过量氟化物对软骨基质糖胺多糖,蛋白多糖代谢的影响

在偏食和过量氟喂养条件下，实验大鼠软骨组织ＰＧ、ＧＡＧ代谢受到严重干扰，表现为肋软骨基质中ＰＧ聚合体含量明显降低，低聚合的ＰＧ含量明显增加，ＰＧ单体分子量明显降低。同时观察到软骨基南中ＧＡＧ含量的明显升高，这些结果一致地表明，过量氟化物引起动物慢性中毒辣时软骨组织ＰＧ合成代谢受到抑制而其分解代谢增强     

Prostaglandin E1 and misoprostol increase epidermal growth factor production in 3D-cultured human annulus cells

Background contextEpidermal growth factor (EGF) is a peptide known to modulate a number of cellular responses including embryogenesis, cell proliferation, and cell survival. Little is known about EGF and its regulation in human annulus cells. Previous work has identified EGF and its receptor in control outer annulus disc tissue, but not in herniated tissue.PurposeTo determine if human annulus cells express EGF in vitro, to determine if the epidermal growth factor-receptor (EGF-r) was expressed in vivo and in vitro in disc cells, to test the effect of EGF on annulus cell proliferation and proteoglycan production in vitro, and to test the effect of prostaglandin E1 (PGE1) and misoprostol on disc cell production of EGF in vitro.Study design/settingStudies were approved by the authors' Human Subjects Institutional review Board. Human disc tissue was used for immunocytochemistry, and human annulus cells were tested in vitro.Patient sampleThirty-four disc specimens were used for studies of proteoglycan production, cell proliferation, and EGF production in vitro. An additional nine discs were used for EGF-r immunolocalization.MethodsDisc tissue was used for immunocytochemical studies for the localization of EGF-r and as a source for cultured annulus cells. Monolayer culture was used to test proliferation responses to 0, 25, 50, or 75 ng/mL EGF over a 2-day culture period. Three-dimensional (3D) culture in a collagen sponge was used to test 100,000 cells cultured in a paired experimental design over 14 days for production of EGF and proteoglycans. Cells were exposed to control conditions, or to either misoprostol at 8 ng/mL or PGE1 at 10^?7 M. Conditioned media was harvested and assayed using an enzyme-linked immunosorbent assay (ELISA) assay with the Human Protein Cytokine Antibody Array I kit. Replicate EGF relative intensity values were averaged and normalized to controls assayed on the same membrane. 3D-cultured cells were also used to confirm EGF gene expression using microarray analysis. Standard statistical methods were used to analyze results.ResultsMicroarray analysis of mRNA from annulus cells in 3D culture confirmed expression of EGF, and immunocytochemistry verified the presence of EGF-r in vitro and in vivo. PGE1, at a dose of 10^?7 M, and misoprostol (a synthetic PGE1 analog) at a dose of 8 ng/mL, both significantly increased EGF levels in annulus cells cultured in 3D compared with control levels (p=.031 and .034, respectively). No significant difference, however, was seen in cell proliferation or in total sulfated proteoglycan production in EGF-exposed annulus cells.ConclusionsData showed that EGF is expressed and produced by annulus cells in vivo and in 3D culture, with significantly greater in vitro EGF produced in the presence of PGE1 or the PGE1 analog misoprostol. Misoprostol, developed for prevention/treatment of nonsteroidal anti-inflammatory-induced gastropathy, has now been reported to have some interesting anabolic effects stimulating osteoblasts during fracture healing and during ovariectomy in animal models. Exogenous EGF did not increase cell proliferation in monolayer, or total production of proteoglycans in 3D culture. Additional work is needed to further delineate the role of EGF in the human disc.     

病损关节软骨的磁共振成像与软骨内蛋白多糖和Ⅱ型胶原纤维变化的关系研究

目的探讨MR对关节软骨各期病变的敏感成像序列以及软骨蛋白多糖（proteoglycan,PG）和Ⅱ型胶原纤维（collagen fibers,CF）改变与MR信号变化的关系。方法通过1例新鲜截肢患者和4例正常猪膝关节的T2WI、PDWI、GE、STIR、3D FS-FSPGR等序列的成像研究,选定最佳的序列用于6例猪骨性关节炎（Osteoarthritis,OA）模型膝关节扫描,以MR图像为标准切取标本染色,图像与染色图片对照以判断PG和CF改变与MR信号变化的关系。结果5种序列的组织分辨力、病变检出率以3DFS-FSPGR序列最高分别达67．9％和93．7％;PG和CF主要分布在关节软骨的深层组织。以MR图像为标准切取的各级软骨标本之间染色浓度,PG逐渐减少,CF早中期增加,晚期又出现下降。结论在5种序列中,3DFS-FSPGR是理想的关节软骨成像序列;由于软骨内的PG含量下降和CF的先升后降共同导致了MR信号在不同病变期的改变。     

