一氧化氮对兔关节软骨细胞凋亡的影响

目的　探讨NO对软骨细胞凋亡的影响。方法　以NO供体硝普钠 3mmol/L和 6mmol/L诱导培养的兔关节软骨细胞凋亡 ,在加药处理后 4,8,12 ,16及 2 0小时取样检查 ,利用透射电镜技术、流式细胞术 ,脱氧核苷酸末端转移酶介导的缺口末端标记法 ,观察细胞凋亡现象。结果　在 3mmol/L硝普钠诱导下 ,随着作用时间的延长 ,细胞从核边集到核裂解 ;加药后 12小时 ,凋亡率为 ( 12 .71± 3 .2 6) % ,2 0小时后的凋亡率已达 ( 93 .47± 4.12 ) % ;在 6mmol/L硝普钠诱导下 ,细胞出现肿胀、破裂成碎片等坏死变化。结论　高浓度的NO可诱导兔关节软骨细胞凋亡 ,过高浓度的NO可引起细胞坏死。细胞凋亡率异常增高可能是影响组织工程化软骨修复软骨缺损的原因之一     

一氧化氮对兔关节软骨细胞凋亡的影响

目的探讨NO对软骨细胞凋亡的影响.方法以NO供体硝普钠3?mmol/L和6?mmol/L诱导培养的兔关节软骨细胞凋亡,在加药处理后4,8,12,16及20小时取样检查,利用透射电镜技术、流式细胞术,脱氧核苷酸末端转移酶介导的缺口末端标记法,观察细胞凋亡现象.结果在3?mmol/L硝普钠诱导下,随着作用时间的延长,细胞从核边集到核裂解;加药后12小时,凋亡率为(12.71±3.26)%,20小时后的凋亡率已达(93.49±4.12)%;在6?mmol/L硝普钠诱导下,细胞出现肿胀、破裂成碎片等坏死变化.结论高浓度的NO可诱导兔关节软骨细胞凋亡,过高浓度的NO可引起细胞坏死.细胞凋亡率异常增高可能是影响组织工程化软骨修复软骨缺损的原因之一.     

Modulation of bovine articular chondrocyte gene expression in vitro by oxygen tension

OBJECTIVE: Adult articular cartilage is a physiologically hypoxic tissue with a proposed gradient of oxygen tension ranging from <10% oxygen at the cartilage surface to <1% in the deepest layers. This gradient may be disturbed during diseases of the joint, for example in rheumatoid arthritis when synovial fluid pO(2)falls. We investigated whether changes in oxygen tension modulate gene expression in articular chondrocytes. DESIGN: Bovine articular chondrocytes were cultured in alginate beads in medium maintained at <0.1, 5, 10 or 20% oxygen. A modified RNA arbitrarily primed polymerase chain reaction (RAP-PCR) technique was used to identify several genes whose mRNA abundance in articular chondrocytes was dependent upon oxygen tension. Northern hybridization slot blots were used to quantify changes in mRNA level relative to a housekeeping gene, beta-actin. RESULTS: Genes found by RAP-PCR to undergo up-regulation in hypoxia included TIMP-1 and integrin-linked kinase. Collagen V mRNA levels were down-regulated in hypoxic chondrocytes. This led us to examine mRNA levels for various cytokines, matrix structural molecules and beta1 integrin. Interleukin 1beta, transforming growth factor beta and connective tissue growth factor were all up-regulated by low oxygen tensions, as was beta1 integrin. Collagen II (COL2A1) was down-regulated by hypoxia but aggrecan mRNA levels remained unchanged. The mRNA levels for GAPDH, the archetypal hypoxia responsive gene, were not modulated in articular chondrocytes by changes in oxygen tension. CONCLUSIONS: Oxygen tension modulates the abundance of mRNAs encoding structural molecules, several cytokines, beta1 integrin and integrin-linked kinase in articular chondrocytes. This may be important during disease progression. Chondrocytes are unusual in their response to hypoxia, presumably because they exist physiologically in a low oxygen environment.     

