Gene transfer to mammalian cells using genetically targeted filamentous bacteriophage.

We have genetically modified filamentous bacteriophage to deliver genes to mammalian cells. In previous studies we showed that noncovalently attached fibroblast growth factor (FGF2) can target bacteriophage to COS-1 cells, resulting in receptor-mediated transduction with a reporter gene. Thus, bacteriophage, which normally lack tropism for mammalian cells, can be adapted for mammalian cell gene transfer. To determine the potential of using phage-mediated gene transfer as a novel display phage screening strategy, we transfected COS-1 cells with phage that were engineered to display FGF2 on their surface coat as a fusion to the minor coat protein, pIII. Immunoblot and ELISA analysis confirmed the presence of FGF2 on the phage coat. Significant transduction was obtained in COS-1 cells with the targeted FGF2-phage compared with the nontargeted parent phage. Specificity was demonstrated by successful inhibition of transduction in the presence of excess free FGF2. Having demonstrated mammalian cell transduction by phage displaying a known gene targeting ligand, it is now feasible to apply phage-mediated transduction as a screen for discovering novel ligands.     

Dopamine-Induced Growth of Au and Ag Nanoparticles on ITO Substrate and Their Application in PCPDTBT-Based Polymer Solar Cell

The direct attachment and growth of gold or silver nanoparticles (NPs) on indium tin oxide (ITO) surfaces was demonstrated using a simple and inexpensive successive ionic layer adsorption and reaction (SILAR) method by chemical reduction of the precursor metal salts with dopamine aqueous solution. Ag NPs on ITO substrate were approximately spherical with an average particle size of about 57 nm, but had a wide particle size distribution. Compared with Ag NPs, under the same 10 SILAR cycles, Au NPs have higher density packing and smaller average particle size of about 36 nm. XRD characterization and surface chemistry analysis confirmed the formation of Ag and Au NPs on ITO substrate with small amounts of dopamine-quinone adsorbed on the surface of them. Although Au NPs showed characteristic plasmon absorption, this did not result in performance enhancement in solar cell with the structure of ITO/ZnO/PCPDTBT:[6,6]-phenyl C₇₁/MoO₃/Ag because of the energy level mismatch between ZnO and dopamine molecules adsorbed on the surface of metal NPs.     

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.     

Fibronectin-independent attachment of human gingival fibroblasts to interstitial and basement membrane collagens

We have studied the ability of human gingival fibroblasts (HGF) to attach to different interstitial (types I, II and III) and basement membrane (types IV and V) collagens. HGF cells were plated onto collagen-coated Petri dishes under various conditions and the percentage of cells attaching to the collagen was determined. HGF were found to attach to all the different types of native collagens, but attached poorly to the corresponding denatured collagens. When plated in the presence of 15% fetal bovine serum (FBS) or fibronectin-depleted FBS, similar percentages (approximately 85%) of cells attached to both interstitial and basement membrane collagens, demonstrating an attachment mechanism that is independent of plasma fibronectin. That the attachment in the presence of serum was also independent of cellular fibronectin was shown by the inability of fibronectin antibodies to block attachment to any of the collagen types. HGF were also capable of attaching to all of the collagen types in the complete absence of serum. In previous studies, investigators using cell lines have suggested that cell attachment in the absence of serum is non-physiological. However, the serum-free attachment of HGF to collagen was found to be dependent on cellular protein synthesis indicating that this attachment mechanism has biological significance.     

Identification of asparagine-linked oligosaccharides involved in tumor cell adhesion to laminin and type IV collagen

MDW4, a wheat germ agglutinin-resistant nonmetastatic mutant of the highly metastatic murine tumor cell line called MDAY-D2 has previously been shown to attach to fibronectin and type IV collagen, whereas MDAY- D2 and phenotypic revertants of MDW4 attached poorly to these substrates. The increased adhesiveness of the mutant cells appeared to be closely related to a lesion in cell surface carbohydrate structures. In an effort to identify the carbohydrates involved in cell attachment, glycopeptides isolated from mutant and wild-type cells as well as from purified glycoproteins were tested for their ability to inhibit the attachment of MDW4 cells to plastic surfaces coated with fibronectin, laminin, or type IV collagen. The addition of mannose-terminating glycopeptide to the adhesion assay inhibited MDW4 cell attachment to type IV collagen. In contrast, a sialylated poly N-acetyllactosamine- containing glycopeptide, isolated from wheat germ agglutinin-sensitive MDAY-D2 cells but absent in MDW4 cells, inhibited MDW4 attachment to laminin. None of the glycopeptides used in this study inhibited attachment of MDW4 cells to fibronectin-coated plastic. Peptide N- glycosidase treatment of the cells to remove surface asparagine-linked oligosaccharides inhibited MDW4 adhesion to type IV collagen, but not to laminin, and the same treatment of the wheat germ agglutinin- sensitive cells enhanced attachment to laminin. Tumor cell attachment to, and detachment from, the sublaminal matrix protein laminin and type IV collagen are thought to be important events in the metastatic process. Our results indicate that tumor cell attachment to these proteins may be partially modulated by the expression of specific oligosaccharide structures associated with the cell surface.     

