骨关节炎软骨中衰老相关β-半乳糖苷酶和c-Fos蛋白的表达

背景：文献在对软骨细胞衰老的研究中,常以衰老相关β-半乳糖苷酶的表达作为其细胞老化的一个指标,而c-Fos在骨关节炎软骨中表达情况罕见报道。目的：观察β-半乳糖苷酶、c-Fos蛋白在正常关节软骨和骨关节炎不同退变程度关节软骨中表达水平的差异。方法：选取正常关节软骨标本10个,骨关节炎软骨标本26个,根据大体观察凿取正常和骨关节炎不同退变严重程度软骨块50个,再根据关节软骨改良Mankin病理评分法分为正常关节软骨组、轻度退变组、中度退变组、重度退变组。采用免疫组织化学SABC法,计算各组标本中软骨细胞β-半乳糖苷酶、c-Fos蛋白染色阳性细胞率,比较各组中二者阳性细胞率的差异性并分析其与关节软骨不同退变程度之间的相关性。结果与结论：随着衰老软骨细胞阳性率逐渐增加,软骨退变程度逐渐加重,提示软骨细胞衰老与骨关节炎病程进展有关,软骨细胞衰老是骨关节炎可能的发病机制之一。c-Fos蛋白与骨关节炎软骨退变严重程度和软骨细胞衰老均无直接相关性,但可能在软骨退变早中期促进软骨细胞代偿性分裂增殖活跃。     

细菌β-半乳糖苷酶快速纸片法检测

目前绝大多数细菌生化反应需18-24h,有的甚至要48-72h以上,这不仅延长了细菌鉴定时间,而且也延误了疾病的诊断和治疗,因此在细菌学领域,快速、准确的鉴定已是临床细菌学实验室的迫切要求,为此我们参照国内外有关文献,设计了β-半乳糖苷酶（onitrophenyl-β-galactoside,ONPG）快速试验纸片,经初步试用效果较好。     

β-半乳糖苷酶快速鉴定与定量肠杆菌

[目的]检测大肠埃希菌和肠杆菌属的β-半乳糖苷酶(β-GAL)。[方法]应用对硝基酚-β-D半乳糖苷为底物，以酶的反应来快速检出样本中的肠杆菌，且定量此种酶，并定出菌量。[结果]经实验确定了本法的最佳底物浓度，最适反应时间和pH值，最底检出限达300CFU／ml。[结论]本法快速、简便、敏感，为临床细菌鉴定，食品卫生检验提供了一种新的方法。     

大鼠肾囊内β-半乳糖苷酶基因注入及其在肾脏内的局部表达

目的基因治疗的研究受到学术界广泛关注,治疗性基因定向导入体内、定位性表达、继而发挥局部特异性治疗是基因治疗学的研究热点。本研究以β-半乳糖苷酶基因为实验基因,对基因治疗的定向性、定位性、特异性进行了深入研究。研究基因定向导入定位表达的可行性,探讨一种基因定向导入局部表达治疗肾脏病的新技术。方法选用正常雄性SD大鼠15只,应用β-半乳糖苷酶基因,以脂质体为载体进行肾周脂肪囊内注射,分别在注射后12、24、36、48、72h及7天观察肾脏内的基因表达,及肾外脏器:心脏、肝脏、脾脏、肺脏、肌肉的表达情况。结果肾囊内注射β-半乳糖苷酶基因后12h可见肾脏内有半乳糖苷酶表达,表达持续存在,直至7天;全身其它脏器未见表达。基因表达仅限于肾小管内,肾小球内未见表达。结论基因定向导入肾囊内后可在肾脏组织内表达。本方法简便易行,为肾脏病的基因治疗开拓了一条新途径。     