Induction of macrophagic prostaglandin E2 synthesis by glioma cells

OBJECT: It has been reported that glioma cells produce prostaglandin (PG)E2, which promotes the growth of tumor cells and possesses immunosuppressive activity, and that cyclooxygenase (COX) inhibitors impede tumor growth and infiltration. Macrophages in tumor-bearing hosts are activated to produce PGE2, which induces an immunosuppressive state. Note, however, that the precise mechanism by which PGE2 induces an immunosuppressive state is still unclear. In this study, the authors investigated the mechanism of PGE2 production in glioma-bearing hosts. METHODS: The human and murine glioma cells that were studied did not produce a significant amount of PGE2. However, the coculture of human peripheral blood mononuclear cells or murine peritoneal macrophages with glioma cells or conditioned glioma medium led to the production of a large amount of PGE2. In contrast, production of tumor necrosis factor and interleukin (IL)-12p70 by macrophages and cytotoxic T lymphocyte induction were suppressed by culturing with conditioned glioma medium; this suppression was abrogated by the addition of the COX inhibitor indomethacin. The macrophagic expression of COX-2, and particularly the expression of microsomal PGE synthase (mPGES)-1, a terminal enzyme of the arachidonate cascade, was enhanced by the glioma-derived soluble factors. Furthermore, IL-12p70 production was not clearly suppressed in macrophages from mPGES-1-deficient mice. The glioma-derived soluble factors were sensitive to treatment with heat and papain. CONCLUSIONS: These results indicated that PGE2 production by macrophages is enhanced by glioma-derived soluble factors, which induce an immunosuppressive state in glioma-bearing hosts. Therefore, the inhibition of PGE2 synthesis, targeting COX-2 and mPGES-1, is an effective treatment for the induction of antiglioma immune responses.     

骨关节炎软骨细胞凋亡调控基因的研究

目的　比较分析正常人及老年性骨关节炎患者软骨细胞bax和bcl 2的表达及细胞凋亡状况。　方法　取 9例骨关节炎患者的关节软骨做实验标本 ,以 6例无骨关节炎病史的意外死亡者关节软骨作为正常对照 ;采用逆转录 /聚合酶链反应 (RT PCR)方法检测bax和bcl 2mRNA表达 ,免疫组化检测bax和bcl 2蛋白 ;应用TUNEL方法进行凋亡细胞原位检测。　结果　骨关节炎患者和正常对照软骨细胞都能表达bax和bcl 2mRNA ;骨关节炎关节软骨细胞baxmRNA表达量较正常对照显著增高 (P <0 0 1) ,bcl 2mRNA表达量也高于正常对照组 (P <0 0 5 ) ,两组间bax/bcl 2表达量的比值差异无显著性意义 (P >0 0 5 ) ;免疫组化可检测到相应表达水平的蛋白 ;骨关节炎软骨细胞凋亡 (4%～ 14% )多于正常对照 (0～ 2 % )。　结论　软骨细胞凋亡受bax和bcl 2共同调节 ;bax和bcl 2的共同调节结果可能是OA患者软骨细胞凋亡增加 ,但凋亡率不高、病理过程进展缓慢的一个重要的原因     

Induction of chondrocyte apoptosis following impact load

OBJECTIVE: To investigate the presence and extent of chondrocyte apoptosis following impact load of articular cartilage in an in vivo model. DESIGN: An in vivo animal model, using a pendulum device and New Zealand White rabbits, was designed to study the effects of impact load on the development of chondrocyte apoptosis. Animals were placed into either a High Impact group or a Low Impact group, and the right medial femoral condyle was impacted with a single impact load. A sham operation was performed on the left limb, and this cartilage served as the control. SETTING: Academic medical center. PARTICIPANTS: New Zealand White rabbits (3 months). INTERVENTION: Impact load to the right medial femoral condyle. MAIN OUTCOME MEASURES: Three different methods were used to assess the presence and extent of chondrocyte apoptosis: 1) light microscopy (hematoxylin and eosin and terminal dUTP nick end labeling staining); 2) transmission electron microscopy; and 3) fluorescent microscopy with Hoechst 33342 staining. Secondary outcome measures included determination of the magnitude of impact force and time to peak force. RESULTS: Light microscopy demonstrated chondrocytes with changes consistent with apoptosis including condensed nuclei, deep eosinophilic cytoplasmic staining, and vacuolization within the impacted specimens. Terminal dUTP nick end labeling staining-stained specimens had a high degree of positively stained cells (60%) in both injured and uninjured specimens. Transmission electron microscopy of the impacted specimens demonstrated numerous chondrocytes with changes characteristic of apoptosis, including nuclear and cellular fragmentation, volume shrinkage, and cytoplasmic vacuolization. Eleven percent of the cells in the High Impact group had changes consistent with apoptosis, versus 3% for the low impact group and <1% for the sham specimens. The High Impact group received a statistically significant greater stress than the Low Impact group.Impact group (P < 0.05), and the average time to peak force was 0.021 seconds for each impact group. CONCLUSIONS: The current data strongly indicate that in vivo chondrocyte apoptosis can be stimulated by the application of a single, rapid impact load and that the extent of chondrocyte apoptosis is related to the amount of load applied. The contribution chondrocyte apoptosis makes to the development of posttraumatic arthritis following joint injury or intra-articular fracture still remains to be determined.     

