周期性张应变力对不同年龄兔关节软骨细胞产生糖胺多糖的影响

目的 观察周期性张应变力(CTS)对体外培养不同年龄兔软骨细胞产生糖胺多糖(GAG)的影响。方法 选取雄性新西兰大白兔9只,按照年龄分为幼年组（2月龄）、成年组（8月龄）及老年组（31月龄）（每组3只）,无菌条件下取双膝关节,将各年龄组兔膝软骨细胞进行消化分离和体外培养。将每个年龄组兔原代软骨细胞分别培养于2个BioFlex 6孔培养板上,随机分为CTS组和对照组（每组6个样本）,同置于培养箱内,CTS组每天CTS (sin1 0％,0.5 Hz,6h/次)作用6h,对照组不予特殊处理。CTS组与对照组在首次作用后第12、24、36、48、60、72小时分别吸取培养细胞上清液,Alcian blue染色沉淀法测定上清液GAG含量,比较两组GAG分泌的变化。结果 CTS组与对照组相比,各年龄组兔软骨细胞GAG增加量在不同时间]点差异均有统计学意义(P＜0.05)。老年组在起始时,两组GAG的增加量与年轻组的增加量无明显差异,但从第48小时起老年组增加量开始低于年轻组,差异有统计学意义(P＜0.05)。结论 CTS可以促进兔软骨细胞产生GAG,且CTS刺激后年轻细胞比老年细胞产生更多的GAG。     

人肋软骨细胞体外培养中生长代谢及功能的变化

目的　通过检测体外培养的人肋软骨细胞老化过程中Ⅰ、Ⅱ型胶原和蛋白聚糖(aggrecan)的表达变化 ,为组织工程软骨构建的种子细胞选择适宜的回植时机。方法　取体外培养的P1 ～P5人肋软骨细胞 ,通过观察细胞形态 ,细胞增殖率 ,Alcianblue法定量检测每代Aggrecan中GAG含量 ,免疫组化蛋白水平观察Ⅰ、Ⅱ型胶原表达及RT PCR从mRNA水平分析Ⅰ、Ⅱ型胶原及Aggrecan基因表达量。结果　人肋软骨细胞从第 3代起逐渐向成纤维样细胞形态转换 ;每代软骨细胞Aggrecan中GAG含量随传代次数逐渐降低 ,第 3代后处于较低水平 ;Ⅰ、Ⅱ型胶原的表达在蛋白水平及mRNA水平基本一致 ,第 2代以前Ⅱ型胶原表达较强 ,Ⅰ型胶原表达较弱 ,之后随传代Ⅱ型胶原表达减弱 ,Ⅰ型胶原表达逐渐增强 ;而Aggrecan在第 3代以前表达较高 ,从第 4代后明显下降。结论　人软骨细胞体外培养老化过程中综合细胞扩增及细胞功能因素 ,第 2代的软骨细胞 (体外扩增约 18 32 6倍 )可作为构建人组织工程软骨的种子细胞。     

An immunohistochemical study of enthesis development in the medial collateral ligament of the rat knee joint

The changing distributions of collagens and glycosaminoglycans have been studied at the attachments of the medial collateral ligament during postnatal development. The ligament is of particular interest because it has a fibrocartilaginous attachment to the femoral epiphysis, but a fibrous one to the tibial metaphysis. Ligaments were examined in rats killed at birth and at 2, 4, 6, 8, 10, 20, 30, 45, 60, 90 and 120 days after birth. Cryosections were immunolabelled with monoclonal and polyclonal antibodies against types I and II collagen, chondroitin 4 and 6 sulfate, dermatan and keratan sulfate. Although the ligament is attached at both ends to bones that develop from cartilage, there was a striking difference in collagen labelling. Type II collagen was only found in spicules of calcified cartilage in bone beneath the tibial enthesis after ossification had commenced, but there was a continuous band of labelling at all stages of development at the femoral enthesis. Initially, the cartilage at the femoral attachment lacked type I collagen, but by 45 days labelling was continuous from ligament to bone. Continuity of labelling was seen much earlier at the tibial enthesis, as soon as bone had formed. There were also marked changes in glycosaminoglycan distribution. Keratan sulfate was present at both entheses up to 45 days, but only at the femoral enthesis thereafter. Both attachments labelled throughout life for dermatan sulfate, but chondroitin 4 and 6 sulfate were only found at the femoral end. The results suggest that enthesial cartilage at the femoral attachment was initially derived from the cartilaginous bone rudiment but was quickly eroded on its deep surface by endochondral ossification as bone formed at the attachment site. It was replaced by fibrocartilage developing in the ligament. This mechanism allows enthesis cartilage/fibrocartilage to contribute to the growth of a bone at a secondary centre of ossification in addition to dissipating stress at the ligament-bone junction.     

