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Cell Adhesion & Migration
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Collagen matrix as a tool in studying fibroblastic cell behavior
Ji ?í Kanta
a
a
Department of Medical Biochemistry , Medical Faculty in Hradec Králové, Charles University in Prague, Czech Republic
Accepted author version posted online: 03 Mar 2015.
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Collagen matrix as a tool in studying fibroblastic cell behavior
Ji ?í Kanta
Department of Medical Biochemistry, Medical Faculty in Hradec Králové, Charles University
in Prague, Czech Republic
Keywords : extracellular matrix, collagen, fibroblasts, myofibroblasts, cell culture, cell proliferation, integrins, metalloproteinases, fibronectin, substrate stiffness
Abbreviations : AP-1, activator protein 1; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; GT, granulation tissue; HSC, hepatic stellate cells; JNK, c-Jun N-terminal kinase; MFB, myofibroblasts; MKL1, megakaryoblastic leukemia
1; MMP, metalloproteinases; NF-κB, nuclear factor kappa B; PI3K/Akt, phosphatidylinositide
3-kinase/Ak strain transforming; PEG, polyethylene glycol; α-SMA, alpha-smooth muscle actin; 3D, three-dimensional; TGF β-1, transforming growth factor beta 1; TIMP, tissue inhibitor of metalloproteinases; TNF-α, tumor necrosis factor α
Correspondence to: Ji ?í Kanta; Email: kanta@lfhk.cuni.cz
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Type I collagen is a fibrillar protein, a member of a large family of collagen proteins. It is present in most body tissues, usually in combination with other collagens and other components of extracellular matrix. Its synthesis is increased in various pathological situations, in healing wounds, in fibrotic tissues and in many tumors. After extraction from collagen-rich tissues it is widely used in studies of cell behavior, especially those of
fibroblasts and myofibroblasts. Cells cultured in a classical way, on planar plastic dishes, lack
the third dimension that is characteristic of body tissues. Collagen I forms gel at neutral pH
and may become a basis of a 3D matrix that better mimics conditions in tissue than plastic dishes.
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Introduction
Cells in a tissue are surrounded with other cells and with ECM, which is a network containing proteins, glycoproteins, proteoglycans and glycosaminoglycans. ECM provides chemical and mechanical signals whose effects are interdependent. Chemical signals may originate in the
chemical structure of ECM components or may be provided by cytokines and growth factors
stored in the ECM and released under certain circumstances. ECM is more pliable than hard
plastic surface and its mechanical properties contribute to diversity of physiological and pathological situations in the tissue. ECM is degradable and cells can migrate through it.1,2 Cell culture on thin collagen film covering plastic substrate is useful in studies of some aspects of cell behavior, e.g. interaction with integrins,3 but the contact with 3D environment makes the cells in tissue behave differently than the cells in conventional tissue culture on stiff plastic dishes do, as far as their morphology, differentiation, migration, and proliferation is concerned. The cells surrounded with an appropriate scaffold, usually
collageneous, acquire tissue-like phenotype not observed in cells in monolayer. Mechanical
signals from the ECM to the cells and contractile forces to the ECM are transmitted by
protein complexes called focal adhesions. Three-dimensional matrix adhesions differ from
adhesions on 2D substrates in protein composition and biological activity.4 Force applied to
integrins is transmitted through focal adhesions to the cytoskeleton.5 The cells may integrate global signals coming from the entire surface and sense the spatial organization of activated adhesions.6
Fibroblasts and myofibroblasts are cells involved in the healing of various tissues.
Fibroblasts in the tissue surrounding the wound are activated and migrate into the provisional matrix containing fibrin and plasma fibronectin. Fibrin is a major component of
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the provisional matrix formed during wound healing and enables migration of inflammatory cells and fibroblasts. Collagen production becomes the main fibroblast function and the provisional matrix is gradually replaced with a collagenous ECM.7,8,9 A part of fibroblasts may differentiate to protomyofibroblasts and further to MFB characterized by prominent stress fibers that contain α-SMA associated with non-muscle myosin. These proteins endow MFB
with high contractional force that is combined with synthetic abilities of the MFB.10,11 The
wound contracts and the provisional matrix is replaced with GT that is gradually converted
to a scar. Most cells then die by apoptosis. However, the reparative process may be dysregulated and result in fibrosis. The most abundant ECM component providing a scaffold that binds other proteins and proteoglycans is collagen I.12,13
Solid tumors contain stroma that resembles GT in many aspects. The stroma is highly
vascularized. Fibrin is formed by clotting extravasated fibrinogen and together with other plasma proteins it gives rise to a provisional matrix. Fibroblasts settle in the matrix and produce collagen I and other ECM components. Cancer-associated fibroblasts promote the
growth of cancer cells and vice versa they respond to signals from epithelial cells by
increased synthesis of collagen and other fibrogenic factors.14,15
Three-dimensional matrices used as a model to study cell behavior in the tissue-like
environment in normal and pathological situations are therefore often made of collagen.2,16
Fibrin gels can also be used to provide 3D environment for cells but they have much smaller influence on cell behavior and their effects may be opposite to those of collagen.17,18
Various models of collagen matrix aiming to mimic in vivo situations will be discussed
in this review. Fibroblastic cells transferred from plastic to collagen gel change their morphology and functions and in response they modify their environment. Secretion of MMP by the cells has a particular role in these interactions. The contact of cells with collagen
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is mediated by specific receptors, integrins. Plasma fibronectin interacts both with collagen and cells in vivo and it is therefore often added to collagen matrix in vitro.
Collagen matrix models
Rodent tail tendons contain almost pure collagen I that can be extracted with diluted
acids.19,20,21 Collagen forms gel when its solution is neutralized. The concentration of 1 to 2
mg collagen/ml is frequently used to form matrix. Collagen contained in bovine skin is more crosslinked and its extraction requires the use of pepsin. This enzyme cleaves off telopeptides, the nonhelical amino acid sequences at the C- and N-ends of the collagen molecule. The lack of telopeptides may interfere with gel formation and change the properties of the gel. The pores in gel made of acid-extracted rat tail collagen are 1 – 2 μm in diameter. The pores in the gel from pepsin-digested collagen are larger and allow easier migration of the cells.16 Cells can be seeded on top of the gel or be suspended in collagen
solution. The cells on collagen may be covered with a second layer of collagen gel to add the
cells the third dimension. Fibroblasts placed between two collagen layers migrate into
them.22,23
Fibroblasts cultured for a few weeks in the presence of L-ascorbic acid 2-phosphate
form a multilayered structure surrounded by hydroxyproline-rich ECM.24 The self-produced dermis-like structure formed in the presence of ascorbic acid in a long term culture contains collagens I and VI.25
Fibroblasts embedded in collagen gel cause its contraction. When collagen gel formed in tissue culture dishes remains attached to the walls of the dish, fibroblasts can contract it only in the vertical direction. When the gel is detached from the dish immediately
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after gelation, it floats in the culture medium and contracts in all directions. The resulting matrix is called floating. The gel may be maintained under tension for 1 or more days before it is detached. This type of matrix is called stressed or stress-released. Contraction of floating collagen matrices gives rise to mechanically relaxed tissue resembling dermis, attached matrices resemble granulation tissue.26,27,28 The shape of liver MFB growing on hard plastic
and on collagen gels is shown in Figs. 1 – 3. The cells respond to changing stiffness and
tension of the substrate.
Collagen properties can be modified by crosslinking collagen molecules and fibrils.