Induction of labour with oral prostaglandin E2.

Oral prostaglandin E2 was used for inducing labour in 37 patients without initial rupture of membranes. Two dosage regimens were used: in the one 0.5 mg of oral PGE2 was given hourly, and in the other the dose of oral PGE2 was doubled hourly, starting with a dose of 0,5 mg and increasing to a maximum single dose of 2,0 mg. The incremental dosage regimen was found to be more successful in inducing labour than the constant dosage regimen. The success rate was 94,95%. Side-effects were found to occur rarely and to be dose-related. Fetal distress did not occur in this study.     

关节软骨撞击伤后软骨细胞凋亡的实验研究

[目的]用TUNEL标记法和透射电镜来观察软骨撞击伤后软骨细胞凋亡的演变情况。[方法]56只健康成年新西兰大白兔,随机分高、低能量两组,每组28只,随机选取一侧后肢为实验组,另一侧为对照组;不同重量的击锤撞击股骨内侧髁软骨面致不同的损伤。术后4 d,1、2、4、8、16、32周收集标本,每个时间组4只兔子,进行TUNEL标记法和透射电镜来观察软骨细胞的凋亡。[结果]对照组标本中在软骨的中间层和钙化层,出现凋亡细胞。低能量组中,伤后4 d,1、2周的标本中,软骨的移形层和过渡层出现凋亡细胞,细胞凋亡率与对照组之间无统计学意义;4周后的标本中凋亡细胞明显减少,细胞凋亡率与对照组之间无统计学意义。高能量组中,伤后4 d软骨浅表层和移形层中出现凋亡细胞,而且伤后时间越长凋亡细胞所占比率越大,细胞凋亡率与对照组之间有统计学意义。[结论]软骨高、低能量撞击伤后早期软骨细胞均出现凋亡,凋亡率高于对照组;但低能量伤后4周软骨细胞凋亡率恢复到对照组水平;高能量伤后软骨细胞凋亡率随时间逐渐增高。     

Induction of labour with prostaglandin E2 tablets.

The induction of labour with prostaglandin E2 (PGE2) tablets in two dosage regimens, and with desamino-oxy-tocin, has been studied in association with amniotomy. In multiparas at or near term and with a high Bishop score. PGE2 appears superior with regard to the induction-to-delivery interval and the duration of labour, but both preparations are highly effective in this respect. In nulliparas with a low Bishop score, however, intravenous oxytocin after amniotomy is the method of choice.     

Effect of prostaglandin E2 on adult pig articular cartilage slices in culture

Confirming earlier work by other investigators in isolated chondrocyte cultures, prostaglandin E2 (PGE2) (25 micrograms/ml) causes rapid inhibition of in vitro articular cartilage proteoglycan synthesis in adult pig cartilage slices. The potential impact of E prostaglandins on articular cartilage should be considered in clinical situations associated with elevated intra-articular prostaglandin levels (trauma, meniscal injury, arthritis, etc.).     

Induction of apoptosis of articular chondrocytes and suppression of articular cartilage proteoglycan synthesis by heat shock

We investigated cellular and matrix responses of articular cartilage to heat shock. Rat articular cartilage was pretreated at 37 degrees C for 24 h before being exposed to 48 degrees C for 10 min and subsequently incubated at 37 degrees C for 1, 2, 4, 7, 10, and 14 days. Following heat shock, a terminal deoxynucleotidyl transferase nick end labeling assay showed that articular chondrocyte apoptosis appeared at day 1, peaked at day 7, and declined by day 14. Analysis by transmission electron microscopy confirmed that the chondrocytes had characteristic morphological features of apoptosis; immunohistochemical staining revealed that caspase-3 activity in chondrocytes increased, 3-B-3-positive articular chondrocytes decreased in number, and the expression of 3-B-3 native epitope in articular chondrocytes was reduced. Safranin-O staining revealed that depletion of proteoglycans in the matrix was not found in any group. Morphological and biochemical evidence from this study suggested that heat shock at 48 degrees C induced articular chondrocyte apoptosis and suppressed proteoglycan synthesis of articular cartilage in vitro. This study thus provides evidence of the onset of osteoarthritis induced by heat shock and a basis for choosing a temperature at which malignant bone tumor cells can be killed with minimal damage to articular cartilage.     

