卵泡基膜中Ⅳ型胶原,层粘连蛋白来源的初步探讨

通过离体培养猪、大鼠卵泡颗粒细胞、内泡膜细胞，应用免疫组化技术，观察细胞外基质与卵泡细胞功能关系，初步探讨卵泡基膜中Ⅳ型胶原、层粘连蛋白的源及其生物学意义。结果表明：内泡膜细胞合成、分泌大量的ＩＶ型胶原，颗粒细胞不合成ＩＶ型胶原；颗粒和泡细胞合成、分泌大量ＬＮ。由于ＩＶ型型原属于不溶性蛋白，ＬＮ属于微溶性蛋白，因此卵泡基膜中的ＬＮ主要来源于基膜两侧的颗粒细胞、内泡膜细胞，ＩＶ型胶在娅源于内泡膜细胞     

三个细胞外基质成分在人卵泡分布的免疫组织化学观察

应用ＡＢＣ免疫组织化学方法，观察了细胞外基质的主要成分ＩＶ型胶原、纤维粘连蛋白和层粘连蛋白在人卵泡的分布。结果发现：ＩＶ型胶原分布在泡膜细胞和分隔颗粒细胞、泡膜细胞的基膜上，纤维粘连蛋白分布在卵泡颗粒细胞和泡膜细胞，而层粘连蛋白则分布在基膜以及基膜两侧的颗粒细胞和泡膜细胞。这３个细胞外基质成分分布规律不同，提示它们由不同细胞产生，并在卵泡发育的微环境中发挥着既互相协同又各自独特的作用，对卵泡发育及功能具有重要意义。     

纤维粘连蛋白与卵泡发育关系的探讨

采用酶联免疫吸附试验(ELISA)法和 ABC 免疫组化法对人卵巢内纤维粘连蛋白(FN)的分布、层粘连蛋白(LN)的分布、FN 与激素的关系进行了观察。结果表明:FN 水平药物诱发排卵的卵泡液(321.3+16.6mg/L)与正常卵泡液(376.9±49.1mg/L)差异无显著性(P>0.05),与同期静脉血清FN(325.0+34.2mg/L)差异亦无显著性(P>0.05),卵泡液 FN 与 E_2水平呈负相关(r=-0.254,P<0.05),FN 与其他激素无相关性(P>0.05)。由此推论:随着卵细胞成熟,E_2增高,FN 降低;FN 与卵泡颗粒细胞分化有关,FN 降低可使卵细胞周围卵丘细胞疏散,有利于卵子成熟与排出。     

层粘连蛋白、纤粘连蛋白、IV型胶原在人月经周期子宫内膜中的表达

目的　研究细胞外基质蛋白层粘连蛋白 (LN)、纤粘连蛋白 (FN)和IV型胶原 (IVCL)在正常人月经周期子宫内膜中的表达。　方法　采用免疫组织化学方法检测 4 9例正常月经周期子宫内膜中LN、FN、IVCL蛋白的表达。　结果　LN主要分布于腺上皮及腔上皮基底膜 ,分泌中期表达最强。FN主要分布于间质 ,腔上皮及腺上皮基底膜也均有表达 ,分泌中期含量丰富。IVCL在腺上皮基底膜的表达分泌中期最强 ,在间质的表达分泌早期最强 ,分泌中、晚期表达逐渐减弱。LN、FN、IVCL在血管基底膜上均有表达 ,无周期性变化。　结论　LN、FN、IVCL在月经周期子宫内膜中的表达随月经周期而变化。     

米非司酮对早孕蜕膜组织中IV型胶原及纤维粘连蛋白的影响

目的 :探讨米非司酮 (Ru486 )对早孕蜕膜组织中 IV型胶原、纤维粘连蛋白(FN)的影响 ,进一步认识米非司酮的抗早孕机理。方法 :孕 <6 0 d妇女 ,随机分为对照组和药物组。药物组口服米非司酮总量为 1 5 0 mg。采用免疫组织化学 (ABC)法测定早孕蜕膜组织 IV型胶原 (对照组 1 5例 ,药物组 2 0例 )和纤维粘连蛋白 (对照组 2 5例 ,药物组 30例 )的分布状况。结果 :与对照组相比 ,药物组早孕蜕膜组织中 IV型胶原、FN均明显减少(P<0 .0 1 ,P<0 .0 5 )。尤其是 IV型胶原在腺体基膜 ,FN在蜕膜细胞间质中降低最为明显 (P<0 .0 1 ) ,上皮基膜、血管周围 IV型胶原含量也明显降低 (P<0 .0 5 )。结论 :米非司酮明显影响早孕蜕膜组织中这两种细胞外基质 (ECM)的含量及分布 ,可使 IV型胶原、FN的含量减少 ,而起到抗早孕作用。早孕蜕膜组织中这两种 ECM成分可能受孕激素及糖皮质激素的调控。     

