纤维粘连蛋白及层粘连蛋白与肿瘤细胞的浸润和转移

纤维粘连蛋白(fibronectin,FN)及层粘连蛋白(laminin,LN)是近年来受到广泛重视的细胞外基质非胶原糖蛋白。FN主要由中胚层来的细胞产生,亦可由上皮细胞及内皮细胞生成;LN则主要由上皮细胞及内皮细胞合成。FN以可溶及不溶两种形式分别存在于各种体液及细胞外基质(包括某些基膜);LN则以不溶的形式     

层粘连蛋白糖肽对实体型瘤细胞粘着于层粘连蛋白基质的影响

 本文报告由小鼠EHS瘤提取的层粘连蛋白(Laminin,LN)经链霉蛋白酶(Pronase)消化,再经Sephadex-G50层析分离得到LN总糖肽。它可显著抑制黑色素瘤细胞B16-MBK及S180肉瘤细胞与LN基质的识别及粘着,并具有明显剂量依赖性。与五肽(YIGSR),卵清蛋白及其糖肽,胎球蛋白及其糖肽比较,LN总糖肽的抑制效果显著高于YIGSR及胎球蛋白糖肽,而其它三者均无抑制作用。因而提示:LN分子中一定结构的糖链特异地参与了LN对肿瘤细胞表面LN受体的识别与结合。     

层粘连蛋白(LN)和纤维连接蛋白(FN)在植入期地鼠子宫内膜中的表达——形态学和免疫组织化学研究

选用未孕及怀孕4－8天地鼠子宫内膜,用组织学、免疫组织化学染色及图像分析法,研究植入前后地鼠子宫内膜中LN、FN分布及含量的变化,探讨其与植入过程的相关关系。结果：在未孕地鼠,LN主要分布于宫内膜各类上皮基膜内,FN存在于基膜及细胞外基质中。孕4－8天,各类上皮基膜中LN、FN表达明显增多,与未孕地鼠相比,差异显（P＜0.01）。随着蜕膜形成,蜕膜（基质）细胞周围LN及FN表达增多。上述结果表明,LN、FN与植入过程及蜕膜功能密切相关。     

小鼠Lewis肺癌组织中糖肽的分离纯化及其对肿瘤细胞与层粘连蛋白基质粘着的影响

小鼠Lewis肺癌组织经氯仿甲醇去除脂类,用木瓜蛋白酶消化,再经Sephadex柱层析分离得到总糖肽。它有明显地抑制小鼠Lewis肺癌细胞,S180肉瘤细胞及人巨细胞肺癌细胞与层粘连蛋白(Laminin,LN)基质粘着的作用;对层粘连蛋白受体(LN-R)与其配体的识别及结合也具有同样明显的阻断效应。糖肽的上述作用均具有剂量依赖性。进一步经ConA-Sepharose CL-4B亲和层析将总糖肽分为三个部分。与ConA不结合的糖肽部分对Lewis肺癌细胞与LN基质的粘着也具有剂量性抑制作用。     

层粘连蛋白与疾病相关性的研究进展

层粘连蛋白(lanminin,LN)是由3条多肽链组成的异源三聚体,是基膜的主要成分,存在于多种组织中,它可识别细胞表面受体从而使基膜与细胞紧密结合。LN参与基膜的构建及细胞的黏附、生长、增殖、迁移和分化,是机体正常生长发育所必需的。LN的不正常表达常常与肿瘤以及皮肤、神经-骨骼肌、肝、肾等组织疾病的发生发展密切相关。通过深入探讨LN与疾病及肿瘤发生、发展的关系,有望为相关疾病及肿瘤的治疗开拓新的思路。     

