自由基对体外培养人胚关节软骨细胞的影响

采用体外培养人胚关节软骨细胞的方法，研究黄嘌呤－黄嘌呤氧化酶系产生的自由基对软骨细胞生长的影响。倒置显微镜下，软骨细胞生长缓慢、形态异常、没有集落形成。通过测定软骨细胞ＤＮＡ、蛋白质、蛋白多糖及胶原的含量变化，发现自由基要显著抑制ＤＮＡ、蛋白质及蛋白多糖的合成。采用流式细胞仪对软骨细胞周期进行了分析，发现ＤＮＡ合成后期（Ｇ２＋Ｍ期）细胞数显著增加。显然，自由基通过对软骨细胞ＤＮＡ的损伤，从而影响细     

自由基清除剂对氧自由基致人胚软骨细胞损伤的保护作用

本实验采用体外软骨细胞培养方法，观察了维生素Ｃ、维生素Ｅ、硒、超氧化物歧化酶等自由基清除剂对受黄嘌呤－黄嘌呤氧化酶系自由基损伤人胚软骨细胞的保护作用。通过对软骨细胞合成ＤＮＡ，基质蛋白多糖及胶原能力的测量和超微结构改变的观察，作者认为上述自由基清除剂对黄嘌呤－黄嘌呤氧化酶系自由基造成的人胚软骨细胞损伤无明显防御作用。     

硒锌对培养兔和人胚软骨细胞生长的影响

本实验在培养液中加入一定量的硒，锌对一代兔和人胚关节软骨细胞进行培养１９２小时。结果显示：补充适量的硒或锌均能促进培养软骨细胞的生长代谢，但硒过量则损害于软骨细胞，若同时补给适量的锌和硒，其效果有相加的作用。补锌和补硒的效果可能为硒＋锌＞锌＞硒。同时表明体外培养软骨细胞的基质代谢能很快进入平衡状态。人胚软骨细胞生长慢于兔，基质胶原代谢慢于蛋白多糖     

补肾行气活血法在骨髓基质干细胞诱导软骨细胞修复兔关节软骨缺损中的作用

[目的]探讨补肾行气活血法在骨髓基质于细胞诱导软骨细胞修复兔关节软骨缺损中的作用. [方法]将胶原凝胶包埋的骨炎定含药血清培养的骨髓基质干细胞诱导的软骨细胞和异体软骨细胞接种CPPf/PLLA支架构建的复合物体外培养3周,行倒置显微镜和扫描电镜观察,并将复合物异体移植入兔关节软骨缺损,术后4、8、12周取材,从大体、组织学和Ⅱ型胶原免疫组织化学分别对再生软骨组织进行评价. [结果]诱导软骨细胞组在支架内分布均匀、透明软骨样组织的形成、表面光滑度、与周围组织整合程度及基质内有Ⅱ型胶原分布等方面明显优于软骨细胞组. [结论]补肾行气活血法在关节软骨缺损修复的作用中较传统的方法有其优越性.     

低强度脉冲式超声促软骨修复及其基因表达研究进展

细胞外基质中蛋白聚糖和Ⅱ型胶原合成减少、降解增加及X型胶原的出现.是血运差、自身修复能力低下的关节软骨伤痛及退变的主要原因.一定条件的低强度脉冲式超声波(LIPUS)可促进股骨骨折大鼠模型骨痂中软骨细胞聚合素与Ⅱ型胶原mRNA表达,但后期经骨膜诱导可发生骨化.LIPUS对关节软骨细胞及基质是否产生类似效应并抑制软骨细胞的成熟与退变.则成为关节软骨伤痛无创修复的研究热点.该文通过总结近年来相关文献,就LIPUS对关节软骨伤病动物在体模型的软骨组织学及基因改变.LIPUS对体外培养软骨一软骨细胞的细胞活性、增殖、表型分化及基质合成分泌功能的影响,LIPUS的临床应用研究.LIPUS促进软骨基质合成的调控机制等进行综述,并提出目前研究存在的问题及进一步研究方向.     