Collagen I is a substrate of transglutaminase that introduces ?-(γ-glutamyl) lysine cross-links into its molecule at 37o C.29,30 Fibroblast attachment, spreading and proliferation is enhanced on collagen polymerized as a result of transglutaminase treatment.31 Collagen can also be crosslinked by 0.2% glutaraldehyde and used as a matrix for cell culture. No toxicity to fibroblasts is observed when collagen is treated with this glutaraldehyde concentration.32
Mechanical properties of the matrix play a significant role in determining cell
behavior. The stiffness of isotropic material is characterized by Young ’s modulus (elastic
modulus). Its unit is Pascal (Pa). The stiffness of soft tissues is low; the stiffness of liver is 0.1
to 1 kPa, dermis 1 – 5 kPa and fibrotic tissues 20 to 100 kPa. Young ’s modulus of the
provisional matrix in healing wounds (0.01 to 0.1 kPa) is comparable to that of collagen gel.33
Collagen fibrils in 0.2-2.0% gel are similar to those in tumor ECM.34
Collagen stiffness can be increased when a portion of liquid is removed by
ultracentrifugation,34 gel compression 35 or by evaporating the solvent.36 Fibroblasts seeded in matrix containing 20 or 40 mg collagen/ml are viable, migrate and proliferate. They reach a density similar to that found in human dermis.36,37
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The differences between cell growth on hard plastic surface and on soft collagen gel suggest that the rigidity of the substrate plays a crucial role. Cells can be placed between two sheets of collagen-coated polyacrylamide gel. The stiffness of polyacrylamide gel can be controlled by changing the percentage of the crosslinking compound, bis-acrylamide, in the reaction mixture and can be adjusted to correspond to the physiological stiffness of tissues.
This treatment makes fibroblasts that are well spread in 2D culture change their morphology
into bipolar or stellate characteristic of fibroblasts in vivo.38 Increased rigidity affects not only
stress fibers formation but also integrin expression on the cell surface.39,40
Polyethylene glycol (PEG) can be covalently bonded to collagenous matrix extracted from porcine heart. PEG gels retain fibrillar structure, are more resistant to enzymatic degradation and do not inhibit metabolic activity of incorporated fibroblasts.41
Interaction of fibroblasts with collagen gel
The cells can be plated on the surface of collagen gel or incorporated into it to form a tissue-
like structure.19,20 Fibroblasts are rounded when they are embedded into collagen gel; they
adopt stellate morphology within a few hours and they are spindle-shaped later.42 Fibroblast
and MFB morphology in 3D matrix is comparable to that in their original tissue but much
different from polygonal appearance they adopt on a planar substratum.18,43 Fibroblasts on collagen gel aggregate; this tendency decreases with increasing collagen concentration.18,44
Fibroblasts embedded in collagen remodel surrounding matrix. They have few cell
adhesions on their surface but they produce dendritic extensions that interact with collagen fibrils.45,46 Fibroblasts align the flexible collagen meshwork around themselves and hold collagen fibrils in place. The fibrils are then stabilized by noncovalent interactions that do not
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require cell presence.47,48 Fibroblasts in attached matrices develop isometric tension. The forces they generate do not depend on the stiffness of the substrate.49 The forces produced by fibroblasts not expressing α-SMA increase rapidly within the first six hours after embedding the cells into collagen.50
When collagen matrix is attached to the walls of the culture dish, distinct actin stress
fibers that develop in fibroblasts can be visualized by phalloidin staining.51,52 When
fibroblasts are embedded in collagen layer cast on polyacrylamide gels, actin fibers staining
with rhodamine phalloidin appear if Young ’s modulus is adjusted to 1.6 to 3.6 kPa. Stress fibers formation is facilitated by cell-cell contact. Direct linkage of the cytoskeleton stress fibers mediated by cell surface cadherins maintains tension between neighbouring cells.39,53 When tension generated by the cells reaches a critical level, α-SMA that is at first diffusely distributed in the cytosol is incorporated to preexisting β-actin-containing stress fibers.54 Transcription factor MKL1 attached to globular actin (G-actin) is released after G-actin polymerization and translocated to the nucleus. It binds to the α-SMA gene promoter and
initiates α-SMA expression. Matrix stiffening also activates the small GTPase RhoA and Rho
kinase (ROCK) that control the balance between polymerized and depolymerized actin.55
These changes correspond to the increasing tension in the forming GT in healing wounds.
Cytoplasmic actin microfilament system containing α-SMA characterizes MFB.56
Profibrogenic cytokine TGF-β1 and smad signaling are involved in gel contraction by
fibroblasts derived from normal skin or hypertrophic scars.57 TGF-β-pretreated fibroblasts cause significantly more rapid gel contraction.58 The degree of substrate stiffness determined by underlying polyacrylamide gel modulates TGF-β-induced transdifferentiation of fibroblasts.59 Both substrate stiffness and the presence of TGF-β are required for the differentiation of liver portal fibroblasts to MFB.60
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Generation of the threshold tension necessary for α-SMA incorporation requires formation of large, …supermature “ adhesion sites.54 Contractility of both muscles and non-muscle cells is dependent on the interaction of actin and myosin. Non-muscle myosin II is closely associated with actin stress fibers in cochlear fibrocytes and the contraction of collagen matrix can be prevented by an inhibitor of myosin II function.61 Aging dermal
fibroblasts lose the ability of force generation in collagen gel which may be caused by
decreased expression of myosin light chain kinase and Rho kinase.62
Increasing collagen concentration supports cell proliferation and suppresses apoptosis.63 Fibroblasts migrate along collagen concentration gradient to the stiffer regions of a collagen construct. This effect is called durotaxis.64 α-SMA-positive MFB appear in wound GT when Young ’s modulus is about 20 kPa.54 The stiffness of collagen matrices containing 1-2 mg collagen is about 50 Pa and the stiffness of plastic used in tissue culture is about 1 GPa.2
Fibroblasts, MFB and other cells involved in wound healing are affected by intrinsic
forces produced by the ECM and extracellular fluid.65 The final outcome of ECM remodeling
is determined both by tissue stiffness and by mechanical loading of the tissue, i.e. the force
applied to tissue. Mechanical forces influence both cell proliferation and gene expression.
Prolonged mechanical loading may result in higher tissue stiffness.66,67
Most cells grow only if their surfaces are attached to the ECM. The attachment of
cells to ECM molecules is mediated by integrins. These receptors consisting of subunits α and β link ECM with the actin cytoskeleton and transmit signals from the outside to the cell and vice versa.68 Integrins α1β1, α2β1, α10β1 and α11β1 are collagen receptors.69 The engagement of integrins leads to the activation of signaling cascades, focal adhesion kinase (FAK), extracellular signal-regulated protein kinase (ERK) and Rho GTPases.70,71 Discoidin
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domain receptors (DDR) 1 and 2 represent another family of cell-surface receptors. They are activated by collagens and regulate cell proliferation and ECM synthesis.They are expressed on fibroblasts in healing wounds and in tumors.72,73
…Synthetic “ phenotype of fibroblasts in mechanically stressed collagen matrices
Attached matrix . Physiological levels of tissue stiffness function as a brake on fibroblast
proliferation and collagen I synthesis. Fibrotic diseases are accompanied by tissue stiffening which is no longer regarded as a mere consequence of the disease; it has become clear that it may drive the whole process.74
Fibroblasts switch between proliferative and quiescence phenotypes. Fibroblasts in attached gels assume a …synthetic “ phenotype.52 They proliferate and synthesize collagen. The number of cells in attached gels increases rapidly while the culture in floating gels regresses. However, the cells remaining in the floating gels are viable and divide at the same
rate when they return to standard culture conditions. Fibroblasts in attached gel are bipolar,
fibroblasts in floating gel are stellate.75 DNA synthesis measured by 3H-thymidine
incorporation into DNA is almost one order of magnitude higher in the cells on plastic than in
the cells in attached matrix and about two orders higher than in the cells in floating matrix.76
Fibroblast proliferation is proportional to collagen concentration in the matrix when the gel is compressed and its Young ’s modulus increased.77 DNA synthesis in attached gel is dependent on the ERK pathway. This signaling pathway is disrupted when the gels are released.78 The cytoskeleton is then disorganized and DNA synthesis is inhibited. However, these two events are independent because only DNA synthesis in adhering gel is affected by an ERK inhibitor.79
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The release of attached matrix from the walls of the culture dish 24 hours after casting, (stress-released matrix), induces secretion of cytokines IL-6 and IL-8 by the embedded fibroblasts. The response of cells to TGF-β1 and TNF-α changes, growth inhibition is less severe in stress-released matrix than in the attached one. The signaling networks that include these cytokines are modified.80
Collagen synthesis is downregulated on both RNA and protein levels when fibroblasts
are transferred from plastic to 3D collagen matrix.81 Both total protein synthesis and
collagen synthesis measured by 3H-proline incorporation is several times higher in attached gels than in floating gels.75,82 The expression of fibrillar collagens I and III in liver portal fibroblasts increases on a stiff substrate in paralel with α-SMA, while the expression of net-forming collagen IV decreases.60 Tensile strength also controls the expression of collagen type XII that is associated with collagen fibrils.83 Collagen α1(I) mRNA synthesis and steady-state level is decreased in fibroblasts transferred from plastic into collagen gel. No change is observed in the expression of fibronectin mRNA.84 Total protein synthesis and collagen
synthesis are high in the cells on plastic and in attached fibrin gel but low in floating collagen
and fibrin gels. Mechanical forces seem to play a dominant role in this case.85
Floating matrix . Contraction of freely-floating matrix by cells is dependent on α-SMA
expression.86,87 Fibroblasts in relaxed collagen gel lose stress fibers and focal adhesions and
do not proliferate. They form dendritic extensions that have microtubule cores and actin rich-tips.88,89 The extent of contraction is dependent on initial collagen concentration; lower density gels contract more rapidly. The release of mechanical tension triggers fibroblast apoptosis.90,91 This effect is specific of collagen, apoptosis is not observed in contractile fibrin gels.92 Signal transduction from the ECM is disturbed.93 Ribosomal RNA content is lower in collagen matrices than in the cells in monolayer.94 mRNA expression of TGF-β1 increases in
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the order of plastic, attached matrix, stress-relaxed matrix and floating matrix. The expression of collagenase mRNA is higher in collagen matrix than in the cells on plastic.95 The release of stressed matrix is followed by a burst of c-fos expression and ERK 1/2 kinase activation.96
Cell survival, collagen synthesis and degradation are regulated by integrins.