III型前胶原、血清透明质酸和层粘连蛋白在糖尿病肾病中的临床意义

目的:探讨III型前胶原、血清透明质酸和层粘连蛋白在糖尿病肾病中的临床意义. 方法:11例糖尿病肾病病人,15例慢性肾炎病人采血测定血肌酐(SCr),血尿素氮(BUN)等生化指标以及III型前胶原(procol lagen typeIII, PCIII)、血清透明质酸(hyaluronic acid, HA)、层粘连蛋白(LN)等放免指标与正常组进行对比.结果:与正常对照组比较※P<0.01,糖尿病肾病组与正常对照组HA、LN、PCIII比较有显著性差异(P<0 01).慢性肾炎与正常组比较无显著差异(P>0.05).结论:HA、LN、PCIII在对糖尿病肾病的进程中有一定的作用,并针对情况进行有效防治对预防终末期肾病有着十分重要的意义.     

透明质酸、层粘连蛋白及Ⅳ型胶原水平与肾移植急性排斥反应的关系

目的探讨移植肾组织及血清中透明质酸(HA)、层粘连蛋白(LN)及Ⅳ型胶原(C_(-Ⅳ))含量与急性排斥反应的关系。方法以雄性SD大鼠为供者,Wistar大鼠为受者,采用改进的Blom法制作大鼠原位左肾移植模型,设同种移植对照组、同种移植用药组(术后腹腔注射环孢素A 10 mg·kg~(-1)·d~(-1)),另设同系移植组(供、受者均为Wistar大鼠)和假手术组(Wistar大鼠仅切除右肾,不行移植),每组20只大鼠。每组分别于术后3、5、7及9 d各随机选取5只大鼠,从下腔静脉采血2 ml,随后切取移植肾(假手术组切取左侧肾脏),用放射免疫分析法测定肾组织匀浆及血清中HA、LN及C_(-Ⅳ)的含量,并行肾组织切片,根据“Banff 97”标准进行病理诊断。结果从术后5d开始,同种移植对照组的肾组织匀浆及血清中HA、LN及C_(-Ⅳ)的浓度均显著高于同系移植组、假手术组及同种移植用药组(P＜0．01)；血清中HA、LN及C_(-Ⅳ)的浓度与肾组织匀浆中HA、LN及C_(-Ⅳ)的浓度呈正相关(r分别为0．960、0．934和0．847,P值均为0．000)。病理检查证实,同种移植对照组共有17个肾脏发生急性排斥反应,同种移植用药组共有4个肾脏发生急性排斥反应,它们的Banff急性排斥反应指数与移植肾组织匀浆中及血清中HA、LN及C_(-Ⅳ)的浓度均呈正相关(P＜0．01)。结论HA、LN及C_(-Ⅳ)在肾移植急性排斥反应的发生及发展中起重要作用,肾移植术后监测血清中HA、LN及C_(-Ⅳ)的浓度有助于急性排斥反应的诊断。     

肾纤康汤对系膜增生性肾小球肾炎患者血清层粘连蛋白及Ⅳ型胶原的影响

目的：探讨经验方肾纤康汤对系膜增生性肾小球肾炎（Mesangial Proliferative Glomerulonephritis MsPGN）患者血清细胞外基质（Extacelluar Matrix ECM）成分的抑制作用。方法：60例MsPGN患者随机分成肾炎对照组、苯那普利组和肾纤康组，治疗前后采用放射免疫法检测血肖层粘连蛋白（Laminin LN）和Ⅳ型胶原（Collagen Ⅳ Col-Ⅳ）并观察各组差异。结果：MsPGN患者血清LN、Col-Ⅳ水平显著高于健康对照组（P＜0.01）。肾纤康汤能明显下调MsPGN患者血清LN和Col-Ⅳ水平（P＜0.01），其作用强度珉本那普利相近（P＞0.05）。结论：肾纤康汤能减少MsPGN患者血清LN、Col-Ⅳ的水平，其效果和作用机制有待于进一步的研究。     