重组PcDNA3-hBMP3转染兔关节软骨细胞和对其增殖的影响

目的研究将人骨形成蛋白３（ＢＭＰ３）的ｃＤＮＡ构建于真核,表达载体ＰｃＤＮＡ３,形成重组ＤＮＡ ＰｃＤＮＡ３－ｈＢＭＰ３,转染兔关节软骨细胞,以探讨基因转染对关节软骨细胞增殖的影响。方法利用重组ＤＮＡ和基因克隆技术构建重组ＤＮＡ;用细胞培养和基因转染技术体外转染兔关节软骨细胞;用核酸杂交和蛋白电泳检测细胞ＢＭＰ３的表达,最后通过ＦＣＭ、ＤＮＡ和葡糖醛酸含量检测以及Ⅱ型胶原探针原位杂交分析其对增殖的影响。结果软骨细胞６次传代后,Ⅱ型胶原原位杂交的灰度值降低,同样条件下转染的软骨细胞仍保持较高水平Ⅱ型胶原的表达;转染的软骨细胞经流式细胞仪的分析,软骨细胞Ｓ期细胞比例增多,说明细胞ＤＮＡ的合成增加;经ＤＮＡ和葡糖醛酸含量测定,转染的软骨细胞ＤＮＡ和葡糖醛酸含量较转染前有明显提高。结论ｈＢＭＰ３对维持软骨细胞的表型具有十分重要的作用,在脂质体的介导下,ＤＮＡ ＰｃＤＮＡ３－ｈＢＭＰ３转染兔关节软骨细胞获得成功。     

骨形态发生蛋白对体外培养胎儿关节软骨细胞胶原基因表达…

为了解骨形态发生蛋白对关节软骨细胞的作用特点及意义，本研究用部分纯化的牛骨形态发生蛋白对胎儿关节软骨细胞进行诱导，并用人Ⅰ、Ⅱ、Ⅲ、Ⅳ型胶原的前胶原ｃＤＮＡ探针对被诱导关节软骨细胞进行原位杂交。结果发现，ｂＢＭＰ可明显增加被诱导软骨细胞中Ⅰ型胶原的表达，中断Ⅱ型胶原的表达，并轻度增加软骨细胞中Ⅲ型胶原的表达，但对Ⅳ型胶原的表达影响不大。本研究认为，ｂＢＭＰ诱导的软骨细胞胶原基因表达的这种改变是软骨     

双相支架负载软骨细胞修复兔关节软骨缺损

目的研究自固化磷酸钙/纤维蛋白凝胶(CPC/FG)双相支架负载软骨细胞修复兔关节软骨缺损的可行性和有效性.方法将分离培养的第3代软骨细胞包埋在CPC/FG双相支架的FG中,体外培养1周后,将软骨细胞-支架复合体移植修复兔膝关节股骨髁的软骨缺损(φ4 mm,深3.5 mm,达软骨下骨质).然后对软骨缺损的修复情况进行大体、光镜和电镜观察.同时对移植后第12周的修复软骨进行胶原含量测定,并与正常的关节软骨细胞胶原含量进行比较.结果移植的软骨细胞能在双相支架上良好地生长,软骨缺损以透明软骨的形式被修复,而对照组为纤维组织修复.多孔自固化磷酸钙在软骨修复过程中能起软骨下骨的临时替代作用.胶原含量测定显示:移植术后12周的修复软骨胶原含量为(43.25±0.85)%;正常的关节软骨胶原含量为(55.69±0.76)%,两者差异有显著性(P<0.01).结论 CPC/FG双相支架负载软骨细胞能以透明软骨的形式修复兔关节软骨缺损.新环境中移植的软骨细胞生长的不适应和FG降解过快,可能是导致新生修复软骨与自身正常关节软骨胶原含量有差异的原因.     