Antibodies to α2β1 integrins prevent the contraction and reduce apoptosis.97,98,99 Integrin
α11 mRNA and protein are up-regulated in attached collagen gel and down-regulated in
fibroblasts grown in floating gel.100 Rat liver MFB utilize α1β1 integrin for collagen matrix contraction as α2 subunit is not expressed in HSC, their precursors in vivo.101 The expression of α2 subunit in fibroblasts cultured in collagen gel is dependent on NF-κB activity that is induced by contact of the cells with collagen.102 The expression of the two receptors is regulated differentially and their functions are not identical. α1β1 mediates downregulation of collagen gene expression and α2β1 mediates induction of collagenase.103 Both of them are able to mediate gel contraction but their expression in vitro depends on the
environment and in vivo on the physiological state of the tissue.101 Matrix contraction may
be enhanced by collagen V that binds integrin αv β3.104
Increased synthesis of collagen is observed in the skin of α1-null mice. Col1(I) mRNA
levels in both granulation tissue and fetal fibroblasts are higher in the cells isolated from α1-
null animals and embedded in collagen gel than in wild-type cells. Integrins α1β1 provide a feedback inhibition of collagen synthesis.105 Blocking of β1 subunit by a monoclonal antibody, which abrogates phoshorylation of Akt/protein kinase B, protects cells from contraction-induced apoptosis. Phosphatidyl-inositol 3-kinase (PI3K)/Akt signaling is a regulator of cell survival. Downregulation of PI3/Akt survival signal results in apoptosis.
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Constitutive expression of phophatidylinositol 3-kinase (PI3K) protects fibroblasts from both apoptosis and anoikis.106
Secretion of metalloproteinases
MMP are a family of zinc-dependent proteinases that are secreted to the extracellular space
or localized to the cell surface. They are collectivelly able to cleave all components of ECM
and they can modify other biologically active molecules. A subgroup of collagenases comprises MMP-1, -8, -13 and -14 that degrade fibrillar collagens. Gelatinases MMP-2 and -9 cleave denatured collagen and collagen type IV.107
Both mechanical forces and the chemical nature of collagen play an important role in regulating MMP expression. Collagenase mRNA expression and activity are higher in fibroblasts cultured on type I collagen gel when compared to cells on plastic.108 Releasing stress by treating fibroblasts on plastic with cytochalasin D that disrupts cell cytoskeleton
enhances expresion and secretion of MMP-1, -2, -3, -13 and membrane-bound MMP-14. The
active form of MMP is more strongly expressed in cells cultured in floating matrices than in
cells in monolayer.109 Increased expression of MMP-2 and its inhibitor TIMP-2 mRNAs is
observed in fibroblasts when collagen gel with cultured cells is prestrained.110 Contact of
human MFB with collagen I gel results in the activation of proMMP-2 that is not observed in the cells grown on plastic or plastic coated with a thin layer of collagen I or IV, laminin or Matrigel. The induction of MMP-2 by accumulating collagen I may contribute to the remodeling of ECM in fibrotic liver.111,112 Increased expression of the active form of MMP-2 on collagen gel is accompanied by up-regulation of MT1-MMP protein. Metalloproteinase MT1-MMP (MMP-14) is known to activate MMP-2.113
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Collagen degradation is more rapid in floating matrices than in attached gels.114 Active forms of MMP-1 and MMP-2 can be detected around human HSC cultured on collagen gel by in situ zymography and in the culture medium, respectively.115 The expression of MMP-13 increases in rat liver MFB when the cells are embedded in attached collagen gel. However, the observed collagen degradation is a result of a joint action of a few
proteinases.18
Collagen contraction is enhanced by MMP activity and impaired by MMP inhibition.
MMP activity is stimulated in floating gel.116,117,118 Gel contraction by fibroblasts is greatly accelerated when the matrix is treated with plasmin that may activate MMP-1 secreted by the cells. Fibroblasts in healing wounds are in close proximity to keratinocytes that produce plasminogen activator in response to cytokines. Plasmin may play a role in provisional matrix remodeling.119 Contraction of floating collagen matrix gives rise to a mechanically relaxed tissue resembling dermis. The cells in floating matrix show low capacity to synthesize DNA and proliferate, decreased responsiveness to growth factors and decreased ability to
synthesize collagen.26 Three-dimensional matrix, especially at higher stiffness, impedes cell
proliferation and migration. The cells may secrete proteinases and degrade adjacent matrix
to create space for these activities. Interconnected multicellular networks are formed in gels
with low Young ’s modulus.120
Integrins α1β1 and α2β1 have different functions in the regulation of MMP
expression. MMP-1 mRNA level in fibroblasts cultured in retracting collagen gel is higher than that in cells on plastic. The diference can be eliminated when α2β1 integrin is blocked by a specific antibody. In contrast, the difference is much larger when α1 subunit is blocked. Changes in MMP expression are paralleled by changed expression of Ets-1 transcription factor.103,121 The induction of MMP-13 in periodontal ligament fibroblasts is dependent on
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integrin α11β1.122 The induction of MMP in collagen gel may be affected by signaling pathways downstream of integrin ligand binding. Contact of human skin fibroblasts with 3D collagen results in simultaneous activation of three groups of mitogen-activated protein kinases, ERK 1/2, JNK and p38. Tyrosine kinase inhibition suppresses MMP-13 expression, ERK1/2 inhibition enhances the expression.123 Collagen activates a member of protein kinase
C family PKC ξ and NF κB DNA binding.124 The expression of MMP-3, -9, -13, and -14 mRNA as
well as the activation of MMP-9 is enhanced in activated rat HSC embedded in collagen I gel.
The stimulation of MMP-9 expression requires NF-κB and AP-1 activities.125,126
The influence of fibronectin on collagen properties
Type I collagen is a major component of connective tissues but its action can be modified by other components of the ECM as well as by cytokines and agents used for treating the cells. Fibronectin contained in blood plasma and to a smaller extent in fetal bovine serum used in
cell culture influences the events in collagen matrix substantially.
