertonic mannitol. Brain hNGFbeta concentration was measured by ELISA and GFP in brain sections was examined by laser scan confocal microscope.

RESULTSAfter 48 hours, hNGFbeta content in supernatant was up to (188.0 +/- 28.6) pg/ml when BHK-21 cells were infected by rAAV-2/hNGFbeta at multiplicity of infection (MOI) 1.0 x 10(6) vector genome. Neurone fibre outgrowths were obvious in dorsal root ganglion neurone assays by adding serum free culture medium harvested from BHK-21 cells exposed to rAAV-2/hNGFbeta. Whole brain hNGFbeta content in rAAV-2/hNGFbeta transferred group was up to (636.2 +/- 140.6) pg/ml. hNGFbeta content of BBB disruption in rAAV-2/hNGFbeta infused group increased significantly compared to the control group (P <0.05). GFP expression was clearly observed in brain sections of rAAV-2/GFP transferred group.

CONCLUSIONrAAV-2/hNGFbeta successfully expresses in the CNS after BBB disruption induced by hypertonic mannitol.

Animals ; Blood-Brain Barrier ; Brain ; metabolism ; Cricetinae ; Dependovirus ; genetics ; Genetic Vectors ; genetics ; Humans ; Nerve Growth Factor ; genetics ; Recombination, Genetic

BACKGROUNDAccumulating evidence indicates that both innate and adaptive mechanisms are responsible for the postnatal development of the mammalian visual cortex. Most of the studies, including gene expression analysis, were performed on the visual cortex during the critical period; few efforts were made to elucidate the molecular changes in the visual cortex during much earlier postnatal stages. The current study aimed to gain a general insight into the molecular mechanisms in the developmental process of the rat visual cortex using microarray to display the gene expression profiles of the visual cortex on postnatal days.

METHODSAll age-matched Sprague-Dawley rats in various groups including postnatal day 0 (P0, n = 20), day 10 (P10, n = 15), day 20 (P20, n = 15) and day 45 (P45, n = 10) were sacrificed respectively. Fresh visual cortex from the binocular area (Area 17) was dissected for extraction of total RNA for microarray analyses. Taking advantage of annotation information from the gene ontology and pathway database, the gene expression profiles were systematically and globally analyzed.

RESULTSOf the 31 042 gene sequences represented on the rat expression microarray, more than 4000 of the transcripts significantly altered at days 45, 20 or 10 compared to day 0. The most obvious alteration of gene expression occurred in the first ten days of the postnatal period and the genomic activities of the visual cortex maintained a high level from birth to day 45. Compared to the gene expression at birth, there were 2630 changed transcripts that shared in three postnatal periods. The up-regulated genes in most signaling pathways were more than those of the down-regulated genes.

CONCLUSIONSAnalyzing gene expression patterns, we provide a detailed insight into the molecular organization of the developing visual cortex in the earlier postnatal rat. The most obvious alteration of gene expression in visual cortex occurred in the first ten days. Our data were a basis to identify new relevant candidate genes that control visual cortex development.

Animals ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; genetics ; physiology ; Oligonucleotide Array Sequence Analysis ; Rats ; Visual Cortex ; metabolism