Induction of cyclooxygenase-2 by mechanical stress through a nitric oxide-regulated pathway

OBJECTIVE: Biomechanical signals play important roles in regulating the homeostasis of articular cartilage, but under abnormal conditions may be a critical factor in the onset and progression of arthritis. Prostaglandin E(2) (PGE(2)) and nitric oxide (NO), derived from the enzymes cyclo-oxygenase 2 (COX2) and NO synthase 2 (NOS2), are inflammatory mediators that modulate numerous physiological and pathophysiological processes and are potentially important pharmacological targets in osteoarthritis. The goal of this study was to determine the effect of mechanical compression on PGE(2) production in the presence of selective NOS2 and COX2 inhibitors. METHODS: Articular cartilage explants harvested from 2-3-year-old pigs were subjected to intermittent compression at 0.5Hz over a range of stress magnitudes. PGE(2) and NO production into the media were determined in the presence and absence of the NOS2 inhibitor 1400W or the COX2 inhibitor NS398. COX2 protein levels were determined by immunoblot analysis. RESULTS: Mechanical compression significantly increased NO and PGE(2) synthesis in a manner that was dependent on the magnitude of stress. The selective COX2 inhibitor blocked compression-induced NO and PGE(2) production. Compression in the presence of 1400W further increased COX2 expression resulting in a 10-fold increase in PGE(2) production compared to uncompressed explants with 1400W and a 40-fold increase in PGE(2) compared to uncompressed explants without 1400W. CONCLUSION: Mechanical compression of articular cartilage increased COX2 and PGE(2) production through a NO-dependent pathway, and therefore pharmacological agents that target the NOS2 pathway in cartilage may have a significant influence on prostanoid production in the joint.     

前列腺素E2受体在前列腺素E2诱导H9c2心肌细胞肥大中的作用

目的 评价前列腺素E2受体(EP受体)在前列腺素E2(PCE2)诱导H9c2心肌细胞肥大中的作用.方法 培养H9c2心肌细胞,以4×104个/ml的密度接种于培养瓶(每瓶3ml)、24孔(每孔1 ml)或6孔(每孔2 ml)培养板.采用随机数字表法,将细胞随机分为4组(n=24):空白对照组(C组)不予任何处理,继续培养48 h;PGE2组在细胞培养液中加入PGE2(终浓度1μmol/L);AH6809组(A组)在细胞培养液中加入PGE2(终浓度1μmol/L)和AH6809(EP1及EP2受体拮抗剂,终浓度10μmol/L);GW627368X组(G组)在细胞培养液中加入PGE2(终浓度1μmol/L)和GW627368X(EP4受体拮抗剂,终浓度10 μmol/L).孵育48 h后采用免疫荧光观察心肌细胞形态,Image J医学图像分析系统测量心肌细胞直径,BCA法检测心肌细胞总蛋白含量,RT-PCR法测定胞浆ANP mRNA及BNP mRNA的表达水平.结果 与C组比较,PGE2组、A组和G组心肌细胞总蛋白含量和心肌细胞直径增加,胞浆ANPmRNA及BNPmRNA表达上调(P＜0.05).与PGE2组比较,G组心肌细胞总蛋白含量和心肌细胞直径降低,胞浆ANP mRNA及BNP mRNA表达下调(P＜0.05),A组上述各指标差异无统计学意义(P＞0.05).结论 EP4受体介导了PGE2诱导的心肌细胞肥大效应,而该效应与EP1和EP2受体无关.     

Influence of oxygen tension on nitric oxide and prostaglandin E2 synthesis by bovine chondrocytes