Medium containing fetal bovine serum is procontractile. Collagen gel contraction by
human dermal fibroblasts is inhibited when serum used in culture medium is depleted of
fibronectin by affinity chromatography.127 The inhibition can be abolished by adding plasma
fibronectin or vitronectin to culture medium containing fibronectin-depleted fetal bovine serum.128 Fibronectin is much more efficient. The stimulated gel contractility is inhibited by peptides containing arg-gly-asp (RGD) sequences.129 Fibronectin fibrils are associated with stress fibers formed in the cells in attached collagen gel. Fibroblasts in floating gel do not form stress fibers or form fibronectin fibrils.130 Fibronectin added to collagen sponges
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accommodating chick fibroblasts enhances DNA synthesis in the cells. Fibronectin-coated sponges enhance wound healing in vivo.131
F-actin and α5 integrin are induced by fibronectin in trabecular meshwork cells cultured in collagen gel.132 Fibronectin promotes gel contraction by human corneal fibroblasts. It stimulates the formation of stress fibers in the cells and increases the amounts
of integrin subunits α5 and β1 and of paxillin, a component of focal adhesions.133 Fibroblasts
cultured on collagen matrix form clusters that are stabilized by fibronectin fibrils. Cell
clustering requires α5β1 integrins and can be prevented by blocking Rho kinase or myosin II activity.44,134 A succession of events taking place in the first hours of matrix contraction has been proposed. The earliest stage involving fibronectin and the integrin subunit α5 is followed by vitronectin-mediated cell attachment and the sequence is completed by the appearance of α2 integrin subunit and its interaction with collagen.135 Fibronectin produced by the fibroblasts themselves may enhance matrix contraction.136,137 Fibronectin is present on cell surfaces and associated with collagen in attached matrices. It disappears from the cell
surface after the matrix is released. Protein and DNA synthesis decrease.138 Fibronectin-null
mouse embryonic fibroblast adhere to collagen gel in the absence of serum but do not
spread or proliferate. Addition of plasma fibronectin results in an increase in the cell number
and formation of multicellular structures. Inhibition of fibronectin polymerization prevents
cell proliferation.139
Conclusion and Perspectives
The number of studies of cell behavior utilizing various 3D tissue models is increasing. Three-dimensional structure produces cells that differ in many aspects from their counterparts
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cultured on flat plastic dishes. Both morphology and metabolism of the cells are changed. Type I collagen as the most abundant protein found in tissues is the basis of many 3D models. Its chemical properties, the ability to interact with cells and to bind other ECM components, contribute a distinct specificity to the contact with cells. Collagen can be further modified by crosslinking or by casting on a stiffer substrate to better mimic matrix
development in healing wounds or in other tissues under physiological or pathological
situations. Future collagen matrix models may come even closer to situations in vivo by
better controling physico-chemical properties of the matrix, by incorporating into it other ECM proteins, glycoproteins and proteoglycans and by allowing fibroblastic cell interaction with inflammatory cells, epithelial and endothelial cells that also participate in the events going on in tissues.
Acknowledgement
This research was supported by PRVOUK P37/1.