衰老相关β-半乳糖苷酶在老年大鼠肾脏组织中的变化及意义

【目的】研究大鼠肾脏组织随增龄衰老相关β-半乳糖苷酶（SA-β-Gal）活性的变化规律,并探讨其与肾脏衰老的关系,以寻找可用于评判肾脏衰老的相关指标。【方法】留取3,12,24月龄Wistar大鼠肾脏（每组各6只）。采用PAS和Masson染色观察肾组织病理变化;β-gal染色观察大鼠肾组织SA-β-Gal活性的变化。【结果】随增龄,老年大鼠肾组织出现肾小球肥大,节段性肾小球硬化,局灶性肾小管萎缩、间质纤维化和炎性细胞浸润;肾脏病理评分显示肾小球和小管间质病变评分随增龄逐渐增高,24月龄组评分值显著高于3月龄和12月龄组（P〈0．05）。大鼠肾组织SA-β-gal染色阳性面积百分比随增龄逐渐升高,24月龄组阳性百分比显著高于3月和12月龄组（P〈0．05）,并与同月龄组肾脏病理评分（肾小球和肾小管间质）呈正相关（r=0．585和0．612,P〈0．05）。【结论】随增龄大鼠肾组织SA-β-Gal表达逐渐增强,可能作为大鼠肾脏衰老的一项检测指标。     

α-半乳糖苷酶酶解制备通用红细胞的动物实验研究

目的　观察酶解对猕猴红细胞形态及功能的影响 ,评价酶解后通用红细胞的安全性。方法　以基因重组的α 半乳糖苷酶体外酶解猕猴类人B型抗原 ,并以改良吸收放散试验鉴定酶解效果 ,检测酶解前后猕猴红细胞的形态和功能变化 ,流式细胞仪测定酶解红细胞在受体内的 2 4h存活率及半衰期 ,监测受体回输前后不同时期的血液、尿液生化指标。结果　经α 半乳糖苷酶酶解后的红细胞其B抗原被清除 ,形态及功能与酶解前无明显变化 ,在受体内的 2 4h存活率为 84 .6 %和 6 8.1 % ,半衰期为 7d和 8d ,血液、尿液等主要生化指标亦正常。结论　α 半乳糖苷酶酶解对猕猴红细胞的形态和功能无不良影响 ,酶解后红细胞输给异体猕猴是安全的     

急性脑出血患者N-乙酰-β-D-氨基葡萄糖苷酶:β-D-半化糖苷酶变化及意义

目的：了解急性脑出血对肾脏的损害。方法：检测９４例急性脑出血患者血ＢＵＮ和尿ＮＡＧ、ＧＡＬ。结果：发现ＮＡＧ、ＧＡＬ全部升高。成人组较婴儿组升高明显（Ｐ＜０．０１）。ＢＵＮ升高者２４例，占２５．５３％，ＢＵＮ升高组与ＢＵＮ正常组两种尿酶升高程度相差显著（Ｐ＜０．０１，＜０．０５）。结论：提示在急性脑出血患者有不同程度的肾功能损害，尿酶ＮＡＧ、ＧＡＬ比ＢＵＮ敏感     

利用新型α-半乳糖苷酶进行B→O血型改造

目的利用原核生物来源的α-半乳糖苷酶进行B→O血型改造研究。方法应用PCR的方法从脆弱类杆菌(Bacteriodes fragilis)中克隆了新型α-半乳糖苷酶基因,构建原核生物表达载体,转化大肠杆菌BL21(DE3),IPTG诱导α-半乳糖苷酶的胞内可溶性表达,应用重组酶的上清液进行B型红细胞(RBC)初步酶解试验。结果重组酶分子量约为64.6 kD,超声破碎的菌液上清中的酶活力约为2 U/ml。在26℃及pH 6.8的等渗柠檬酸-磷酸氢二钠缓冲体系中2,h内完全酶解200μl B型RBC需要30μl的上清液,酶解后的B型RBC与抗-B和抗-A单克隆抗体不凝集,流式细胞术检测结果显示酶解后B型RBC的血型抗原呈现O型特征。结论重组的α-半乳糖苷酶可以有效的将B型RBC转变为O型RBC。     