Glomerular basement membrane type IV collagen antigens in Goodpasture's and Alport's syndromes

Type IV collagen, the main constituent of the renal glomerular basement membrane, is involved in Goodpasture's syndrome, and autoimmune disease, and in Alport's syndrome, a genetic disease. There are 6 alpha chains, α1(IV) through α6(IV), in type IV collagen in mammals. Immunohistochemical studies, using α-chain-specific monoclonal antibodies on tissue specimens from healthy people and patients with Alport's syndrome, have shown that there are 3 forms of type IV collagen molecules in mammalian basement membranes, namely α1/α1/α2, α3/α4/α5, and α5/α5/α6. Antibody specificity analysis of sera from patients with Goodpasture's syndrome show that all sera have autoantibody, with the highest titer against α3(IV)NC1, although they also have titers against the other 5α chains. This indicates that α3(IV)NC1 is the major target antigen of the disease, although the glomerular basement membrane contains the α1 through α5 chains. Experimental glomerulonephritis in rats, induced by the injection of 6 recombinant α(IV)NC1s with an adjuvant, has shown that α3(IV)NC1 and α4(IV)NC1 are nephritogenic. The lack of, or very poor, nephritogenicity of the other 4 α(IV)NC1s can be explained by the high immunologic tolerance against these chains, which are distributed widely in basement membranes of the whole body. This paper was presented at the 2nd International Forum “The Frontiers of Nephrology,” Tokyo, May 10, 1998.     

Alport syndrome,basement membranes and collagen

Alport syndrome, an inherited disorder of the kidney, eye and ear, has fascinated nephrologists, pathologists, and geneticists for nearly a century. With the recent application of molecular biochemical and genetic techniques, this mysterious disease has begun to yield some of its secrets. Alport syndrome can now be viewed as a generalized disorder of basement membranes that appears to result from mutations in an X-chromosome-encoded basement membrane collagen chain. This chain, along with two other novel collagen chains, is absent from Alport basement membranes, in contrast to the classical chains of collagen IV. Phenotypic heterogeneity in Alport syndrome probably arises from allelic mutations at a single genetic locus. The phenomenon of post-transplant anti-glomerular basement membrane nephritis may be a manifestation of specific mutations at the Alport locus that prevent synthesis of the gene's protein product and the establishment of immunological tolerance.     

基膜黏连蛋白和Ⅳ型胶原对培养许旺细胞贴壁及增殖的作用

目的：了解基膜黏连蛋白和Ⅳ型胶对体外培养条件下许旺细胞的贴壁及增殖作用的影响。方法：用基膜黏连蛋白和Ⅳ型胶原包被培养板，以结合了相应抗体的培养板及包被Ⅰ型胶原和多聚赖氨酸的培养板为对照，将培养的许旺细胞以相同浓度加入各组培养板内。测定2、6、24h细胞贴壁率，培养72h进行^3H-TdR掺入试验，双道液体闪烁计数器测定各组的放射性计数。结果：基膜黏连蛋白和Ⅳ型胶原组早期贴壁率较Ⅰ型胶原和多聚赖氨酸组高，抗基膜黏连蛋白和抗Ⅳ型胶原且贴壁率较低。基膜黏连蛋白和Ⅳ型胶原组^3H-TdR掺入量高于其它组。结论：基膜黏连蛋白和Ⅳ型胶原对体外培养条件下的许旺细胞贴壁及增殖有促进作用。     

Laminin modified infection-preventing collagen membrane containing silver sulfadiazine-hyaluronan microparticles

The newly developed laminin modified infection-preventing collagen membrane consists of a 3 component laminate, comprising 2 outer collagen layers and a central laminin layer. The 2 outer collagen layers (dense and porous layers) were fabricated by air-drying and freeze drying, respectively, and the laminin layer was formed by a straightforward liquid coating method. In addition, hyaluronan based microparticles containing silver sulfadiazine (AgSD) were incorporated into the 2 collagen layers (AgSD content 50 microg/cm2). Laminin coated collagen surfaces did not promote fibroblast attachment but showed a retarded fibroblast proliferation rate and an increased rate of collagen synthesis versus pure collagen surfaces. In an animal study, a laminin coating on a nonmedicated collagen membrane significantly increased both wound size reduction and vessel proliferation 7 days after application versus polyurethane film. Interestingly, the laminin coated AgSD medicated collagen membrane demonstrated higher wound size reduction and vessel proliferation and lower inflammation than the polyurethane control, suggesting that the laminin AgSD medicated collagen membrane substantially improves dermal wound healing.     