重组PcDNA3—hBMP3转染兔关节软骨细胞和对其增殖的影响

目的 研究将人骨形成蛋白３（ＢＭＰ３）的ＣＤＮＡ构建于真核，表达载体ＰＣＤＮＡ３，形成重组ＤＮＡＰｃＤＮＡ３－ｈＢＭＰ３，转染兔关节软骨细胞，以探讨基因转染对关节软骨细胞增殖的影响。方法 利用重组ＤＮＡ和基因克隆技术构建重组ＤＮＡ；用细胞培养和基因转染技术体外转染兔关节软骨细胞；用核酸杂交和蛋白电泳检测细胞ＢＭＰ３的表达，最后通过ＦＣ几葡糖醛酸含量检测以及Ⅱ型胶原探针原位杂交分析其对增殖的影响。结     

关节软骨磁共振生理性成像临床应用

关节软骨主要由软骨细胞和细胞外基质组成,软骨基质构建了稳定而复杂的耐压框架结构.关节软骨磁共振生理性成像是评价关节软骨基质成分变化的新型影像学技术.按照观察对象不同,可将其分为蛋白多糖相关技术(延迟动态增强、T1ρ成像和Na谱成像)、胶原含量相关技术(T2地图成像和磁化转移成像)、软骨基质内水含量相关技术(质子密度地图成像和弥散加权成像).延迟动态增强和T2地图成像已开始应用于软骨病变的早期诊断和治疗评价中.随着磁共振设备和分析软件的不断进步,关节软骨磁共振生理性成像将成为骨关节影像研究的重点之一.     

Free radical damage to chondrocytes and protection by selenium, tocopherol and ascorbic acid using serum-free medium method]

Free radical derived from xanthine oxidase damage to rabbit articular chondrocytes cultured in serum-free medium and antioxidant defense factors protecting chondrocytes from free radical were studied. The results showed that free radical mediated an inhibition of DNA as well as proteoglycan synthesis in cultured chondrocytes and selenium, a-tocopherol and L-ascorbic acid failed to protect chondrocytes from free radicals.     

Effect of oxygen free radicals on rabbit and human erythrocytes. Studies on cellular deformability

Changes in deformability of rabbit and human erythrocytes caused by exposure in vitro to the oxygen free radical generator hypoxanthine and xanthine oxidase were studied. The deformability reduction observed after 30 min of exposure to hypoxanthine-xanthine oxidase could be prevented by pretreatment with SOD, while after only 5 min of such exposure allopurinol and catalase also appeared to have a protective effect. Exposure of human erythrocytes to hypoxanthine and xanthine oxidase in Krebs solution prevented an otherwise occurring hemolysis. Exposure to both substances or to xanthine oxidase alone in Dulbeccos phosphate solution produced a reduction in deformability. The results indicate that exposure of erythrocytes to free oxygen radicals reduces deformability and that this effect may contribute to the myocardial dysfunction and the epicardial erythrostasis observed during open-heart surgery.     

Effects of Tamm-Horsfall protein and albumin on the inhibition of free radicals.

INTRODUCTION: Oxalate in urine can cause tubular cellular damage by the production of free radicals. Then, cell death and cellular debris may promote the retention of calcium oxalate crystals and finally the formation of stones. The two most abundant urinary proteins, Tamm-Horsfall protein (THP) and albumin, were tested for the effects of antioxidants. MATERIALS AND METHODS: By using xanthine-xanthine oxidase reaction, purified THP and albumin were tested for the inhibitory effect. OD(295) was used as a spectrophotometric method to measure the production of uric acid during the reaction. RESULTS AND CONCLUSIONS: Both proteins can inhibit the reaction of xanthine oxidase on xanthine, although the effect was decreased after enzymatic deglycosylation of sialic acid. Albumin has an IC(50) of 10.7 nM in native condition and 11.9 nM after deglycosylation, whereas THP has 69.6 nM in native condition and 102.0 nM in deglycosylated condition. The data indicates that THP and albumin have an antioxidant effect. Sialic acid in THP has partly an inhibitory effect and is associated with calcium oxalate formation. Studies have indicated that further investigation of the role of free radicals in the formation of urolithiasis and of sialic acid in protein function is needed.     