Ⅰ型胶原、内皮素对肾小球内皮细胞及系膜细胞产生细胞外基质的影响

本文观察了Ⅰ型胶原、内皮素对肾小球内皮细胞、系膜细胞增殖的影响,同时探讨了Ⅰ型胶原和内皮素对培养的内皮细胞及系膜细胞产生层粘连蛋白、纤连蛋白和Ⅳ型胶原的影响。结果提示:Ⅰ型胶原可以明显促进内皮细胞的增殖(P<0.01),内皮素对系膜细胞的增殖有一定作用:Ⅰ型胶原可以促进内皮细胞产生层粘连蛋白和纤连蛋白,并可以促进系膜细胞产生Ⅳ型胶原;内皮素可以促进系膜细胞产生FN增多(P<0.05)。     

小鼠Lewis肺癌组织中层粘连蛋白受体的分离及其性质的研究

层粘连蛋白(Laminin,LN)是基膜(basement membrane)中的一种主要大分子糖蛋白。一些研究资料表明肿瘤细胞的浸润转移可能与LN有关。肿瘤细胞与LN的作用可能是通过细胞表面LN受体进行的。本文采用亲和层析法从小鼠Lewis肺癌组织中分离LN受体并对其理化性质进行研究。Lewis肺癌LN受体的表观分子量为70,000,还原后SDS电泳图为一条较宽的条带。氨基酸组成中疏水氨基酸占38％,苏氨酸、絲氨酸、门冬氨酸(包括门冬酰胺)占23.5％,通过硝酸纤维素膜片法用HRP-LN测定受体与LN的结合特性,证明具有配基结合专一性,饱和性及高亲和性(Kd=0.95×10~(-9)mol/L)。     

双峰驼睾丸和隐睾细胞外基质相关蛋白的组织化学特征及超微结构比较

为探索细胞外基质相关蛋白在隐睾双峰驼的分布情况及其组织化学特征,应用电镜技术和多种组织化学方法比较了隐睾和正常睾丸的超微结构,组织化学特点及层粘连蛋白（LN）、Ⅳ型胶原（Col Ⅳ）和硫酸乙酰肝素糖蛋白（HSPG）的分布特征。结果显示：(1)与正常睾丸间质结构相比,光镜下隐睾生精小管发育不全,间质内胶原纤维稀疏,网状纤维分布明显,间质血管及生精小管固有膜PAS及AB-PAS阳性反应较弱。电镜下,隐睾生精上皮基膜明显增生,外围I型胶原纤维较少,管周肌样细胞不典型;间质毛细血管及Leydig细胞周围纤维细胞多见,而正常睾丸在间质毛细血管及Leydig细胞周围多分布有成纤维细胞。(2) 免疫组织化学染色显示,正常睾丸组织的Col Ⅳ、LN及HSPG在Leydig细胞内均为强阳性表达,Col Ⅳ和LN在毛细血管内皮细胞强阳性表达,后者在Sertoli细胞的表达尤为明显,HSPG在精原细胞无表达;隐睾时Col Ⅳ、LN及HSPG在Leydig细胞内阳性表达均明显减弱,Col Ⅳ、LN在管周肌样细胞及毛细血管内皮细胞阳性表达也减弱明显,HSPG在精原细胞较强阳性表达,且在精子细胞呈强阳性表达。免疫组织化学图像分析结果显示,双峰驼正常睾丸组织中Col Ⅳ和LN的分布显著高于隐睾组织（P<0.05）,HSPG检测结果在正常睾丸与隐睾之间无统计学差异（P>0.01）。该研究表明,双峰驼隐睾生精小管发育异常,间质组织中合成胶原纤维的能力下降,睾丸细胞外基质的重要成分Col Ⅳ,LN与正常组差异显著与生精小管及Leydig细胞异常发育有关,而HSPG在隐睾生精上皮的强阳性表达与精原细胞发育不成熟密切相关。     