Enhanced cell attachment using a novel cell culture surface presenting functional domains from extracellular matrix proteins

Many factors contribute to the creation and maintenance of a realistic environment for cell growth in vitro, e.g. the consistency of the growth medium, the addition of supplements, and the surface on which the cells grow. The nature of the surface on which cells are cultured plays an important role in their ability to attach, proliferate, migrate and function. Components of the extracellular matrix (ECM) are often used to coat glass or plastic surfaces to enhance cell attachment in vitro. Fragments of ECM molecules can be immobilised on surfaces in order to mimic the effects seen by whole molecules. In this study we evaluate the application of a novel technology for the immobilisation of functional domains of known ECM proteins in a controlled manner on a surface. By examining the adherence of cultured PC12 cells to alternative growth surfaces, we show that surfaces coated with motifs from collagen I, collagen IV, fibronectin and laminin can mimic surfaces coated with the corresponding whole molecules. Furthermore, we show that the adherence of cells can be controlled by modifying the hydropathic properties of the surface to either enhance or inhibit cell attachment. Collectively, these data demonstrate the application of a new technology to enable optimisation of cell growth in the tissue culture laboratory.     

Characteristics of a BHK cell variant defective in the cell-substratum contact process

In this paper we document the phenotypic characteristics of a novel BHK cell adhesion variant designated FN-2. Unlike parental cells, FN-2 cells did not attach to fibronectin (pFN)-coated dishes, even after 4-hr incubations on dishes treated with 100 micrograms/ml of pFN. Mixing experiments with the variant and parental cells revealed that the parental cells attached normally in the presence of a ninefold excess of variant cells and the variant cells failed to attach in the presence of a ninefold excess of parental cells. Therefore, the defect in FN-2 cells could not be explained by secretion of a factor inhibiting attachment or lack of secretion of a factor required for attachment. Also, the inability of FN-2 cells to attach to pFN-coated dishes could not be explained by an absence of cell pFN receptors since the variant cells bound normal numbers of small (ca. 0.8 micron) pFN-coated latex beads, although they phagocytosed the beads poorly compared to parental cells. Also, the variant cells were not able to bind large (5.7 or 16.8 microns) pFN-coated beads. When tested on dishes coated with ligands that, unlike fibronectin, have a high affinity for cell surface receptors, e.g., lectins and anti-BHK antibodies, FN-2 cells were observed to attach at a rate similar to that of parental cells but spread much more slowly. The phenotypic characteristics of FN-2 cells suggest that they are deficient in what previously has been called the "cell contact" process in cell adhesion. It is proposed that the cell contact process is the initial formation by an individual cell of a sufficient number of cell-substratum bonds to resist the shear forces operationally used to define "attachment," and that more cell-substratum bonds are necessary for cell attachment to large substrata (dishes or large beads) than for attachment to small substrata (small beads). The molecular defect in FN-2 cells was studied by electroblotting analysis. A high molecular weight (ca. 370 kd) glycoprotein detected by blotting with anti-BHK antibodies and ConA that was present in parental cell membranes was reduced or absent in the variant cells.     

Kinetics of adhesion and spreading of animal cells

The adhesion and spreading of cells at surfaces are well established phenomena which have been extensively observed using light or electron microscopy. The kinetics of both processes can be quantitatively measured by allowing the cells to attach themselves to the surface of a planar waveguide allows the number of cells per unit area and a parameter uniquely characterizing their shape, such as the area in contact with the surface, to be determined. Cells suspended in nutrient medium or pure phosphate buffer were allowed to attach to and spread on a metal oxide surface or a layer of fibronectin, and the kinetics of attachment and spreading have been measured. Attachment kinetics are the same in all cases, but spreading on the metal oxide requires nutrient medium, without which it does not occur. Spreading of cells attached to a layer of fibronectin in the absence of nutrient medium occurs at about a tenth of the rate of cells spreading on metal oxide in the presence of nutrient. (c) 1994 John Wiley & Sons, Inc.     