α-半乳糖苷酶A缺乏对凝血因子V Leiden小鼠组织纤维蛋白沉积和血栓形成的影响

目的 评价α-半乳糖苷酶A(Gla)缺乏与凝血因子V(FV)Leiden(FVL)在体内对血栓形成的影响.方法 建立Gla与FVL基因复合突变小鼠,并分析其器官组织纤维蛋白沉积和血栓形成情况.结果 在FVL存在情况下,野生型小鼠组织器官纤维蛋白沉积[Gla+/0 FV Q/Q,(0.08±0.05)%]较Gla缺乏型[Gla-/0 FV Q/Q,(0.24±0.07)%)]显著降低(P＜0.01),Gla缺乏型纯合子小鼠组织器官纤维蛋白沉积[Gla-/-FV Q/Q,(0.32±0.03)%)]较Gla野生型[Gla+/+FV Q/Q,(0.06±0.01)%)]显著增加(P＜0.05);同时Gla缺乏型小鼠(Gla-/-FV Q/Q和Gla-/0 FV Q/Q)血栓数目[(1.9±0.7)个]亦显著高于Gla野生型小鼠(Gla+/+FV Q/Q和Gla+/0 FV Q/Q)的血栓数目[(0.3±0.1)个](P＜0.05).结论 Gla缺乏加重FVL突变小鼠的组织纤维蛋白沉积和血栓形成,提示Cla缺乏可能是重要的增强FVL患者血栓形成的遗传因素,临床上FVL患者出现早发和严重的血栓疾病可能合并其他遗传性血栓相关的基因缺陷.     

新型α-半乳糖苷酶清除动物红细胞表面αGal抗原的研究

异种抗原αGal是引起异种移植免疫排斥反应的重要原因之一。脆弱类杆菌来源的α-半乳糖苷酶是一种新型的糖苷酶,能够切除支链多糖和直链多糖末端的αGal残基。本研究探讨新型α半乳糖苷酶清除红细胞表面αGal抗原的作用。利用新型的基因重组α-半乳糖苷酶酶解牛、猪、狗、兔红细胞上的异种抗原,并用流式细胞术分析细胞表面的αGal抗原。结果表明,动物红细胞表面的异种抗原αGal被完全或部分清除。结论:新型α-半乳糖苷酶可以用来清除异种红细胞上的αGal抗原,对降低异种移植引起的超急性排斥反应具有潜在意义。     

Collagen synthesis in normal and osteoarthritic human cartilage.

Collagen metabolism in osteoarthritic human articular cartilage was compared to that in normal cartilage and was also correlated with the degree of severity of the osteoarthritic lesion as determined by a histological-histochemical grading system. No correlation was apparent between the concentrations of DNA, hydroxyproline, and hydroxylysine and the degree of severity of the osteoarthritic lesion (except in far-advanced lesions). Similarly, there was no correlation in levels of these components in tissues from the normal vs. osteoarthritic group. The similarity of the values of the ratio hydroxylysine/hydroxyproline in osteoarthritic tissue compared with normal, and the lack of variation in these with increasing severity of the disease process argues against the possibility that osteoarthritis is associated with a major shift in the synthesis of type II collagen to type I. [3H]Proline incorporation into osteoarthritic cartilage was increased fourfold as compared to normal cartilage and varied with advancing histological-histochemical grade. Measurement of the specific activity of insolubilized hydroxyproline-containing material of the cartilage matrix, as an index of the turnover of collagen, showed a sixfold increase in osteoarthritic cartilage which also varied with grade. These data suggest that collagen synthesis in these tissues is substantially greater than in nonosteoarthritic tissues and varies directly with the severity of the disease process up to a point and then varies inversely as the lesion becomes more severe.     

Collagenase and collagenase inhibitors in osteoarthritic and normal cartilage.