人工基膜对鼻咽癌上皮细胞株（CNE—2）生长的影响

人工基膜（ＡＢＭ）主要以Ⅰ型胶原的水合性胶原丝网为网架,辅上纤维连结蛋白,Ⅳ型胶原和层粘连蛋白等主要基膜糖蛋白制备而成,具海绵状的形态结构。ＡＢＭ可减少胎牛血清用量１０％,提高细胞生活力和延长细胞传代周期。在２－５％血清浓度的情况下,ＡＢＭ可提高ＣＮＥ－２细胞的生长效率,克隆形成率和克隆生长率而抑制细胞的＾３Ｈ－ＴｄＲＡ掺入。提示在体外研究细胞外基质对细胞的影响时应使用低血清培养液。ＡＢＭ是体外诱     

Ⅰ型胶原,内皮素对肾小球内皮细胞及系膜细胞产生细胞外基…

本文观察了Ⅰ型胶原，内波素对肾小球内皮细胞、系膜细胞增殖的影响，同时探讨了Ⅰ型胶原和内皮素对培养的内皮细胞及系膜细胞产生层粘连蛋白、纤连蛋白和Ⅳ型胶原的影响。结果提示：Ⅰ型胶原可以明显促进内皮细胞的增殖，内皮素对系膜细胞的增殖有一定作用；Ⅰ型胶原可以促进内皮细胞产生层粘连蛋白和纤连蛋白，并可以促进系膜细胞产生Ⅳ型胶原；内皮素可以促进系膜细胞产生ＦＮ增多。     

HeLa cell adhesion to microcarriers in high shear conditions: evidence for membrane receptors for collagen but not laminin or fibronectin

HeLa-S3 cells were analyzed for their ability to attach and spread on cell culture microcarriers that were made either positively or negatively charged with polymeric plastics or were coated with BSA, gelatin, fibronectin or laminin. The cells stuck to all microcarriers under low shear, i.e. low stirring conditions with similar rates of attachment. Except in the case of gelatin microcarriers where cells fully spread, cells did not or only partially spread on the others. Under high shear, cells attached with the following rates: positive = negative = gelatin = BSA greater than laminin greater than fibronectin. Cells detached from all but the gelatin and BSA coated beads. However, the cells did not fully spread on BSA beads. The observation that cells not only attached but also spread on gelatin beads indicated that gelatin could be a specific substratum adhesion protein while the other surfaces were 'non-specific'. It should be noted that neither antibodies to laminin nor fibronectin interfered with attachment to gelatin. Protein synthesis inhibitors reduced the attachment and spreading on gelatin beads under high but not low shear conditions. With low shear, attachment and spreading appeared normal. We concluded that the density of the cell surface attachment proteins was reduced by the protein synthesis inhibitors and there were not enough present to facilitate attachment under high shear. The results also indicated that protein synthesis was not essential for cell spreading. Proteolysis of the cell surface with low concentrations of trypsin abolished the attachment of cells to gelatin-coated beads. The reappearance of attachment ability took several hours and was inhibited by actinomycin-D.     

Concomitant loss of cell surface fibronectin and laminin from transformed rat kidney cells

Both fibronectin and laminin were found by immunofluorescence as a matrix at the surface of normal rat kidney cells. These matrices were absent from the surface of virally transformed rat kidney cells. Soluble fibronectin and laminin were detected in the culture media of the transformed as well as the normal cells. Culture supernates of the transformed cells contained even more fibronectin than the supernates of the transformed cells contained even more fibronectin than the supernates of the normal cells while laminin was present in similar amounts in both culture media. This shows that the loss of fibronectin and laminin from the surface of the transformed cells is caused by failure of the cells to deposit these proteins into an insoluble matrix and not caused by inadequate production. Fibronectins isolated from culture media of the normal and transformed cells were similar in SDS polyacrylamide gel electrophresis. Laminin isolated from culture media by affinity chromatography on heparin-Sepharose followed by immunoprecipitation was composed of three main polypeptides, one with a molecular weight of 400,000 and two with a molecular weight close to 200,000 in both cell types. Fibronectins from both cell types were equally active in promoting cell attachment. Rat fibronectin from transformed cells, like normal cells, when applied to culture dishes coated with fibronectin, readily attached and spread on the substratum, requiring approximately the same amount of fibronectin as the normal cells. On the basis of these results it seem that the failure of the transformed cells to incorporate fibronectin into an insoluble cell surface matix is not a consequence of a demonstrable change in the functional characteristics of the fibronectin molecule or in the ability of the cells to interact with fibronectin. It may depend on as yet unidentified interactions of the cell surface. Similar interactions may be needed for the deposition of laminin into the matrix, because laminin was also absent from the surface of transformed cells, despite its being synthesized by these cells.     