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简述张洁作品中女性意识的研究 摘要：张洁是一位在中国当代文学史上占有重要位置的女作家，随着90年代中期以来女性文学研究地位的不断提升，其作品中的女性意识受到越来越多的批评者的关注，本文列举并简要分析了研究张洁作品中女性意识的经典性论著，在此基础上进一步提出了未来研究的可能路径。 关键词：张洁作品女性意识研究两性声音 自1978年凭借《森林里来的孩子》初登文坛，张洁的作品就引起了文坛的广泛关注与争议，仅1978到1987十年间国内对张洁的评论文章就达到了160多篇。这一时期的批评关注点是其作品风格、主题等方面。随着90年代中后期，西方女性主义理论的不断输入，更多研究者尝试着以这种新的理论来解读女性作家的创作。此时，作为新时期女作家旗手的张洁再次受到了研究界的关注，她的创作也在更大程度上被纳入到了中国当代女性主义文学的范畴，考察其作品中女性意识及其发展变化成为了近十年张洁研究的焦点。 一、张洁作品中女性意识研究现状评述 张洁创作中的女性意识在其作品中的显现程度不尽相同，在《沉重的翅膀》、《谁生活得更美好》、《条件尚未成熟》这类作品中，女性意识呈现隐匿状态，因此女性批评的研究者们更倾向于选取张洁作品中女性意识相对凸显的作品进行研究。著名批评家荒林认为张洁前期代表作品《爱，是不能忘记的》、《方舟》和《祖母绿》三篇小说“呈现了作家对女性问题思想的连续深入和统一性，表现了作家对女性新的价值确立的强大热情”。如果说《爱，是不能忘记的》所喧寓的，是一个关于理想爱情与理想男性的神话，那么《方舟》则称得上是一篇预示着理想破灭的“愤世之作”。80年代中期，张洁作品风格转型之巨，不禁让人哗然，此时的张洁以老辣、尖酸的形象极尽讽刺，理想的爱情神话破灭了，理想的男性偶像倒塌了，对此王绯认为，“张洁的审丑意识有着很强烈的性别色彩，在男/女二项对立中，她故意让他们原形毕露，让他们丑态百出。”度过这段偏激、锋利的创作时期，张洁的以其特有的女性姿态书写了两部现实主义作品《红蘑菇》和《她吸的是薄荷味儿的烟》，这两部作品既不同于早期的纯情之作，又有别于她前一阶段的狂怒与怨愤，蕴涵深刻的女性觉悟。有论者曾这样评价到，“此间，张洁的女性书写是在更高一级的层面上对妇女自身和男性弱处的现代审视。”在经历了由“审美”到“审丑”的转变后，张洁的创作逐渐进入了文学”老年期”，此时张洁的心态渐趋平和。随着长篇记事小说《世界上最疼我的那个人去了》的发表，张洁自身的女性意识也得到了升华，小说着眼于探索祖孙三代女性绵绵不绝的血缘之爱。批评家徐坤将这篇作品归入了“母亲谱系的梳理与母女关系重新书写”的范畴。 二、张洁研究中两性批评者的不同声音 以女性意识为切入点来研究张洁作品最早可以追溯到1991年董瑾发表的评
F0/23B(C)、H3/36B、C7030电气系列 F0/23B(C)、H3/36B、C7030Electrical series 使 用 说 明 书 成都久和传动机械有限责任公司 地址:成都市双流县彭镇燃灯社区5组 电话(Phone):（028）67028807 传真(FAX):（028）85847360 邮编(ZIP code):610203
一．使用环境 1．周围空气温度 周围空气温度不超过+40℃,周围空气温度的下限为-25℃。且在24h周期内平均温度不超过+30℃。 2．海拔高度 安装地点的海拔不超过2000m。 3．大气条件 空气清洁，而其相对湿度在最高温度为+40℃，不超过50%，在较低温度时，亦允许有较大的相对湿度，如最湿月平均温度为+20℃，月平均最大相对湿度不超过90%，并注意因温度变化产生在产品表面的凝露。 4．供电电网质量 供电电网容量应保证满足塔机功耗，进线电压波动范围须保证不超过额定电压值的±10%。起升电控柜(L柜)适用于交流50Hz/380V、60Hz/440V三相电源。 5．安装条件 垂直安装倾斜度不超过5°；安装牢固，在主机工作过程中不会发生相对于主机的平移和垂直跳动；安装部位最高震动条件为：5～13Hz时,位移为1.5mm；13～15Hz时，震动加速度为1.0g。 二．阅读电气原理图的方法 1. 符号表示 各个部分字母表示见下列表格: a)操作,检测,指示
b) Ⅰ部分 c)Ⅱ或Ⅲ部分
d）方向或速度 2 . 工作顺序、工作原理及符号 不同的工作阶段用下面两种不同的形式表示： 在开关转换顺序中 A）在工作顺序示意图中，采用下面符号： 接触器或继电器进入“工作状态”：PV 接触器或继电器进入“停止状态”：PV PV表示两种工作状态。 B）在开关转换顺序中，采用下面符号： 接触器或继电器进入“工作状态”并通过同一机械或电气连锁保持：● 接触器或继电器进入“停止状态”：○ 3. 动作特性和各机构功能 F0/23B(C)、H3/36B、C7030等塔式起重机电气控制柜可工作在交流50Hz/380V、60Hz/440V的额定电压条件下。电气控制柜分A、L、HF柜，分别有供电，吊钩升降，小车变幅、回转几大系统。供电系统（A柜）供电源给塔机各机构的用电、并起电路的短路、过载保护作用。吊钩升降（L柜）控制塔机的吊钩起升、下降；小车变幅系统（HF柜）控制塔机的小车变幅（前后）；回转系统（HF柜）控制塔机的回转。
荧光法鉴别纤维 发布日期：2007-1-20 23:11:19 中国纺织检测网 https://www.doczj.com/doc/5310319421.html,/ ·利用紫外线荧光灯照射纤维，根据各种纤维发光的性质不同，纤维的荧光颜色也不同的特点来鉴别纤维。各种纤维的荧光颜色具体显示： (1)、棉、羊毛纤维：淡黄色 (2)、丝光棉纤维：淡红色 (3)、黄麻（生）纤维：紫褐色 (4)、黄麻、丝、锦纶纤维：淡蓝色 (5)、粘胶纤维：白色紫阴影 (6)、有光粘胶纤维：淡黄色紫阴影 (7)、涤纶纤维：白光青天光很亮 (8)、维纶有光纤维：淡黄色紫阴影。 牛奶纤维纺织品定性 检验方法 发布日期：2007-1-22 9:45:58 中国纺织检测网 https://www.doczj.com/doc/5310319421.html,/ ·SHCIQH 0003—200l 牛奶纤维纺织品定性检验方法 4．1 红外光谱分析 从红外光谱分析可以知道牛奶纤维中含有N—H，一CH3，，一C≡N，>C=O等基团，牛奶纤维是由从牛奶中提取的氨基酸与丙烯腈接枝而成，但其红外图谱既不同于丝、毛等天然蛋白质纤维，也不同于腈纶。 4．2 切片投影法 用哈氏切片器作该纤维的纵向和横截面切片，置于500倍投影仪中观察结果：纵向有隐条纹，边缘光滑；横截面呈圆形，似合成纤维。 4．3 燃烧法 靠近火焰：熔融并卷曲；接触火焰：卷曲，融化，燃烧；离开火焰：燃烧，有时自灭；燃烧时气味：毛发燃味；残留物特征：黑色状，基本松脆，但有极细微量硬块。 从以上的燃烧特征看，极似真丝等蛋白质纤维。 4．4 熔点法 300％，以下无熔点，同麻、棉等无熔点纤维。 4．5 溶解法
条件和结果见表1。 根据以上试验情况，我们提出以下两种鉴别方法： (1)切片投影法与燃烧法相结合。 纵向无鳞片(区别于羊毛)，横截面呈圆形(区别于真丝)，燃烧时有蛋白质臭味(区别于化纤、棉、麻等非蛋白质纤维)，可确认为是“牛奶纤维”。 该方法的特点是快速、简便。能鉴别目前横截面呈圆形的牛奶纤维。若横截面为非圆形时，则宜用方法(2)。 (2)燃烧法与化学试剂溶解法相结合。 在100％：下用2．5％NaOH溶解30 min，纤维溶胀成冻胶状(区别于羊毛和真丝)，燃烧时有蛋白质臭味(区别于化纤、棉、麻等非蛋白质纤维)。 以上方法是对牛奶蛋白纤维的定性分析方法。目前，对于我们生产的牛奶蛋白混纺纱线也有了相应的定量分析方法标准，在这里因篇幅所限不赘述。
岗位技能要求矩阵填写指导意见一、目的技能要求矩阵的核心是明晰团队能力现状与需求的差距，用以确定未来的发展方向，是一项 非常重要的基础性工作，为课件、培训、技能评估、晋升做好准备工作。: 二、编制 1、技能水平评分标准 0——不作要求； 1——学习知晓：参加过培训，测试合格；但需要在别人的帮助与指导下进行工作。 2——独立应用：接受培训，进行实际工作半年以上，能力评估达标，能够独立上岗。年能力评估达标，并没有发生因能力缺失而造成——熟练应用：达到独立应用的水平，连续23 3次以上成功应急操作的经验。事故发生，或具有年以上该技能的实践经验，具备一定的培训与辅导——指导他人：达到熟练应用的水平，有54 技巧。、责任目标2主要从宏观、微观角度阐述员工对责任目标的知晓、应用、理解与执行。 公司总经理及总经理办公会议成员作为政策的制定者和推行者，应具备指导他人如何有序开展工作的能力标准；专业部门的部门负责人作为政策实施的组织者、策划者、监管者，也应具备指导他人的能力；其他部门部长、车间主任应具备熟练应用能力，领会并组织团队进行执行公司的政策、方针、目标、计划等；各级管理人员应在职责权限范围内，领会公司政策和发展方向，独立运用到本职工作中；基层岗位，包含班组长、主操、副操等，需要知晓公司宏观的责任目标方面内容。 根据技能因素与岗位需求的紧密程度需要特别指出。 （1）方针、政策与目标 主要包括公司的经营方针、经营目标、安全\环保\质量等政策与目标，主要是指宏观方面。 公司总经理及总公司办公会议成员需要达到指导他人的能力标准； 部门部长、车间主任——熟练应用； 管理人员——独立应用； 基层岗位（班长、主操、副操）——学习知晓。 （2）目标与指标 主要指公司级年度/月度计划、目标，如质量目标、环境目标等。 部门部长、车间主任——熟练应用； 管理人员、班长——独立应用； 主操、基层岗位（副操）——学习知晓。 （3）激励机制 主要指激励制度、薪资考核制度、福利政策、奖惩制度等。 部门部长、车间主任——熟练应用； 管理人员——独立应用； 基层岗位——学习知晓。 （4）安全文化 主要包括安全文化的宣导、推行、监督等。