增生性瘢痕中纤维连接蛋白和层粘连蛋白的分布

取临床病人的增生性瘢痕和相邻的正常皮肤作组织切片，分别行纤维连接蛋白（ｆｉｂｒｏｎｅｃｔｉｏｎ，ＦＮ）免疫组织化学染色和层粘连蛋白（ｌａｍｉｎｉｎ，ＬＮ）免疫组织化学染色。结果发现：①增生性瘢痕中，基底膜ＦＮ呈弱阳性；真皮全层由浅及深ＦＮ呈弱阳性－强阳性－弱阳性分布；中层含有大量ＦＮ阳性的成纤维细胞；部分瘢痕最深层ＦＮ分布呈“树根”状突入皮下组织内。提示增生性瘢痕增生最旺盛的部位在中层。真皮ＦＮ阳性与否和强弱是判断瘢痕增生程度的一种指标。②增生性瘢痕中基底膜ＬＮ呈阳性；真皮内分布有密度不均、疏松紊乱、呈条索状或丛状的ＬＮ阳性结构。增生性瘢痕真皮内ＬＮ的存在可能是瘢痕长期增生的另一个原因。     

Molecular mechanisms in basement membrane complications of diabetes. Alterations in heparin, laminin, and type IV collagen association

We report alterations in the ligand-binding properties of laminin, the major noncollagenous protein of basement membranes, resulting from nonenzymatic glycosylation in vitro. Mouse laminin was incubated in vitro with 500 mM glucose, and the level of nonenzymatically glycosylated amino acids increased 2-, 6.2-, and 12-fold after incubation for 1, 3, and 12 days, respectively. Ligand binding assays conducted in solution with varying concentrations of [3H]heparin and a constant amount of control or nonenzymatically glycosylated laminin showed a reduction in heparin binding proportional to laminin glycosylation. An analysis of the stoichiometry of [3H]heparin binding to control and nonenzymatically glycosylated laminin at saturating levels of heparin was performed. The results indicated that the total number of heparin binding sites on laminin decreased 4.7- and 14.7-fold due to nonenzymatic glycosylation of laminin for 1 and 3 days, respectively. [3H]heparin binding to 12-day nonenzymatically glycosylated laminin was abolished. Scatchard analyses of the binding data for heparin and laminin gave a dissociation constant (Kd) of 3.4 X 10(-8) M. The data for nonenzymatic glycosylation of laminin for 1 day in vitro resulted in a biphasic heparin binding curve. Both high- and low-affinity binding was observed (Kd values of 3.3 x 10(-8) and 2.6 x 10(-7) M, respectively). Similar high- and low-affinity binding sites were seen on 3-day nonenzymatically glycosylated laminin (Kd values of 2.1 x 10(-8) and 3.9 x 10(-7) M, respectively). [3H]heparin binding at a fixed concentration to a constant amount of control or 12-day nonenzymatically glycosylated laminin and type IV collagen was also studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural basement membrane topography of the bladder epithelium

The basement membrane underlies epithelium and separates it from deeper tissues. Recent studies suggest that nanoscale topography of the surface of basement membrane may modulate adhesion, migration, proliferation and differentiation of overlying epithelium. This study was performed to elucidate nanoscale topographic features of basement membrane of the bladder. Bladder tissues were obtained from three adult female rhesus macaques. A process was developed to remove the epithelium while preserving the underlying basement membrane, and tissues were evaluated by immunohistochemistry and scanning electron microscopy (SEM). Detailed measurements were made of stereo SEM images to quantitatively define topographic features. Measurements made from multiple SEM images of bladder basement membrane provided the following values for topographic features: mean feature height, 178±57 nm; mean fiber diameters, 52±28 nm; mean pore diameter, 82±49 nm; and mean interpore distance (center to center), 127±54 nm. These dimensions are similar to those reported previously for basement membranes of other species and anatomical locations. This information provides a rational basis for design of nanostructured biomaterials to produce composite grafts for repair or replacement of segments of the urinary tract.