雌激素在大鼠颈椎间盘退变中的作用

目的:研究雌激素在SD大鼠颈椎间盘退变过程中的作用。方法:30只8月龄雌性SD大鼠随机平分为对照组、模型组、实验组,通过切除双侧卵巢和颈后肌肉创建雌激素缺乏大鼠颈椎动静失衡模型,实验组补充外源性雌激素。造模后3月通过观察颈椎间盘形态、细胞凋亡率及测量蛋白多糖含量来比较3组之间颈椎间盘退变程度。结果:形态学观察比较实验组椎间盘退变程度重于对照组而轻于模型组;椎间盘细胞凋亡率模型组>实验组>对照组,3组之间具有显著差异(P<0.01);蛋白多糖含量模型组<实验组<对照组,3组之间具有显著差异(P<0.01)。结论:雌激素对大鼠颈椎间盘退变具有抑制作用,能够延缓椎间盘的退变进程。     

The Relation Between Sox9, TGF-��1, and Proteoglycan in Human Intervertebral Disc Cells

Objective

The aim of this study is to elucidate the effects of transforming growth factor-β (TGF-β)1 and L-ascorbic acid on proteoglycan synthesis, and the relationship between Sox9, proteoglycan, and TGF-β1 in intervertebral disc cells.

Methods

Human intervertebral disc tissue was sequentially digested to 0.2% pronase and 0.025% collagenase in DMEM/F-12 media and extracted cells were cultured in 37℃, 5% CO₂ incubator. When intervertebral disc cells were cultured with TGF-β1 or L-ascorbic acid, the production level of sulfated glycosaminoglycan (sGAG) was estimated by dimethyl methyleneblue (DMMB) assay. The changes of Sox9 mRNA and protein levels via TGF-β1 were detected by RT-PCR and Western blot analysis in each.

Results

The amount of sGAG was increased with the lapse of time during incubation, and sGAG content of pellet cultured cells was much larger than monolayer culture. When primary cultured intervertebral disc cells in monolayer and pellet cultures were treated by TGF-β1 20 ng, sGAG content of experimental group was increased significantly compared to control group in both cultures. L-Ascorbic acid of serial concentrations (50-300 ug/ml) increased sGAG content of mono layer cultured intervertebral disc cells significantly in statistics. The co-treatment of TGF-β1 and L-ascorbic acid increased more sGAG production than respective treatment. After treating with TGF-β1, Sox9 mRNA and protein expression rates were significantly increased in disc cells compared with the control group.

Conclusion

This study suggests that TGF-β1 would increase sulfated glycosaminoglycan (sGAG) and other proteoglycans such as versican by elevating Sox9 mRNA and protein expressions in order.     

Real-time ultrasonic assessment of progressive proteoglycan depletion in articular cartilage

The loss of proteoglycan (PG) is regarded as one of the early signs of osteoarthritis (OA), thus observing the progress of PG loss would be useful for the early detection of OA. In this study, high-frequency ultrasound was used to monitor and analyze the trypsin-induced progressive degeneration in articular cartilage. Full thickness cartilage-bone specimens (n = 10) prepared from normal bovine patellae were digested using 0.25% trypsin solution for different periods of time to evaluate the dynamics of the digestion process. The trypsin penetration front was observed in M-mode image, which was acquired using a nominal 50 MHz focused transducer. The transient speed of the digestion process was estimated from the image. The digestion fraction, which represents the ratio of the digestion depth to the total cartilage thickness, was estimated from ultrasound data and histology sections. With ultrasound, the digestion fraction observed in the 10 specimens ranged from 64% to 99% and was correlated to that measured by histology (R(2) > or = 0.63, p < 0.05). It was found that the digestion speed decreased nonlinearly with depth from 0.61 +/- 0.16 microm/s (mean +/- SD) in the superficial zone to 0.04 +/- 0.02 microm/s in a region located at 70% of the cartilage thickness in depth. The relationship between the digestion depth and the exposure duration in trypsin could be described using a third order polynomial function. The full thickness of digested and undigested tissues was also measured using caliper, estimated from ultrasound data and histology sections, and compared. These findings indicate that ultrasound could provide useful information about the trypsin-induced progressive PG depletion in articular cartilage. Therefore, ultrasound represents a useful tool to evaluate the dynamics of models of OA in vitro in cartilage specimens in a research environment and this would ultimately help the in vitro examination of articular cartilage for research related to model of OA from the early stages of tissue degradation.     