Cell surface localization of tissue transglutaminase is dependent on a fibronectin-binding site in its N-terminal beta-sandwich domain.

Increasing evidence indicates that tissue transglutaminase (tTG) plays a role in the assembly and remodeling of extracellular matrices and promotes cell adhesion. Using an inducible system we have previously shown that tTG associates with the extracellular matrix deposited by stably transfected 3T3 fibroblasts overexpressing the enzyme. We now show by confocal microscopy that tTG colocalizes with pericellular fibronectin in these cells, and by immunogold electron microscopy that the two proteins are found in clusters at the cell surface. Expression vectors encoding the full-length tTG or a N-terminal truncated tTG lacking the proposed fibronectin-binding site (fused to the bacterial reporter enzyme beta-galactosidase) were generated to characterize the role of fibronectin in sequestration of tTG in the pericellular matrix. Enzyme-linked immunosorbent assay style procedures using extracts of transiently transfected COS-7 cells and immobilized fibronectin showed that the truncation abolished fibronectin binding. Similarly, the association of tTG with the pericellular matrix of cells in suspension or with the extracellular matrix deposited by cell monolayers was prevented by the truncation. These results demonstrate that tTG binds to the pericellular fibronectin coat of cells via its N-terminal beta-sandwich domain and that this interaction is crucial for cell surface association of tTG.     

Sustained high-yield production of recombinant proteins in transiently transfected COS-7 cells grown on trimethylamine-coated (hillex) microcarrier beads

The present study shows that COS-7 cells transiently transfected and maintained on positively charged (trimethylamine-coated) microcarrier beads synthesize recombinant protein at higher levels and for longer periods of time than cells transfected and maintained on polystyrene flasks in monolayer culture. Sustained, high-level synthesis was observed with secreted chimeric proteins (murine E-selectin- and P-selectin-human IgM chimeras) and a secreted hematopoietic growth factor (granulocyte-macrophage colony-stimulating factor). Studies with green fluorescent protein indicated that the transfected cells attached more firmly to the trimethylamine-coated microcarriers than to polystyrene flasks. After 10-14 days in culture, most of the transfected cells detached from the surface of the polystyrene flasks, whereas most transfected cells remained attached to the microcarriers. The transiently transfected microcarrier cultures produced higher levels of protein per transfected cell due to this prolonged attachment. The prolonged attachment and higher output of transfected cells on microcarriers resulted in a 5-fold increase in protein production from a single transfection over two weeks. Thus, microcarrier-based transient transfection yields quantities of recombinant proteins with a significant savings of time and reagents over monolayer culture.     

The role of fibronectin in adhesion of metastatic melanoma cells to endothelial cells and their basal lamina

Vascular endothelial cells synthesize an extracellular matrix or basal lamina composed of collagens, proteoglycans and glycoproteins such as fibronectin (FN). Using affinity-purified anti-FN, we have examined the role of FN in adherence of metastatic B16 melanoma cells to endothelial cell monolayers which lack FN on apical cell surfaces and to their basal lamina which contains FN. B16 melanoma cells, which do not contain significant amounts of FN, attached at much higher rates to endothelial basal lamina and polyvinyl-immobilized FN compared with intact endothelial cell monolayers. Anti-FN failed to inhibit attachment of melanoma sublines of low (B16-F1) or high (B16-F10) metastatic potential to intact endothelial cell monolayers, inhibited slightly B16 cell attachment to basal lamina and completely abolished attachment of B16 cells to polyvinyl-immobilized FN. The antibiotic tunicamycin which inhibits glycosylation of B16 cell surface glycoproteins and blocks experimental metastasis [18] inhibited B16 attachment to endothelial cells, basal lamina and immobilized FN. The results suggest that FN mediates, only in part, the adhesion of B16 melanoma cells to basal lamina through glycoprotein receptors on B16 cells.     