Minimal role of xanthine oxidase and oxygen free radicals in rat renal tubular reoxygenation injury

The role of xanthine oxidase and oxygen free radicals in postischemic reperfusion injury in the rat kidney remains controversial. Proximal tubules, the focal segment affected by ischemic renal injury, were isolated in bulk, assayed for xanthine oxidase activity, and subjected to 60 min of anoxia or hypoxia and 60 min of reoxygenation to evaluate the participation of xanthine oxidase and oxygen radicals in proximal tubule reoxygenation injury. The total xanthine oxidase in isolated rat proximal tubules was 1.1 mU/mg of protein, approximately 30% to 40% of the activity found in rat intestine and liver. Lactate dehydrogenase release, an indicator of irreversible cell damage, increased substantially during anoxia (39.8 +/- 2.3 versus 9.8 +/- 1.8% in controls) with an additional 8 to 12% release during reoxygenation. Addition of 0.2 mM allopurinol, a potent xanthine oxidase inhibitor, and dimethylthiourea, a hydroxyl radical scavenger, failed to protect against the reoxygenation lactate dehydrogenase release. Analysis of xanthine oxidase substrate levels after anoxia and flux rates during reoxygenation indicates that hypoxanthine and xanthine concentrations are in a 15-fold excess over the enzyme Km and 0.3 mU/mg of protein of xanthine oxidase activity exists during reoxygenation. Hypoxic tubule suspensions had a minimal lactate dehydrogenase release during hypoxia and failed to demonstrate accelerated injury upon reoxygenation. In conclusion, although xanthine oxidase is present and active during reoxygenation in isolated rat proximal tubules, oxygen radicals did not mediate reoxygenation injury.     

Role of xanthine oxidase in small bowel mucosal dysfunction after surgical stress

BACKGROUND: The small intestine is highly susceptible to surgical stress even at remote locations. An earlier study using a rat model indicated that oxidative stress plays an important role in this process. The enzyme xanthine oxidase is an important source of free radicals in the small intestine. The role of this enzyme in intestinal damage after surgical stress was examined. METHODS: Rats pretreated with xanthine oxidase inhibitors were subjected to surgical stress by opening the abdomen and handling the intestine, as done during laparotomy. Enterocytes at various stages of differentiation were isolated and the protection offered by xanthine oxidase inhibitors against damage due to surgical stress was determined and compared with normal controls. Protection against ultrastructural changes to the mucosa, as well as mitochondrial function was examined. RESULTS: Surgical stress affected both the villus as well as crypt cells, causing increased superoxide generation, accompanied by increased activity of xanthine oxidase. Xanthine oxidase inhibitors ameliorated the increased superoxide generation, and protected against mitochondrial damage and ultrastructural changes in the intestine. CONCLUSION: Surgical stress affects both the villus and crypt cell populations in the small intestine. The enzyme xanthine oxidase maybe an important mediator of surgical stress in the intestine.     

Reactive Oxygen Species Activate the Human Elastin Promoter in a Transgenic Model of Cutaneous Photoaging

BACKGROUND: Generation of free radicals has been shown to play a role in cutaneous alterations resulting from ultraviolet radiation.OBJECTIVE: Cells from a previously described in vitro transgenic model of cutaneous photoaging were exposed to reactive oxygen species to determine if this results in elastin promoter activation.METHODS: Reactive oxygen species were generated using a hypoxanthine and xanthine oxidase system, and elastin promoter activation was measured using cells derived from transgenic mice containing the human elastin promoter.RESULTS: Free radical generation resulted in a greater than sixfold increase in elastin promoter activity, and this increase was blocked with the addition of catalase.CONCLUSION: Elastin promoter activation may play a role in the generation of solar elastosis in photoaged skin. Utilizing hypoxanthine and xanthine oxidase with the in vitro transgenic photoaging model results in a sensitive system for evaluating agents that may prevent oxidative damage.     

Role of xanthine oxidase in passive Heymann nephritis in rats

Passive Heymann nephritis (PHN) in rats is a model of human membranous nephropathy characterized by formation of subepithelial immune deposits in the glomerular capillary wall and complement activation. Oxygen radicals have been implicated in the subsequent glomerular damage which leads to proteinuria. This study examines the involvement of xanthine oxidase in this process. Xanthine oxidase activity was increased nearly twofold in glomeruli isolated 1 and 12 d after induction of PHN, and this was associated with increased glomerular superoxide anion generation. Analysis of glomerular samples by Northern and Western blotting revealed no quantitative changes in xanthine oxidoreductase expression in PHN, suggesting conversion of xanthine dehydrogenase to the oxidase form as the cause of increased activity. Treatment of rats with tungsten, an inhibitor of xanthine oxidase, before induction of PHN resulted in a marked decrease in glomerular xanthine oxidase activity and superoxide anion generation, and decreased proteinuria by 80% (day 12: 423+/-245 mg/d in PHN versus 78+/-53 mg/d in tungsten-treated PHN animals, P < 0.01). These findings point to a pivotal role of xanthine oxidase in the pathophysiology of PHN and could be of importance in the therapy of human membranous nephropathy.     