CNS injury, glial scars, and inflammation: Inhibitory extracellular matrices and regeneration failure

Spinal cord and brain injuries lead to complex cellular and molecular interactions within the central nervous system in an attempt to repair the initial tissue damage. Many studies have illustrated the importance of the glial cell response to injury, and the influences of inflammation and wound healing processes on the overall morbidity and permanent disability that result. The abortive attempts of neuronal regeneration after spinal cord injury are influenced by inflammatory cell activation, reactive astrogliosis and the production of both growth promoting and inhibitory extracellular molecules. Despite the historical perspective that the glial scar was a mechanical barrier to regeneration, inhibitory molecules in the forming scar and methods to overcome them have suggested molecular modification strategies to allow neuronal growth and functional regeneration. Unlike myelin associated inhibitory molecules, which remain at largely static levels before and after central nervous system trauma, inhibitory extracellular matrix molecules are dramatically upregulated during the inflammatory stages after injury providing a window of opportunity for the delivery of candidate therapeutic interventions. While high dose methylprednisolone steroid therapy alone has not proved to be the solution to this difficult clinical problem, other strategies for modulating inflammation and changing the make up of inhibitory molecules in the extracellular matrix are providing robust evidence that rehabilitation after spinal cord and brain injury has the potential to significantly change the outcome for what was once thought to be permanent disability.     

Ultrasound Biomicroscopy Imaging for Monitoring Progressive Trypsin Digestion and Inhibition in Articular Cartilage

This study reports an ultrasound biomicroscopy (UBM) imaging approach to monitor the progressive trypsin-induced depletion of proteoglycan (PG) and its inhibition in articular cartilage. Three fresh, normal bovine patellae were obtained and four full-thickness cartilage-bone specimens were prepared from the lower medial side of each patella. One sample was used as a control and the other three were divided into three groups: Groups A, B and C (n = 3 for each group). After a 40 min 0.25% trypsin digestion, samples from group A were continuously digested in trypsin solution, while those in groups B and C were immersed in physiologic saline and fetal bovine serum (FBS), respectively, for another 280 min. The trypsin penetration front was observed by UBM and M-mode images were acquired using 50 MHz focused ultrasound and custom-developed software. The results show that the 40 min trypsin digestion degraded nearly the whole surface layer of the cartilage tissue. Further digestion in trypsin or residual digestion in saline for 280 min depleted most of the PG content, as observed in groups A and B. The replacement of trypsin with a physiologic saline solution only slightly slowed the digestion process (group B), while trypsin inhibitors in FBS stopped the digestion in approximately 1.5 h (group C). The normalized digestion fractions of the digested tissues were calculated from ultrasound data and histology sections, and then compared between the groups. Without the use of FBS, 80% to 100% of the full thickness was digested, while this number was only approximately 50% when using FBS. Our findings indicate that the UBM imaging system could provide two-dimensional (2-D) visual information for monitoring progressive trypsin-induced PG depletion in articular cartilage. The system also potentially offers a useful tool for preparing cartilage degeneration models with precisely controlled PG depletion. (E-mail: ypzheng@ieee.org)     

高应力环境导致腰椎软骨终板蛋白聚糖含量改变

采用组织切片的计算机图像分析系技术，测量双后肢大鼠增龄过程中腰椎软骨终板蛋白聚糖（ＰＧ）的含量与分布。８月龄正常大鼠软骨终板外区浅层ＰＧ含量已呈下降趋势，而双后肢大鼠１２月龄组ＰＧ含量开始下降。在此以前，外区浅层ＰＧ含量随年龄增长而加大，尤以双后肢大鼠增加为著，４、８月龄组平均较正常大鼠组增加１７％。１、２月龄双后肢大鼠外区深层和内区ＰＧ含量明显小于对照组，但该差别在４月龄后即不明显。双后肢大鼠软骨终板的过度钙化可能与其ＰＧ含量减少有关。     

Attenuation of apoptosis and enhancement of proteoglycan synthesis in rabbit cartilage defects by hyperbaric oxygen treatment are related to the suppression of nitric oxide production.

Proinflammatory cytokine, nitric oxide (NO) and localized hypoxia-induced apoptosis and proteoglycan (PG) degradation are thought to be correlated to the degree of cartilage injury. This study evaluated hyperbaric oxygen (HBO)-induced changes in joint cavity oxygen tension, antigenickeratan sulfate (KS) content, inducible nitric oxide synthase (iNOS) expression, PG synthesis, and cell apoptosis in full-thickness defects of rabbit cartilage. The HBO group was exposed to 100% oxygen at 2.5 atm for 2 h daily, 5 days per week. Meanwhile, the control group was kept in housing cages with normal air. The joint cavity oxygen tension was determined with an oxygen sensor. Blood serum KS was quantified by competitive indirect enzyme-linked immunosorbent assay (ELISA). After sacrifice, specimen sections were sent for histological and histochemical examination with a standardized scoring system. In situ analysis of iNOs expression and apoptosis detection were performed using immunostaining and TUNEL staining, respectively and quantified by a computerized imagine analysis system. This study demonstrated that HBO treatment increased joint cavity oxygen tension but decreased blood KS content. Histological and histochemical score results showed that HBO treatment significantly increased the cartilage repair. Moreover, immunostaining and TUNEL staining showed that HBO treatment suppressed the iNOs expression and apoptosis of chondrocytes, respectively. Accordingly, HBO offers a potential treatment method for cartilage injury.