In advanced osteoarthritis, all of the cartilaginous components are lost from the joint surface. Although mechanisms exist for proteoglycan degradation, there is not known to be any system for removal of the collagen. This study suggests that the loss of the collagen components may be a function of articular cartilage collagenase. The enzyme in normal human cartilage is bound to an inhibitor and appears to be present in very small amounts. Attempts to demonstrate collagenase activity in ground human articular cartilage or in its lysosomal fraction were unsuccessful. 7-Day cartilage tissue cultures also failed to demonstrate the presence of the enzyme; but the same culture fluid, incubated with trypsin, showed significant degradation of collagen, suggesting that trypsin destroyed the inhibitor. 7-Day culture fluids were then chromatographed on a heparin-charged Sepharose 4B affinity column that had been activated with cyanogen bromide. This removed the inhibitor, and the chromatographed fluid from osteoarthritic cartilage released 42% of the incorporated counts of the collagen substrate, whereas normal cartilage released 10.1% and a trypsin control, 6.4%. Electrophoresis of the degradation products of the enzyme-collagen complex incubated at 37 degrees C revealed breakdown was complete to small dialyzable fragments, while at 25 degrees C larger fragments were split off.     

Direct extraction and assay of collagenase from human osteoarthritic articular cartilage

A method has been developed for the direct extraction of collagenase from small quantities (5 mg) of human osteoarthritic articular cartilage. The enzyme, which was not detected in normal cartilage, was entirely in a latent form and demonstrated typical properties of mammalian collagenase after activation by trypsin.     

Expression of 92-kD type IV collagenase/gelatinase (gelatinase B) in osteoarthritic cartilage and its induction in normal human articular cartilage by interleukin 1.

We report here that a 92-kD gelatinolytic metalloproteinase is expressed as protein and mRNA in human osteoarthritic cartilage, but not in normal adult articular cartilage. Western immunoblotting demonstrated that the 92-kD gelatinolytic activity corresponded to 92-kD type IV collagenase/gelatinase (gelatinase B); mRNA for gelatinase B was identified by Northern blotting. Chondrocytes from normal cartilage also exhibited mRNA for 72-kD type IV collagenase/gelatinase (gelatinase A), tissue collagenase, and stromelysin-1, and these mRNAs were increased in osteoarthritic cartilage. Regional analysis of osteoarthritic cartilage samples from four individuals revealed that gelatinase B mRNA was expressed in grossly fibrillated areas; two of four nonfibrillated cartilage samples failed to exhibit the mRNA, but did have increased levels of mRNA for other neutral metalloproteinases. IL-1 alpha treatment of normal human cartilage explants or isolated chondrocytes induced increased levels of gelatinase B and increased mRNA for tissue collagenase and stromelysin-1. Under identical conditions, mRNA levels for gelatinase A were not increased indicating that regulation of this enzyme in human articular chondrocytes is distinct from that of other metalloproteinases. Our data showing expression of gelatinase B in fibrillated cartilage suggest that it is a marker of progressive articular cartilage degradation in osteoarthritis.     

Evidence for metalloproteinase and metalloproteinase inhibitor imbalance in human osteoarthritic cartilage.

Cartilage specimens from tibial plateaus, obtained from 13 osteoarthritic (OA) patients and seven controls, were selected from three regions: zone A, center of fibrillated area; zone B, area adjacent to fibrillation, and zone C, remote region of plateau. Acid and neutral metalloproteinases and tissue inhibitor of metalloproteinase (TIMP) were extracted with 2 M guanidine. Methods were developed to selectively destroy either proteinases or TIMP to prevent cross-reaction during assay. Acid and neutral proteinases were elevated approximately 150% in OA; TIMP was elevated approximately 50%. A positive correlation (r = 0.50) was found between acid and neutral proteinase activities in OA, but not in controls. Both proteinases were elevated two-to threefold in zones A, B, and C. However, the self-active form of the acid metalloproteinase was elevated only in zones A and B (200%); it correlated well with the Mankin scores, whereas the total activities did not. TIMP was elevated (50%) only in zones A and B. Both the proteinase levels and the Mankin score were elevated to a greater extent in the medial, than in the lateral, compartment. Titration of TIMP against the two metalloproteinases indicates that there is a small excess of inhibitor over enzymes in normal cartilage. In OA, TIMP does not increase to the same extent as the proteinases; the resultant excess of proteinases over TIMP may contribute to cartilage breakdown.     