Sulfated glycosaminoglycans in amyloid plaques of prion diseases

Summary Brain sections from cases of human Creutzfeldt-Jakob disease, Gerstmann-Sträussler syndrome, kuru, and hamster scrapie containing amyloid were examined for the presence of sulfated glycosaminoglycans (GAGs), the anionic component of proteoglycans, using the sulfated Alcian blue method and Alcian blue technique with 0.3 M and 0.7 M magnesium chloride. These studies suggest that sulfated glycosaminoglycans are part of the CNS amyloid plaques in each of the above human prion disorders as well as in experimental scrapie. All the amyloid plaques stained positively with Alcian blue at 0.3 M, and less so at 0.7 M magnesium chloride indicating the presence of sulfated GAGs. Therefore, the amyloid plaques of prion diseases possess similar histochemical features to those found in Alzheimer's disease.Supported by Grant MT-3153 from the Medical Research Council of Canada, as well as research grants from the John Douglas French Foundation for Alzheimer's disease and the National Institutes of Health (NS22786, AG02132 and NS14069) and gifts from the R.J. Reynolds Industries, Sherman Fairchild Foundation, and Joseph and Stephaine Koret Foundation     

膀胱肿瘤患者尿中葡萄糖氨基聚糖的研究

检测２３例膀胱肿瘤患者尿中的葡萄糖氨基聚糖（ＧＡＧｓ）。尿中的ＧＡＧｓ总量和硫酸软骨素含量与对照组比较无明显变化，但在高级别肿瘤患者，其尿中的ＨＳ明显升高，若肿瘤发生浸润，则ＨＳ、ＨＡ均明显升高，肿瘤切除后，尿中ＨＳ、ＨＡ恢复正常，当尿中ＨＳ排泄量超过１．６ｍｇ／ｄ，则肿瘤预后不良；ＨＡ排泄量超过０．８ｍｇ／ｄ时，则肿瘤已发生了浸润。     

The nature of mucopolysaccharides

Anionic polyelectrolytes complex other ions, changing their physical and chemical properties and are very important for this reason in industry. Compounds of this type are known in medicine and have proved resistant to elucidation by usual biochemical procedures. The most widely used substance of this nature is heparin. By exploiting its anionic polyelectrolyte properties, it has been shown that heparin is an α-linked polymer of 2 disaccharide units of disulfoglucosamine-sulfoiduronic acid and one of disulfoglucosamine-glucuronic acid. Commercial heparin consists of 21 linear chains (of 2 to 22 hexasaccharides) with different biological activities. The related α-linked sulfated polymer, heparitin sulfate, has been shown to be a family of compounds (heparitins A, B, C, D). Chondroitin sulfates which are β-linked galactosamine — uronic acid polymers have now been shown to be an extensive series, the members of which differ in uronic acid and degree of sulfation (from those with little sulfate to multi-sulfated compounds resembling heparin). The semi-synthetic heparinoids also possess the same properties. It is proposed that mucopolysaccharides be used as the general term for anionic polyelectrolytes of biological and medical interest with heparins, heparitins, chondroitins, heparinoids, hyaluronic acids, as the main classes. An appropriate set of abbreviations is suggested for designating members of the classes.     

Glycosaminoglycans,proteins, and stone formation: adult themes and child’s play

The relative infrequency of renal stones in children is probably the main reason for the paucity of literature devoted to the study of urolithiasis in pediatric patients. Nonetheless, when pediatricians do address the issue, the contents of their papers reflect those prevalent in the adult literature; with one notable exception. Papers dealing with the potential role of urinary macromolecules in pediatric stone disease are very scarce indeed; to my knowledge, only four have been published in the English literature in the last 15 years. One of these is to be found in this issue and, like the remaining three, it compares the urinary excretion of glycosaminoglycans in healthy children and those with stones. This article briefly reviews the history of the association of urinary macromolecules, particularly glycosaminoglycans and proteins, with calcium oxalate urolithiasis, and discusses in more detail the published experimental evidence for their fulfilling a determinant role in stone formation. Received May 16, 1996; accepted May 28, 1996     

Effect of solubilized bone matrix components on cultured fibroblasts derived from neonatal rat tissues

Summary Decalcified rat bone matrix was extracted with 4M guanadinium chloride. Extensive dialysis of the solution yielded material which could be, in part, further solubilized in isotonic salt solution and which was particularly enriched in glycoprotein compared to whole bone matrix. The addition of this solubilized material to tissue culture media in concentrations of 25 to 100 g dry weight per milliliter of medium resulted in characteristic morphologic and metabolic alterations of fibroblasts from neonatal rats. These alterations in glycosaminoglycan synthesis were similar to those we had previously reported, resulting from the addition of whole, particulate bone matrix to such cultures. The most prominent change was an increase in hyaluronic acid synthesis and a smaller increase in chondroitin sulfate synthesis. Both intracellular synthesis and secretion into the medium were stimulated, and the magnitude of this stimulation was related to the concentration of the solubilized material in the medium. However, the age of the rat bone appeared to be also an important determinant of the amount of biological activity that could be solubilized. Bone appears to be greatly enriched in this biological activity compared to skin, and fibroblasts derived from neonatal rat muscle are relatively more responsive than fibroblasts derived from neonatal rat skin.     

Human cartilage proteoglycans isolated from normally ossifying and congenitally malformed leg bones

Summary Proteoglycans were extracted with 4 M guanidine HC1 solution containing protease inhibitors from various zones of human epiphyseal cartilages of the normally ossifying fibula and cartilaginous rudiment of the tibia of a 12-month-old boy with congenital absence of the tibia, when the knee disarticulation was performed. All the proteoglycan preparations from the epiphyseal cartilages were separated with a sucrose density gradient centrifugation into two components; a heavy, major component and a light one. The molecular size and the proportion of isomeric chondroitin sulfates of polysaccharides of the heavy component differed from those of the light one. The relative amounts of isomeric chondroitin sulfates in the polysaccharide moieties of the components also varied among these zones. The glycosaminoglycan content in the rudimentary tibia was equal to that of the epiphyseal cartilage of the fibula. However, proteoglycan preparations showed neither the normal sedimentation profile with two peaks nor the zonal differences as to the proportion of isomeric chondroitin sulfates. These results suggest that the alterations in proteoglycan metabolism might be involved in the pathogenetic mechanisms producing the congenital limb defect.