姓名：张永梅 学号：1004224126 系别：生命科学学院<生物技术> 任课教师：胡瑞香 大 众 文 化 与 女 性 文 学 论 文
浅谈女性意识 女性的主体意识即女性作为主体在客观世界中的地位、作用和价值的自觉意识。女性的自我意识，即女性的自身认识，是女性对自身存在的特殊性的探秘，它观察到的不再是男性眼中的女性，而是女性眼中的自己。它既是女性对男性经验的一种积极有效的否定，也是女性的一种自我反思和自我批判。女性的平等意识，即女性对现实生活中从属地位和一切歧视现象与行为的意识敏感性，以及对女性应该享受和男性同等的权利与地位的确切认知。 ——《女性意识》 女性意识随着女性地位的提高也随之增强，对于女性作家作品中女性意识的分析更具有说服力。 张爱玲的小说对读者来说想必已不陌生了，很多人都或多或少的读过她的一些作品，我想，看过她作品的人可能都会不约而同地发现一个问题：张爱玲爱写女人，尤其爱写30、40年代上海的女人，而且写得与众不同，栩栩如生，深入骨髓。如她所塑造的许多特殊女性形象一样，张爱玲的女性意识也是别具一格，独放异彩！ 娇蕊，是张爱玲小说《白玫瑰与红玫瑰》里的人物，她，原来是一个开放热情，充满欲望的女人，把生活的所有都寄托在了男人身上。她，以自己的美貌和妩媚身姿吸引了众多男人的视线，让他们都围绕着她在转，她天真地以为这样就展现了自己的魅力和价值，女人们就是有了独立的地位，可不久她就发现自己错了，振宝，这个自私、无情、虚伪、满脑子假仁义道德、不负责任的男人打碎了她原有的观念，让她清楚的认识到女人的世界不是只有男人的身影。振宝的爱深深地伤害了她，可这个坚强充满活力的女人并没有陷在振宝编制的罗网里一蹶不振，而是重新思考自己的问题，改变原有的幼稚想法，重新寻得希望，组建了一个幸福的家庭，有了一个可爱的儿子。 可以说，和曹七巧相比，娇蕊是张爱玲塑造的另一个典型的女性人物。张爱玲通过对她生活、爱情观转变过程的描写，向读者传达出对女性命运的深沉思索：爱情不是生命的全部，女人不能把所有的希
11.6 疑难解答 危险： 严格遵守11.1章中“危险”一节的要求。 警告： 在（废料）切割器或者料盘分隔板附近工作时不论何时都必须戴厚度适度的保护手套。不论（废料）切割器及料盘分隔板刀片处于固定还是可动状态，甚至贴片机已经断电，都存在高风险的受伤可能性。 严禁从下方进入气压切割装置或者从上方进入空的皮带供料器，甚至是为了解决问题（如供料器卡住时）。 11.6.1 更换气压切割刀片 警告： 佩戴厚度适度的保护手套。 取出刀片时，只能捏住它的外面，左边和右边。 严禁将刀片放置身体上，例如，放到膝盖或者腿上。 不要将脚放到刀片上。你可能会重伤自己或者至少将衣服划破。 拆除刀片后确保没人会因踩到刀片伤到他们自己。 11.6.1.1 移除刀片 运行贴片机，开启压缩空气系统。 中断贴片机菜单中可动器件，然后将它取出。 停止运行贴片机，切断总电源，然后关闭压缩空气。开启位于压缩空气单元的针状阀以使压缩空气流动（查看11.1章中“危险”一节）。 松弛螺丝更换喷嘴，略微将它举起并保持它在这一位置。 拔下电缆和喷嘴气动软管 慢慢的拔出喷嘴。 拧下空供料器各个配件的螺丝（参考图11.4.1 -> 11, 9），然后将这些管道移出机器。 警告： 刀片的刀刃处始终可能伤到你自己。 基于这一原因，挡板、顶盖及保护罩（参见图11.4.3 -> 6,7, 2）必须安装到位。 打开连接电缆顶盖（见图11.6.6 -> 5） 拧下位于连接线缆（见图11.6.6 -> 5）处的气压连接阀（Y型插座：见图11.6.3 -> 9） 拔下电源和控制面板插头插座。（见图：see Fig. 11.6.5 -> 11, 10） 仔细解开外部控制面板箱内（见图11.6.5 -> 15）对应的接线头（向左或者向右）。在此期间不要损坏连线。 将顶盖放回控制面板及连接线缆处。 取出供料器斜槽（它只是扣住而已）。这使得取下刀片变得容易。 警告： 刀片下方必须保持干净。（例如，不要把脚放到下面） 在贴装元器件情况下松弛位于贴片机左右两个侧面的缓冲部件（2头M8六角头两边螺钉，见图 11.4.1 -> 15）。
纺织纤维及再生纤维的鉴别方法 纺织纤维的判别方法 1、手感目测法：此法适用于呈散纤维状态的纺织原料。 (1)、棉纤维比苎麻纤维和其它麻类的工艺纤维、毛纤维均短而细，常附有各种杂质和疵点。 (2)、麻纤维手感较粗硬。 (3)、羊毛纤维卷曲而富有弹性。 (4)、蚕丝是长丝，长而纤细，具有特殊光泽。 (5)、化学纤维中只有粘胶纤维的干、湿状态强力差异大。 (6)、氨纶丝具有非常大的弹性，在室温下它的长度能拉伸至五倍以上。 2、显微镜观察法：是根据纤维的纵面、截面形态特征来识别纤维。 (1)、棉纤维：横截面形态：腰圆形，有中腰；纵面形态：扁平带状，有天然转曲。 (2)、麻（苎麻、亚麻、黄麻）纤维：横截面形态：腰圆形或多角形，有中腔；纵面形态：有横节，竖纹。 (3)、羊毛纤维：横截面形态：圆形或近似圆形，有些有毛髓；纵面形态：表面有鳞片。 (4)、兔毛纤维：横截面形态：哑铃型，有毛髓；纵面形态：表面有鳞片。 (5)、桑蚕丝纤维：横截面形态：不规则三角形；纵面形态：光滑平直，纵向有条纹。 (6)、普通粘纤：横截面形态：锯齿形，皮芯结构；纵面形态：纵向有沟槽。
(7)、富强纤维：横截面形态：较少齿形，或圆形，椭圆形；纵面形态：表面平滑。 (8)、醋酯纤维：横截面形态：三叶形或不规则锯齿形；纵面形态：表面有纵向条纹。 (9)、腈纶纤维：横截面形态：圆形，哑铃形或叶状；纵面形态：表面平滑或有条纹。 (10)、氯纶纤维：横截面形态：接近圆形；纵面形态：表面平滑。 (11)、氨纶纤维：横截面形态：不规则形状，有圆形，土豆形；纵面形态：表面暗深，呈不清晰骨形条纹。 (12)、涤纶、锦纶、丙纶纤维：横截面形态：圆形或异形；纵面形态：平滑。 (13)、维纶纤维：横截面形态：腰圆形，皮芯结构；纵面形态：1~2根沟槽。 3、密度梯度法：是根据各种纤维具有不同密度的特点来鉴别纤维。 (1)、配定密度梯度液，一般选用二甲苯四氯化碳体系。 (2)、标定密度梯度管，常用的是精密小球法。 (3)、测定和计算，将待测纤维进行脱油、烘干、脱泡预处理，做成小球投入平衡后，根据纤维悬浮位置，测得纤维密度。 4、荧光法：利用紫外线荧光灯照射纤维，根据各种纤维发光的性质不同，纤维的荧光颜色也不同的特点来鉴别纤维。各种纤维的荧光颜色具体显示： (1)、棉、羊毛纤维：淡黄色 (2)、丝光棉纤维：淡红色 (3)、黄麻（生）纤维：紫褐色 (4)、黄麻、丝、锦纶纤维：淡蓝色
《伤逝》、《方舟》、《私人生活》之女性形象分析 中文系汉语言文学专业 09050124 王丽菲指导教师：雷振华 摘要：女性寻求自身解放、追求平等自由的斗争从未间断，无论是“五四”时期女性意识的觉醒还是新时期对女性自身价值、命运、感觉方式等精神层面的探讨，都不断演绎和阐释了真正的女性追求与价值。本文以《伤逝》、《方舟》、《私人生活》为题材，论述了20世纪以来女性意识觉醒与回归的进程。 关键字：女性意识；独立；觉醒
目录 引言 (1) 1.女性意识的初步觉醒 (1) 1.1追求恋爱自由、个性解放 (2) 1.2变不了“家庭主妇的角色” (3) 2.女性意识的复苏和深化 (5) 2.1肯定女性的自身价值 (5) 2.2自我救赎的“诺亚方舟” (6) 3.女性性别意识的膨胀 (8) 3.1逃离男权世界 (8) 3.2虚拟私人化的空间 (9) 结语 (11)