Spreading of cells on various substrates evaluated by Fourier analysis of shape

In order to evaluate in mathematical terms the morphological changes occurring in the course of cell spreading, Fourier analysis of shape was applied. Human urothelial Hu 961 b cells plated on type IV collagen, fibronectin, laminin, glass and bovine serum albumin (BSA) were studied. Fourier parameters describing cell shape as well as surface areas covered by the cells on the substrate were subjected to statistical analysis. Using analysis of variance and discriminant analysis it was found that parameters describing cell shape (both gross shape of cells and their fine scale contour foldings) possessed a higher power of discrimination between the cells spread on various substrates than the differences in cell surface areas. In the course of observation (75 and 150 min) the highest number of attached cells and highest degree of spreading were found when cells were plated on type IV collagen. Moderate alterations in cell shape and moderate increase of surface area were seen in the group of cells seeded on fibronectin, whereas the cells plated on laminin, glass and BSA revealed a moderate increase of surface area, but no changes in their shape were observed. The differences in attachment of cells and in the degree of their spreading might be due to the variation in expression of plasma membrane receptors for various substrates. The Fourier analysis of cell shape coupled with measurement of surface area is a good tool for quantitative evaluation of cell spreading and can be used for discrimination between cells spread on different substrates.     

Antagonistic effects of laminin and fibronectin in cell-to-cell and cell-to-matrix interactions in MCF-7 cultures

Summary During morphogenesis, tumor progression and metastasis, cell adhesion, dissociation, and migration result from a complex balance between cell-to-cell and cell-to-matrix interactions. Two different organization patterns of MCF-7 cells were induced by different extracellular matrix proteins. When plated on plastic or polymeric type I collagen gel used as a model of interstitial matrix, MCF-7 cells spread and grew in monolayer. When cultured on a solid gel of basement membrane (BM) proteins (85% laminin) used as a model of BM, cells formed clusters attached to the matrix. Matrix proteins regulated these two types of cell organization by preferentially promoting cell-to-cell or cell-support interactions. On plastic in the presence of soluble laminin or on laminin-coated dishes, cells also formed clusters. Addition of soluble fibronectin induced spreading of the cells, suggesting that laminin and fibronectin have competitive antagonistic effects on MCF-7 cell morphology. Antilaminin antibodies inhibited cluster formation and attachment, emphasizing the important role of this glycoprotein not only in promoting cluster attachment but also in cell-to-cell contact formation. Such effects of extracellular matrix proteins could play significant roles in tumor progression and metastasis. This work was supported by grants 3.4512.85 and 3.4514.85 from the Belgian Fonds de la Recherche Scientifique Médicale and the Fonds Cancérologique de la CGER.     