OBJECTIVES: To determine the in vitro effects of oxygen tension on interleukin (IL)-1beta induced nitric oxide (*NO) and prostaglandin E(2) (PGE(2)) production by bovine chondrocytes. DESIGN: Enzymatically isolated bovine chondrocytes were cultured for different periods in suspension in 21 (atmospheric), 5 or 1% (low) oxygen tension and in the absence or in the presence of increased amounts (0.01 to 1nM) of IL-1beta. Nitrite and nitrate concentrations in the culture supernatants were determined by a spectrophotometric method based upon the Griess reaction. PGE(2) production was quantified by a specific radioimmunoassay (RIA). Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) mRNA steady state levels were also quantified by real-time polymerase chain reaction (PCR). RESULTS: In the absence of IL-1beta, ()NO production remained stable whatever the oxygen tension used. IL-1beta dose-dependently increased *NO production in both atmospheric and low oxygen conditions but the effect was more pronounced in low (1 and 5%) than in atmospheric (21%) oxygen tension (P<0.001). Under low and atmospheric oxygen tension, iNOS gene expression was increased by IL-1beta, but to a lesser extent in 21% than in 1 or 5% oxygen (P<0.01). In the basal condition, bovine chondrocytes spontaneously produced PGE(2) whatever the oxygen tension used. At 21% oxygen, IL-1beta dose-dependently increased PGE(2) production while no significant effect was observed at 1 or 5% oxygen. COX-2 gene expression was significantly upregulated by IL-1beta in both low and atmospheric oxygen tension. No significant difference between oxygen tension conditions was observed. CONCLUSIONS: This study demonstrates that a hypoxic environment fully blocks COX-2 activity but favours iNOS gene expression in chondrocytes culture. These findings indicate that O(2) tension modulates cellular behaviour in culture and supports the concept of chondrocyte culture in low oxygen tension to reproduce in vitro the life conditions of chondrocytes.     

Stimulation of cAMP production and cyclooxygenase-2 by prostaglandin E(2) and selective prostaglandin receptor agonists in murine osteoblastic cells

Prostaglandins (PGs), particularly PGE(2), can stimulate bone resorption and formation and auto-amplify their effects by inducing cyclooxygenase (COX)-2. We examined the role of different PG receptors in stimulating cAMP production and COX-2 expression in murine calvarial osteoblasts. Cells were obtained from PGE(2) receptor (EP2R and EP4R) wild-type and knockout (KO) mice and from mice transgenic for the COX-2 promoter fused to a luciferase reporter. We analyzed effects of selective agonists, EP2A and EP4A, for EP2R and EP4R, which mediate the increase in cAMP in response to PGE(2). We also tested agonists for other PGE(2) receptors (EP1A and EP3A) and for prostacyclin (IPA), prostaglandin D(2) (DPA), thromboxane (TPA), and prostaglandin F(2alpha) (FPA) receptors. PGE(2) and EP2A were the most effective stimulators of cAMP production. EP4A, IPA, and DPA produced smaller responses, and EP1A, EP3A, FPA, and TPA were ineffective. In EP2R KO cells, cAMP responses to PGE(2) were reduced by 80%, and responses to EP2A were abrogated. In EP4R KO cells, cAMP responses to PGE(2) and EP2A showed a small reduction, while the response to EP4A was abrogated. Pretreatment with PGE(2), EP2A, or EP4A down-regulated the subsequent response to the respective ligands. COX-2 induction was measured by increased luciferase activity and mRNA expression. PGE(2) was the most effective agonist; EP2A and another selective EP2R agonist, butaprost, showed similar efficacy, and EP4A was less effective. EP2A and EP4A effects on luciferase activity were additive, and effects of the combination were similar to PGE(2) itself. IPA, TPA, and DPA produced 2- to 6-fold increases in COX-2 expression. FPA was a weak agonist, while EP1A and EP3A were inactive. Treatment with specific inhibitors indicated that PGE(2), EP2A, and EP4A induced COX-2 expression largely through protein kinase A (PKA). We conclude that the PG induction of COX-2 in this system generally paralleled effects on cAMP production and was mediated predominantly via the PKA pathway.     

骨关节炎早中期关节软骨YKL-40表达与软骨细胞凋亡的关系

背景：软骨细胞凋亡是骨关节炎（osteoarthritis,OA）发病过程中重要的病理学特征。YKL-40是壳质酶蛋白家族的一种糖蛋白,但不具有壳质酶活性,在关节炎软骨、滑膜、巨噬细胞等均有表达,可能与炎症的状态、组织重塑等功能有关。YKL-40在OA早中期中的作用尚有待研究。目的：探讨OA早中期关节软骨YKL-40表达与软骨细胞凋亡率（apoptosisindex,AI）的关系。方法：通过前交叉韧带切断术（anterior cruciate ligament transaction,ACLT）建立SD大鼠膝关节OA模型,组织学评估软骨退变程度,采用改良Mankin评分系统进行评估,免疫组织化学法检测YKL-40的表达情况及软骨细胞AI,观察两个指标的表达特点,分析两者在此病变过程中的关系。结果：随造模时间的延长,软骨出现退变并逐渐加重,YKL-40的表达与软骨细胞AI呈正相关。结论：软骨细胞凋亡是OA早中期的重要事件,YKL-40可能为OA早中期病理过程中软骨细胞凋亡的重要影响因子。