大鼠高强度运动后的软骨损伤及寡聚基质蛋白的表达

目的探讨高强度运动对大鼠关节软骨及大鼠膝关节液软骨寡聚基质蛋白(COMP)表达的影响。方法将20只SD大鼠随机分为对照组和高强度运动组各10只。高强度运动组进行8周高强度跑台运动。对照组不进行运动跑台。8周后取大鼠股骨髁病理学检查,采用酶联免疫吸附法检测大鼠关节液中COMP水平。结果高强度运动组大鼠膝关节出现关节软骨损伤表现,关节液中COMP的表达高于对照组(P0.05)。结论高强度运动导致的关节软骨损伤,创伤性关节炎与COMP水平有关。     

c-Jun、c-Fos在皮肤恶性黑素瘤中的表达及意义

目的：探讨c-Jun、c-Fos在皮肤恶性黑素瘤中的表达及意义。方法：采用免疫组化法检测45例皮肤恶性黑素瘤、34例瘤旁组织和20例色素痣中c-Jun、c-Fos的表达。结果：（1）c-Jun、c-Fos在皮肤恶性黑素瘤、瘤旁组织中的阳性表达率明显高于色素痣;（2）c-Jun、c-Fos在皮肤恶性黑素瘤中的表达与浸润深度有关,与淋巴结转移无关;（3）皮肤恶性黑素瘤中c-Jun、c-Fos的表达呈显著正相关。结论：c-Jun、c-Fos可作为皮肤恶性黑素瘤早期诊断的生物学标记。     

Extrusion of pyrophosphate into extracellular media by osteoarthritic cartilage incubates.

The distribution of calcium pyrophosphate mineral phase, almost exclusively confined to articular cartilage in chondrocalcinosis, and the high level of pyrophosphate (PPi) ion relative to serum in synovial fluid in patients with either chondrocalcinosis or advanced osteoarthritis led to an investigation of whether cartilage cells elaborate PPi ions. Incubates of articular cartilage from young rabbits but not from mature rabbits, as well as growth plates cartilage, released PPi into incubation media during a 4h period. Control rabbit ear cartilage and synovial membrane elaborated negligible amounts of PPi. The PPi was shown to be undialyzable but could be dissociated from the alkaline phosphatase by ultracentrifugation. In 16 patients with osteoarthritis, a substantial output of PPi by samples of articular cartilage from the knee was demonstrated. It is postulated that either rapid cell division and matrix synthesis found in the base of ulcerating osteoarthritic cartilage or remodeling calcified sites are the source of the PPi in such osteoarthritic cartilage. It is further hypothesized that this PPi output accounts at least in part for the elevated PPi levels found in synovial fluid of patients with osteoarthritis.     

Enhanced cleavage of type II collagen by collagenases in osteoarthritic articular cartilage.

We demonstrate the direct involvement of increased collagenase activity in the cleavage of type II collagen in osteoarthritic human femoral condylar cartilage by developing and using antibodies reactive to carboxy-terminal (COL2-3/4C(short)) and amino-terminal (COL2-1/4N1) neoepitopes generated by cleavage of native human type II collagen by collagenase matrix metalloproteinase (MMP)-1 (collagenase-1), MMP-8 (collagenase-2), and MMP-13 (collagenase-3). A secondary cleavage followed the initial cleavage produced by these recombinant collagenases. This generated neoepitope COL2-1/4N2. There was significantly more COL2-3/4C(short) neoepitope in osteoarthritis (OA) compared to adult nonarthritic cartilages as determined by immunoassay of cartilage extracts. A synthetic preferential inhibitor of MMP-13 significantly reduced the unstimulated release in culture of neoepitope COL2-3/4C(short) from human osteoarthritic cartilage explants. These data suggest that collagenase(s) produced by chondrocytes is (are) involved in the cleavage and denaturation of type II collagen in articular cartilage, that this is increased in OA, and that MMP-13 may play a significant role in this process.