The attachment of Enteromorpha zoospores to a bacterial biofilm assemblage

This study has investigated the relationship between bacterial biofilms and the attachment of zoospores of the green macroalga Enteromorpha. Zoospore attachment to glass slides was enhanced in the presence of a bacterial biofilm assemblage, and the number attaching increased with the number of bacteria present. Zoospores also attached to control surfaces, but at lower numbers; glass surfaces conditioned in autoclaved seawater had the same number of zoospores attached as new glass surfaces. The spatial relationship between bacterial cells and attached zoospores was quantified by image analysis. The hypothesis tested was that zoospores attached preferentially to, or in the very close vicinity of, bacterial cells. Spatial microscopic analysis showed that more bacteria were covered by zoospores than would be expected if zoospore attachment was a random process and zoospores appeared to attach to bacterial clusters. The most likely explanation is that zoospores are attracted to bacterial cells growing on surfaces and the presence of a bacterial biofilm enhances their settlement. The possibility is discussed that Enteromorpha zoospores respond to a chemical signal produced by bacteria, i.e. that there may be prokaryote‐eukaryote cell signalling.     

Adhesion of mammalian cells to a recombinant attachment factor, CBD/RGD, analyzed by image analysis

A luminance thresholding procedure was developed to quantify cell attachment of a variety of cell lines to CBD/RGD, a hybrid attachment factor comprising a cellulose binding domain and the fibronectin-like RGD attachment peptide. The technique used local thresholding, median filtering, and opening to separate and count cells on each image. Cell lines exhibited three different patterns of attachment to CBD/RGD, depending on whether it was immobilized on polystyrene or cellulose acetate. Vero, COS, HFF, 3T3, 293, and U373 cells attached well to CBD/RGD immobilized on polystyrene or cellulose acetate. CHO, MRC-5, and HEp-2 cells attached to CBD/RGD immobilized on polystyrene, but not to CBD/RGD immobilized on cellulose acetate. BHK and L cells failed to attach to CBD/RGD immobilized on either polystyrene or cellulose acetate. The attachment of many cell lines to CBD/RGD was comparable with attachment of these cells to fibronectin. (c) 1995 John Wiley & Sons, Inc.     

层粘连蛋白促进实体型瘤细胞粘着、铺展及~3H-TdR参人

 恶性肿瘤细胞或粘着于基膜以及在一定的基质上增殖对于肿瘤的浸润转移至关重要。层粘连蛋白(1aminin,LN)是基膜所特有的非胶原糖蛋白。我们研究了LN在肿瘤细胞粘着、铺展及增殖中的作用。通过测定粘着细胞所释放的LDH活性定量分析细胞粘着率。LN可显著增加体外培养的小鼠S180-V肉瘤及B16-MBK黑色素瘤细胞在玻璃及Ⅳ型胶原基质上粘着及铺展。纤维粘连蛋白(fibronectin,FN)亦有类似作用。而非糖蛋白(牛血清清蛋白)或其他糖蛋白(鸡卵清清蛋白或免疫球蛋白)均无类似作用。此外,LN或FN抗体可分别抑制细胞粘着于LN或FN。这些结果表明LN或FN促细胞粘着作用是专一性的。扫描电镜观察表明,粘着在LN敷盖的玻璃表面的瘤细胞呈多突扁平形、膜皱襞及微绒毛十分稀少,而粘着在裸露的玻璃表面者为球形,膜皱襞及微绒毛甚多。再者,~3H-TdR向粘着于LN基质上的瘤细胞群的参入量显著高于粘着在裸露玻璃面者。     

Human osteosarcoma cells resistant to detachment by an Arg-Gly-Asp- containing peptide overproduce the fibronectin receptor