Xanthine:acceptor oxidoreductase activities in ischemic rat skin flaps

Xanthine:acceptor oxidoreductase activities were assayed in free skin flaps following prolonged preservation. In normal rat skin, xanthine dehydrogenase transfers electrons to NAD+ and accounts for 73% of total oxidoreductase activity, and xanthine oxidase transfers electrons to molecular oxygen and accounts for the remaining 27%. Xanthine oxidase activity increased significantly in skin flaps during ischemia: approximately 30 and 100% increases after 6 and 24 hr of ischemia, respectively. Allopurinol inhibited xanthine oxidoreductase activity: free skin flaps obtained from allopurinol-treated animals exhibited a low level of xanthine oxidoreductase activity throughout the period of preservation. Systemic allopurinol significantly improved the survival rate from 32 to 75% of free flaps transferred after 24 hr of preservation at room temperature. These observations suggest that the xanthine oxidase system is a major source of oxygen free radicals following ischemia/reperfusion in skin. The increase in xanthine oxidase is attributable to the conversion of xanthine dehydrogenase to oxidase, a conversion which involves sulfhydryl oxidation in skin flaps.     

Modification of oxidative stress in response to intestinal preconditioning

Previous studies have demonstrated that intestinal preconditioning protects the organ from ischemia reperfusion damage. Xanthine oxidase mediating free radical generation contributes to the development of injury associated to ischemia reperfusion. Thus, any process able to modulate the oxygen free radical generation system could attenuate the injury. Also, it is known that nitric oxide is implicated in the preconditioning response. The aim of this work is to determine: (1) the effect of intestinal preconditioning on the xanthine oxidase system, (2) the relevance of this system in the development of injury, and (3) its relationship with nitric oxide. For this purpose, we have determined the activity of the xanthine dehydrogenase/xanthine oxidase system, the levels of its substrate (xanthine), and end-product (uric acid) and oxidant stress status in rat small intestine subjected to ischemic pre-conditioning. The effects of nitric oxide inhibition have also been evaluated. Results show that the percentage of xanthine dehydrogenase to xanthine oxidase conversion, xanthine, uric acid concentration, lipoperoxides, and reduced glutathione were significantly reduced in preconditioned rats irrespectively of nitric oxide inhibition. In summary, this work shows that oxidative stress in intestinal preconditioning is reduced as consequence of the diminished conversion of xanthine dehydrogenase to xanthine oxidase, and also as a consequence of the reduced availability of xanthine.     

Effects of thapsigargin,an intracellular calcium-mobilizing agent,on synthesis and secretion of cartilage collagen and proteoglycan

The calcium-mobilizing agents thapsigargin and 2.5-di-(tert-butyl)-1,4-benzo-hydroquinone were shown to markedly elevate the intracellular calcium concentration of chick embryo chondrocytes in a dose-dependent manner. Under these conditions, the metabolism of macromolecules was variably affected. The synthesis and secretion of protein in general, and of collagen in particular, were significantly inhibited; in contrast, proteoglycan synthesis (but not glycosaminoglycan synthesis) was inhibited, whereas secretion was unaffected. Flunarizine, which prevented the thapsigargin-induced intracellular calcium elevation, and EGTA, which caused only a transient thapsigargin-induced intracellulr calcium elevation, did not reverse these alterations. It was concluded, therefore, that the observed effects of thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone on chondrocyte macromolecule metabolism were not related to the ability of these drugs to increase the cytosolic free calcium concentration but may have been due to the specific depletion of the calcium sequestered in the endoplasmic reticulum. The differential effect of these drugs on protein and proteoglycan secretion suggests that the intracellular trafficking of these two classes of macromolecules may be controlled independently.