Spreading of cells on various substrates evaluated by fourier analysis of shape

Summary In order to evaluate in mathematical terms the morphological changes occurring in the course of cell spreading, Fourier analysis of shape was applied. Human urothelial Hu 961 b cells plated on type IV collagen, fibronectin, laminin, glass and bovine serum albumine (BSA) were studied. Fourier parameters describing cell shape as well as surface areas covered by the cells on the substrate were subjected to statistical analysis. Using analysis of variance and discriminant analysis it was found that parameters describing cell shape (both gross shape of cells and their fine scale contour foldings) possessed a higher power of discrinunation between the cells spread on various substrates than the differences in cell surface areas. In the course of observation (75 and 150 min) the highest number of attached cells and highest degree of spreading were found when cells were plated on type IV collagen. Moderate alterations in cell shape and moderate increase of surface area were seen in the group of cells seeded on fibronectin, whereas the cells plated on laminin, glass and BSA revealed a moderate increase of surface area, but no changes in their shape were observed. The differences in attachment of cells and in the degree of their spreading might be due to the variation in expression of plasma membrane receptors for various substrates. The Fourier analysis of cell shape coupled with measurement of surface area is a good tool for quantitative evaluation of cell spreading and can be used for discrimination between cells spread on different substrates.Supported in part by a grant (MZ-XIV) from the Polish Ministry of Health and Welfare     

Cranial and trunk neural crest cells use different mechanisms for attachment to extracellular matrices.

We have used a quantitative cell attachment assay to compare the interactions of cranial and trunk neural crest cells with the extracellular matrix (ECM) molecules fibronectin, laminin and collagen types I and IV. Antibodies to the beta 1 subunit of integrin inhibited attachment under all conditions tested, suggesting that integrins mediate neural crest cell interactions with these ECM molecules. The HNK-1 antibody against a surface carbohydrate epitope under certain conditions inhibited both cranial and trunk neural crest cell attachment to laminin, but not to fibronectin. An antiserum to alpha 1 intergrin inhibited attachment of trunk, but not cranial, neural crest cells to laminin and collagen type I, though interactions with fibronectin or collagen type IV were unaffected. The surface properties of trunk and cranial neural crest cells differed in several ways. First, trunk neural crest cells attached to collagen types I and IV, but cranial neural crest cells did not. Second, their divalent cation requirements for attachment to ECM molecules differed. For fibronectin substrata, trunk neural crest cells required divalent cations for attachment, whereas cranial neural crest cells bound in the absence of divalent cations. However, cranial neural crest cells lost this cation-independent attachment after a few days of culture. For laminin substrata, trunk cells used two integrins, one divalent cation-dependent and the other divalent cation-independent (Lallier, T. E. and Bronner-Fraser, M. (1991) Development 113, 1069-1081). In contrast, cranial neural crest cells attached to laminin using a single, divalent cation-dependent receptor system. Immunoprecipitations and immunoblots of surface labelled neural crest cells with HNK-1, alpha 1 integrin and beta 1 integrin antibodies suggest that cranial and trunk neural crest cells possess biochemically distinct integrins. Our results demonstrate that cranial and trunk cells differ in their mechanisms of adhesion to selected ECM components, suggesting that they are non-overlapping populations of cells with regard to their adhesive properties.     

Interleukin 6 promotes epithelial migration by a fibronectin-dependent mechanism.

We investigated the effect of interleukin 6 (IL-6) on the migration of rabbit corneal epithelium in vitro and on the attachment of dissociated corneal epithelial cells to a fibronectin matrix. When corneal blocks were cultured with IL-6 for 24 hours, the length of the path of epithelial migration over exposed corneal stroma increased significantly (p less than 0.005 at the concentration of 10 ng/ml) in proportion to the concentrations of IL-6 (0.1-10.0 ng/ml). The addition of antiserum against fibronectin or of GRGDSP abolished the stimulatory effect of IL-6 on epithelial migration. When corneal epithelial cells were cultured with various concentrations of IL-6, suspended, and plated on wells coated with fibronectin (10 micrograms/ml), the number of cells attached to the wells increased in a dose-dependent manner. The presence of antibody against fibronectin or of GRGDSP during the attachment assay decreased the number of cells attached to the fibronectin matrix, regardless of the fact that the cells had been cultured with IL-6 or not. IL-6 stimulated the attachment of corneal epithelial cells to collagen type IV and to laminin matrices. However, the presence of GRGDSP did not affect the cell attachment to collagen type IV and to laminin. These findings strongly indicate that IL-6 stimulates epithelial migration in the cornea by a fibronectin-dependent mechanism, presumably the increased expression of fibronectin receptors.     