MG-63 human osteosarcoma cells were selected for attachment and growth in the presence of increasing concentrations of a synthetic peptide containing the cell attachment-promoting Arg-Gly-Asp sequence derived from the cell-binding region of fibronectin. Cells capable of attachment and growth in 5-mM concentrations of a peptide having the sequence Gly-Arg-Gly-Asp-Ser-Pro overproduce the cell surface receptor for fibronectin. In contrast, these cells show no differences in the numbers of vitronectin receptor they express as compared with the parental MG-63 cells. In agreement with the resistance of the selected cells to detachment by the peptide, 25-fold more Arg-Gly-Asp-containing peptide is required to prevent the attachment of these cells to fibronectin-coated surfaces than is needed to inhibit the attachment of MG-63 cells to the same substrate. However, similar concentrations of this peptide inhibit attachment of both cell lines to vitronectin-coated surfaces. The increase in fibronectin receptor is due to an increase in the levels of mRNA encoding the fibronectin receptor. Because of the nature of the selection process, we reasoned that this increase might be due to amplification of the fibronectin receptor gene, but no increase in gene copy number was detected by Southern blot analysis. The peptide-resistant cells display a very different morphology from that of the MG-63 cells, one that has a greater resemblance to that of osteocytes. The resistant cells also grow much more slowly than the MG-63 cells. The increased fibronectin receptor and altered morphology and growth properties were stable for at least 3 mo in the absence of peptide. The enhanced expression of the fibronectin receptor on the resistant cells indicates that cells are capable of altering the amount of fibronectin receptor on their surface in response to environmental factors and that this may in turn affect the phenotypic properties of the cell.     

Studies on cell adhesion and recognition. II. The kinetics of cell adhesion and cell spreading on surfaces coated with carbohydrate- reactive proteins (glycosidases and lectins) and fibronectin

The kinetics of cell attachment and cell spreading on the coated surfaces of two classes of carbohydrate-reactive proteins, enzymes and lectins, have been compared with those on fibronectin-coated surfaces with the following results: (a) A remarkable similarity between the kinetics of cell attachment to fibronectin-coated and glycosidase- coated surfaces was found. In contrast, cell attachment kinetics induced by lectin- and galactose oxidase-coated surfaces, in general, were strikingly different from those on fibronectin and glycosidase surfaces. The distinction between fibronectin- or glycosidase- and lectin- or galactose oxidase (an enzyme with lectin-type characteristics)-coated surfaces was further supported by the finding that cytochalasin B and EDTA inhibited cell attachment to fibronectin- and glycosidase-coated surfaces but not lectin-coated surfaces. (b) Fibronectin, if labeled and added to a cell suspension, showed only low or negligible interaction with the cell surface. However, fibronectin absorbed on plastic surfaces showed a high cell-attaching activity. It is assumed that fibronectin coated on plastic surfaces may form polyvalent attachment sites in contrast to its lower valency in aqueous solution. (c) Various inhibitors of cell attachment to both fibronectin- , galactose oxidase-, and lectin-coated surfaces were effective only during the first few minutes of the adhesion assay, after which time the attached cells became insensitive to the inhibitors. It is suggested that the initial specific recognition on either lectin-type or fibronectin-type surfaces is followed by an active cell-dependent attachment process. The primary role of the adhesion surface is to stimulate the cell-dependent attachment response. (d) Cells attached on tetravalent concanavalin A (Con A) spread very rapidly and quantitatively, whereas divalent succinyl Con A and monovalent Con A were effective stimulators of cell attachment but not cell spreading. Cross-linking of succinyl Con A restored the cell spreading activity. Tetravalent Con A surfaces specifically bind soluble glycoproteins, whereas succinyl Con A has a greatly reduced ability to bind the same glycoproteins. These results suggest that cross-linking of cell surface glycoproteins by the multivalent adhesive surface may trigger the cellular reaction leading to cell spreading.     

Maximal Expression of Recombinant cDNAs in COS Cells for Use in Expression Cloning

In the process of establishing an expression cloning system for cell surface receptors we examined parameters which influence the expression of foreign genes in COS cells. The bacterial β-galactosidase gene was chosen as a reporter gene, since it permits the determination of (i) the fraction of cells transfected as well as (ii) the total activity of the synthesized enzyme in parallel experiments. This renders it possible to calculate the enzyme activity per individual cell. In transfected COS cells, the plasmid pXMgal directed a 20- and 10-fold higher β-galactosidase activity than pCH110 and pCDLgal, respectively. DEAE-dextran-mediated DNA uptake and protoplast fusion were found to result in higher expression rates than lipofection and electroporation. A coincubation of the cells with chloroquine during the DEAE-dextran transfection protocol caused, as reported, an increase of β-galactosidase positive cells but considerably reduced the total β-galactosidase activity. However, a 10% DMSO shock at the end of the transfection procedure simultaneously increased the number of transfected cells and the total β-galactosidase activity, thus maintaining the high expression per single cell. Using these optimized conditions, COS-1 cells expressed higher amounts of recombinant protein than COS-7 cells.     