引言 虽然女性对爱有天生的渴望，但几千年的父权制使女性始终处于被统治的地位，自然也失去了追求爱情的权利。事实上“由于女性意识、女性自我与当时主导意识形态相冲突，因此几十年来始终受到强大压抑和斥责，广大女性除了做与男人同样的人以外，不能有任何女性意识、特质或特定的流露，这种讳莫如深把女性降到‘空洞能指’的最低水平，其背后深处深藏着那种千百年来顽固不化的传统文化精神——对女性的鄙视和忽略”。［1］而女性主动对爱情、婚姻的追求更是被主流文学作品所避讳。中国新文学中女性意识的觉醒与确定，发轫于“五四”时期。“五四”文学以人的解放为内核，形成了以争取女性独立地位为标志的女性意识传统，并在创作上表现出明显的性别特征和写作姿态。在以后的发展中，随着民族矛盾和阶级矛盾的上升，文学创作中的女性意识被消解在战争的硝烟之中。50年代以后，特别是“文革”时期，女性意识以及写作中的性别特征被阶级性所抹杀。“文革”以后，人性、人道主义在文学创作中恢复、发展与深化的一个重要标志，就是女性意识的再次觉醒与回归，女性从无差别、男性化的社会中脱颖而出，重新获得了自己独立的地位。 女性寻求自身解放、追求平等自由的斗争从未间断，无论是“五四”时期女性意识的觉醒还是新时期对女性自身价值、命运、感觉方式等精神层面的探讨，都不断演绎和阐释了真正的女性追求与价值。本文以《伤逝》、《方舟》、《私人生活》为题材，论述了20世纪以来女性意识的觉醒与回归的进程。 1.女性意识的初步觉醒 “五四”时期，在西方文学思潮的影响下，中国爆发了声势浩大的“五四”新文学运动，这次文学革命的重要实绩之一，就是“人”的发现，尤其是“女人”的发现。中国男性知识分子们在受到“自由、平等、独立”等民主思想和自由主义思潮影响下，在探讨人的自由及价值个体重要性的同时，也注意到了中国女性更悲惨的社会地位。他们纷纷著书立说，为中国女性“人”的意识的觉醒举起了第一面旗帜。如：郭沫若的《三个叛逆女性》，胡适的《易卜生主义》，鲁迅的《我之节烈观》和《伤逝》等等。妇女问题受到前所未有的重视。在这样的氛围下，一批女性创作者在“人”的发现的浪潮中认识了自我，发现了女性，以高昂的主体意识开始了对女性命运和社会问题的思考和探索，发出了大时代中女性的心声，打开了“女人无史”之后的“有史”的开端，知识女性结
Sequence Analysis Software for Macintosh and Windows GETTING STARTED Introductory Tour of the LASERGENE System MAY 2001
DNASTAR, Inc. 1228 South Park Street Madison, Wisconsin 53715 (608) 258-7420 Copyright . 2001 by DNASTAR, Inc. All rights reserved. Reproduction, adaptation, or translation without prior written permission is prohibited,except as allowed under the copyright laws or with the permission of DNASTAR, Inc. Sixth Edition, May 2001 Printed in Madison, Wisconsin, USA Trademark Information DNASTAR, Lasergene, Lasergene99, SeqEasy, SeqMan, SeqMan II, EditSeq, MegAlign, GeneMan, Protean,MapDraw, PrimerSelect, GeneQuest, GeneFont , and the Method Curtain are trademarks or registered trademarks of DNASTAR, Inc. Macintosh is a trademark of Apple Computers, Inc. Windows is a trademark of Microsoft Corp. ABI Prism are registered trademarks of Pharmacopeia, Inc. Disclaimer & Liability DNASTAR, Inc. makes no warranties, expressed or implied, including without limitation the implied warranties of merchantability and fitness for a particular purpose, regarding the software. DNASTAR does not warrant, guaranty, or make any representation regarding the use or the results of the use of the software in terms of correctness, accuracy, reliability, currentness, or otherwise. The entire risk as to the results and performance of the software is assumed by you. The exclusion of implied warranties is not permitted by some states. The above exclusion may not apply to you. In no event will DNASTAR, Inc. and their directors, officers, employees, or agents (collectively DNASTAR) be liable to you for any consequential, incidental or indirect damages (including damages for loss of business profits, business interruption, loss of business information and the like) arising out of the use of, or the inability to use the software even if DNASTAR Inc. has been advised of the possibility of such damages. Because some states do not allow the exclusion or limitation of liability for consequential or incidental damages, the above limitations may not apply to you. DNASTAR, Inc. reserves the right to revise this publication and to make changes to it from time to time without obligation of DNASTAR, Inc. to notify any person or organization of such revision or changes. The screen and other illustrations in this publication are meant to be representative of those that appear on your monitor or printer.
1、手感目测法：此法适用于呈散纤维状态的纺织原料温下它的长度能拉伸至五倍以上。 (1)、棉纤维比苎麻纤维和其它麻类的工艺纤维、毛纤维均短而细，常附有各种杂质和疵点。 (2)、麻纤维手感较粗硬。 (3)、羊毛纤维卷曲而富有弹性。 (4)、蚕丝是长丝，长而纤细，具有特殊光泽。 (5)、化学纤维中只有粘胶纤维的干、湿状态强力差异大。 (6)、氨纶丝具有非常大的弹性，在室2、显微镜观察法：是根据纤维的纵面、截面形态特征来识别纤维。 2、显微镜观察法：是根据纤维的纵面、截面形态特征来识别纤维 (1)、棉纤维：横截面形态：腰圆形，有中腰；纵面形态：扁平带状，有天然转曲。 (2)、麻（苎麻、亚麻、黄麻）纤维：横截面形态：腰圆形或多角形，有中腔；纵面形态：有横节，竖纹。 (3)、羊毛纤维：横截面形态：圆形或近似圆形，有些有毛髓；纵面形态：表面有鳞片。 (4)、兔毛纤维：横截面形态：哑铃型，有毛髓；纵面形态：表面有鳞片。 (5)、桑蚕丝纤维：横截面形态：不规则三角形；纵面形态：光滑平直，纵向有条纹。 (6)、普通粘纤：横截面形态：锯齿形，皮芯结构；纵面形态：纵向有沟槽。 (7)、富强纤维：横截面形态：较少齿形，或圆形，椭圆形；纵面形态：表面平滑。 (8)、醋酯纤维：横截面形态：三叶形或不规则锯齿形；纵面形态：表面有纵向条纹。 (9)、腈纶纤维：横截面形态：圆形，哑铃形或叶状；纵面形态：表面平滑或有条纹。 (10)、氯纶纤维：横截面形态：接近圆形；纵面形态：表面平滑。 (11)、氨纶纤维：横截面形态：不规则形状，有圆形，土豆形；纵面形态：表面暗深，呈不清晰骨形条纹。 (12)、涤纶、锦纶、丙纶纤维：横截面形态：圆形或异形；纵面形态：平滑。 (13)、维纶纤维：横截面形态：腰圆形，皮芯结构；纵面形态：1~2根沟槽。 3、密度梯度法：是根据各种纤维具有不同密度的特点来鉴别纤维。 (1)、配定密度梯度液，一般选用二甲苯四氯化碳体系。 (2)、标定密度梯度管，常用的是精密小球法。 (3)、测定和计算，将待测纤维进行脱油、烘干、脱泡预处理，做成小球投入平衡后，根据纤维悬浮位置，测得纤维密度。 4、荧光法：利用紫外线荧光灯照射纤维，根据各种纤维发光的性质不同，纤维的荧光颜色也不同的特点来鉴别纤维。各种纤维的荧光颜色具体显示： (1)、棉、羊毛纤维：淡黄色 (2)、丝光棉纤维：淡红色 (3)、黄麻（生）纤维：紫褐色 (4)、黄麻、丝、锦纶纤维：淡蓝色 (5)、粘胶纤维：白色紫阴影 (6)、有光粘胶纤维：淡黄色紫阴影 (7)、涤纶纤维：白光青天光很亮 (8)、维纶有光纤维：淡黄色紫阴影。 5、燃烧法：根据纤维的化学组成不同，燃烧特征也不同，从而粗略地区分出纤维的大类。几种常见纤维的燃烧特征判别对照如下： (1)、棉、麻、粘纤、铜氨纤维：靠近火焰：不缩不熔；接触火焰：迅速燃烧；离开火焰：继续燃烧；气味：烧纸的气味；残留物特征：少量灰黑或灰白色灰烬。 (2)、蚕丝、毛纤维：靠近火焰：卷曲且熔；接触火焰：卷曲，熔化，燃烧；离开火焰：缓慢燃烧有时自行熄灭；气味：烧毛发的气味；残留物特征：松而脆黑色颗粒或焦炭状。 (3)、涤纶纤维：靠近火焰：熔缩；接触火焰：熔融，冒烟，缓慢燃烧；离开火焰：继续燃烧，有时自行熄灭；