Embryonic neural retinal cell response to extracellular matrix proteins: developmental changes and effects of the cell substratum attachment antibody (CSAT)

Cell attachment and neurite outgrowth by embryonic neural retinal cells were measured in separate quantitative assays to define differences in substrate preference and to demonstrate developmentally regulated changes in cellular response to different extracellular matrix glycoproteins. Cells attached to laminin, fibronectin, and collagen IV in a concentration-dependent fashion, though fibronectin was less effective for attachment than the other two substrates. Neurite outgrowth was much more extensive on laminin than on fibronectin or collagen IV. These results suggest that different substrates have distinct effects on neuronal differentiation. Neural retinal cell attachment and neurite outgrowth were inhibited on all three substrates by two antibodies, cell substratum attachment antibody (CSAT) and JG22, which recognize a cell surface glycoprotein complex required for cell interactions with several extracellular matrix constituents. In addition, retinal cells grew neurites on substrates coated with the CSAT antibodies. These results suggest that cell surface molecules recognized by this antibody are directly involved in cell attachment and neurite extension. Neural retinal cells from embryos of different ages varied in their capacity to interact with extracellular matrix substrates. Cells of all ages, embryonic day 6 (E6) to E12, attached to collagen IV and CSAT antibody substrates. In contrast, cell attachment to laminin and fibronectin diminished with increasing embryonic age. Age-dependent differences were found in the profile of proteins precipitated by the CSAT antibody, raising the possibility that modifications of these proteins are responsible for the dramatic changes in substrate preference of retinal cells between E6 and E12.     

Effect of serum,fibronectin, and laminin on adhesion of rabbit intestinal epithelial cells in culture

Rabbit intestinal epithelial cells, obtained after a limited hyaluronidase digestion, were incubated in medium with or without calf serum, on bacteriological plastic dishes. The dishes, either plain or coated with an air-dried type I collagen film, were pretreated with medium alone or with medium containing purified laminin or purified fibronectin. Cells did not attach in significant numbers to untreated bacteriological plastic, even in the presence of serum. Cells did attach to collagen-coated dishes, and were judged viable on the basis of their incorporation of radiolabeled leucine into cell protein. Cell adhesion to the collagen substrate increased in proportion to the concentration of serum in the medium, with maximal attachment at 5% serum or greater. Pretreatment of plain or collagen-coated dishes with increasing amounts of fibronectin enhanced cell adhesion in a concentration-dependent manner. Either serum, or fibronectin-free serum in the medium enhanced cell attachment to substrates pretreated with cither fibronectin or laminin. Thus, intestinal epithelial cells appear to possess surface receptors for both laminin and fibronectin. The evidence further suggests that calf serum may contain factors, other than fibronectin, capable of enhancing intestinal epithelial cell attachment to collagen substrates.     

Effects of laminin, fibronectin and type IV collagen on liver cell cultures

The effects on mouse liver cells of laminin, fibronectin and type IV collagen, all of which are the main matrix of the basement membrane, were studied. Laminin, a glycoprotein isolated from cultures of rat yolk sac carcinoma cells, promoted the attachment of mouse fetal liver cells to laminin-coated dishes, but did not have a strong influence upon the attachment of normal adult liver cells. On the other hand, fibronectin which was purified from mouse plasma promoted the attachment of adult liver cells but not that of fetal liver cells. The number of neonatal liver cells attached to the surfaces coated was intermediate between those of fetal and adult liver cells in each matrix. DNA synthesis and cell proliferation during the culture of full-term fetal liver cells in laminin-coated dishes were higher than those in fibronectin- or type IV collagen-coated dishes. The amount of alpha-fetoprotein secreted in the laminin-coated dishes was more than in other groups. No differences in secretion of albumin into media, however, were observed in either group. These results suggest that laminin may be necessary for cell growth, tissue organization and cell differentiation during the normal development of liver in vivo.