The alpha v beta 1 integrin functions as a fibronectin receptor but does not support fibronectin matrix assembly and cell migration on fibronectin

The fibronectin receptor, alpha 5 beta 1, has been shown to be required for fibronectin matrix assembly and plays an important role in cell migration on fibronectin. However, it is not clear whether other fibronectin binding integrins can take the place of alpha 5 beta 1 during matrix assembly and cell migration. To test this, we expressed the human alpha v subunit in the CHO cell line CHO-B2 that lacks the alpha 5 subunit. We found that the human alpha v combined with CHO cell beta 1 to form the integrin alpha v beta 1. Cells that expressed alpha v beta 1 attached to and spread well on fibronectin-coated dishes, but did so less well on vitronectin-coated dishes. This, along with other data, indicated that alpha v beta 1 functions as a fibronectin receptor in CHO-B2 cells. The alpha v beta 1-expressing cells failed to produce a fibronectin matrix or to migrate on fibronectin, although the same cells transfected with alpha 5 do produce a matrix and migrate on fibronectin. The affinity of the alpha v beta 1-expressing cells for fibronectin was fourfold lower than that of the alpha 5 beta 1- expressing cells. In addition, alpha v beta 1 was distributed diffusely throughout the cell surface, whereas alpha 5 beta 1 was localized to focal adhesions when cells were seeded onto fibronectin-coated surfaces. Thus, of the two fibronectin receptors, alpha v beta 1 and alpha 5 beta 1, only alpha 5 beta 1 supports fibronectin matrix assembly and promotes cell migration on fibronectin in the CHO-B2 cells. Possible reasons for this difference in the activities of alpha v beta 1 and alpha 5 beta 1 include the lower affinity of alpha v beta 1 for fibronectin and the failure of this integrin to localize in adhesion plaques on a fibronectin substrate. These results show that two integrins with similar ligand specificities and cell attachment functions may be quite different in their ability to support fibronectin matrix assembly and cell motility on fibronectin.     

The influence of exogenous fibronectin on blood granulocyte adherence to vascular endothelium in vitro

Fibronectin is a major cell surface and extracellular matrix glycoprotein. It binds to a variety of substrata and supports the attachment and spreading of a number of cell types. We have found that purified human plasma fibronectin can also support blood granulocyte adhesion to cultured human umbilical vein endothelial cells. This activity is protected by treatment of the fibronectin with a sulphhydryl-containing agent. The effect of granulocyte attachment was observed at fibronectin concentration of 100 ng/ml with maximum effect at a concentration of 10 μg/ml. The attached granulocytes retained a rounded appearance, compared with the flattening that occurs on attachment to plastic. Granulocytes attached poorly to cultured human vascular smooth muscle cells and no enhancement occurred when fibronectin was added. Immunofluorescence microscopy using monospecific rabbit anti-human fibronectin demonstrated that the sulphhydryl-treated fibronectin accumulated on the endothelial cell surface, forming aggregates on the apical surface by 3 h of continued incubation. Washed, cultured endothelial cells not exposed to fibronectin or exposed to untreated purified plasma fibronectin did not demonstrate an aggregation of cell-surface fibronectin.     

Mmp2基因克隆及其对肝癌细胞的作用

克隆人基质金属蛋白酶-2基因(Mmp2)编码区并构建重组真核表达载体pEYFP-Mmp2,研究其在肝癌细胞中的作用。以肝癌细胞Hep G2的总RNA为模板,通过RT-PCR获得cDNA,并通过PCR获得Mmp2基因编码区。经TA克隆和测序鉴定将无任何突变的Mmp2基因编码区插入到真核表达载体pEYFPN1中,构建p EYFP-Mmp2重组真核表达载体并进行酶切鉴定和测序鉴定。pEYFP-Mmp2稳定性转染肝癌细胞,经G418筛选获得Mmp2稳转单克隆细胞株,并用Western blotting分析鉴定。用实时无标记细胞增殖分析技术(RTCA)分析Mmp2基因对肝癌细胞生长增殖的作用,细胞划痕愈合实验分析Mmp2基因对肝癌细胞迁移的作用。结果证明成功构建重组真核表达载体pEYFP-Mmp2,Western Blotting证实稳转株中高表达外源融合蛋白MMP2-YFP。细胞增殖和迁移结果表明,Mmp2基因可以抑制肝癌细胞增殖但能够促进肝癌细胞迁移。以上研究表明,Mmp2基因能够促进肝癌细胞迁移。