你将格外的不幸，因为你是女人。 ——浅谈张洁《方舟》《方舟》是张洁最富个人特色的作品，小说描写三个中学时代的同窗好友，经过漫长坎坷的人生道路之后，为了摆脱现实的痛苦，又各自离开丈夫相聚在一个住宅的单元里，以求得暂时的庇护。她们如同乘住在一叶“方舟”上，经受着生活海洋里风浪的打击和颠簸，同时通过现实主义表现了现代知识女性人生道路和精神追求上的焦灼、孤独和悲凉。 小说塑造了荆华、梁倩、柳泉三个个性刚硬、独立自强的知识女性形象，她们都是六十年代的大学生，同时三个独身女人在事业和个人生活上都非常不幸。她们都真诚地爱过，但最后无一不感到彻底的幻灭。她们不能接受没有爱情的婚姻，于是同丈夫分居或离婚，这种举动使她们受尽了世人的嘲弄、冷眼和侮辱。这三个人各有特点，但却殊途同归。 荆华是一位思想与学识都很突出的理论工作者，让很多男子汉也佩服的女性，能够写出真正揭示马克思主义真理的论文，却被“凡是派”视为异端，最后还是过着清贫而孤独的生活；梁倩是电影学院的高材生，是极有抱负的才华出众的导演，吃苦耐劳，为了导演一部片子，四处奔波，但仍免不了周围人们的讥讽、嘲弄，最后还是被审查部门枪毙了；柳泉是大学英语系的优等生，是一位热爱工作、精明能干的翻译，为找一份工作，历尽劫难，在男女纠葛中伤痕累累。 正如文章题记所写：“你将格外地不幸，因为你是女人”。张洁在《方舟》中所极力展示的是这三位知识女性在人生道路的追求上无助悲凉的困境。小说的三位主人公曹荆华、柳泉、梁倩分别是三位不同背景的知识女性。荆华与柳泉曾先后离开，荆华是不愿成为生孩子的机器，而柳泉则是不愿成为别人的工具，于是两人冒着身败名裂的危险，成了独身女人。经过一段严峻的生活磨难后，终于靠当年同宿舍住过的老同学梁倩的帮忙，两人搞到了一套房子。然而一个坎子还没有过完，另一个坎子已经等在那里。离异的女子要无端地受到多少“文雅的侵略者”的来犯，白复山的纠缠，居委会贾主任的“关怀”，魏经理的无耻，以及形形色色陌生的，却又自以为有权干预你的种种一一关于婚姻，关于爱情、工作的设想，生活方式甚至走路的姿态，说话的表情以及许许多多不值一提的个人私事。 所谓“寡妇门前是非多”，在一般人的眼里，离过婚的女人，都是些不正经的女人。“谁丢了什么东西，谁倒了什么霉，谁心理不痛快，谁想满足一下自己高人一等的欲望，全可以发泄到她们头上来”。从来没有人怜悯她们，有的只是恶意的猜忌。女主人公所面临的
MAXIDRIVER3.4数字音频处理器 ALTO MAXIDRIVER3.4数字处理器是集增益、噪声门、参数均衡、分频、压缩限 幅、延时为一体的全功能数字音频处理器，具有2个输入通道和6个输出通道，本机内设10种工厂预设的分频模式，64个用户程序数据库位置以及利用多媒体卡（MMC）进行128个用户程序外置储存的功能。MAXIDRIVER3.4是新一代全数字音 频处理器，采用分级菜单形式，操作非常方便。 功能键介绍 前面板 1、MODE---分级菜单选择，按动时循环选择PRESET（预设）、DELAY（延时）、EDIT（编辑）、UTILITY（系统设置）菜单功能。同时相对应的LED指示灯会被点亮。这时可以进入所选择的菜单进行参数编辑。 2、LED指示灯---当你用MODE键选择需要编辑的菜单时，相对应的LED指示 灯会被点亮。 3、2X16位LCD显示屏---显示正在编辑或查看的系统参数或系统状态。 4、数据轮---转动这个数据轮可以调节需要编辑的参数的数值，顺时针旋转提高数值，逆时针旋转减低数值。 5、PREV/NEXT---前翻/后翻键，每个主菜单下面都有若干个子菜单，通过按动这两个按键可以向前或向后选择所需要进行编辑的子菜单。 6、NAVIGATION CURSOR KEYS---光标移动键，每个子菜单中都有若干个可以 编辑的参数选择，按动这两个键，可以选择需要编辑的参数，选中的参数会闪烁。 7、CARD---储存卡插入口，在这个插口插入MMC储存卡，利用PRESET（预设） 菜单下，可以对该储存卡进行写入、读出等操作。 8、ENTER---确认键，按此键可以对所选择的菜单或编辑的参数数值进行确认。 9、ESC---取消键，按此键可以对所选择的菜单或编辑的参数数值进行取消操作，返回上一级菜单。 10、输入电平指示表，实时指示A/B两个输入通道输入电平的强弱数值。 11、MUTE---静音按键，按下后将关闭相应输出通道的输出信号，相对应的 红色LED指示灯将点亮。 12、输出电平指示表，显示每个输出通道输出电平大小数值，这里显示的数 值不是绝对的输出电平数值，而是与该列LED指示灯中的LIMIT（限幅）指示为基础相比较的数值。
山东师范大学学报(人文社会科学版) JOURNA L OF SH ANDONG NOR M A L UNIVERSITY (Humanities and S ocial S ciences ) 2005年第50卷第2期(总第199期) 2005 V ol.50 N o.2(G eneral N o.199) 张洁小说的女性意识浅谈 3 张 莹1,马 芸2 (1.四川师范大学文学院,四川成都,610066;2.四川大学文学院,四川成都,610061) 摘要: 20世纪80年代中期以来,张洁彻底告别了热情高歌的理想主义文本,始终坚持一方面用中性眼光观照社会生活,超越女性意识感情,创造一种不分性别的文学作品;另一方面又不断地寻找“女性自我”,用笔记录下女性“成长”的历程。本文分析了张洁真爱理想的演变过程、女性自我的寻找历程,试图梳理出张洁女性意识的变化成长。 关键词: 真爱理想;解构;女性自我;定位 中图分类号: I206.7 文献标识码:A 文章编号:1001-5973(2005)02-0071-04 3收稿日期:2004-04-19 作者简介:张莹(1965— ),女,四川成都人,四川师范大学文学院副教授;马芸(1981— ),女,四川峨嵋人,四川大学文学院硕士研究生。 沃林格提出决定艺术活动的“艺术意志”来源于人的应世观物所形成的世界态度,即人面对世界形成的心理态度。沃林格把这种态度界定为“世界感”。所以,个体“世界感”的差异决定了在“艺术活动”中的差异。而女性作为普遍意义的人和纯然的女性在“世界感”上必定是双性的。虽然文学自来不缺关于女性的话题,但长久以来都只限定在男性的视野中。只有在女性作为自觉的书写者,从女性立场出发创作关于女性生活和心理的作品,才形成了真正的女性文学。张洁作为一名女作家,一方面在用中性眼光观照社会生活,超越女性意识、感情,创造一种不分性别的文学作品;另一方面则更是在纯然女性眼光下,观照女性自我世界,创作女性心灵外化的女性文学。像大多数女作家一样,张洁同样将男女情感作为自己女性文学创作的基点,关注着女性的生存状况和心理世界。从第一篇涉及到两性关系的小说《爱,是不能忘记的》开始,张洁始终没有放弃探讨“爱”以及“爱”中的男女两性,尤其是女性。 一 虽然女性对爱有天生的渴望,但几千年的父权制使女性始终处于被统治的地位,自然也失去了追求爱情的权利。事实上,“由于女性意识、女性自我与当时主导意识形态相冲突,因此几十年来始终受到强大压抑和斥责,广大女性除了做与男人同样的人以外,不能有任何女性意识、特质或特定的流露,这种讳莫如深把女性降到‘空洞能指’的最低水平, 其背后深处深藏着那种千百年来顽固不化的传统文化精神 ———对女性的鄙视和忽略”[1](P56) 。而女性主动对爱情、婚姻 的追求更是被主流文学作品所避讳。作为一名女性,张洁必定在生活中感受到了历史、社会对于女性的压制,她开始试图用笔探索女性心理、思考女性问题,延续自“五四”后断裂了的对女性的思考。 张洁的女性文学创作是从女性最初“寻找男性”、“寻找爱”开始的,长久以来压抑在女性心中对于理想爱情的呼唤在张洁的笔下终于喷薄而出。既然女性已经获得了经济上的独立,已不在物质上依靠男性,那么,对于爱情她们更希望精神上的契合,追求心灵价值的美好。这种对精神生活的追求相应地造就了一种“理想男性”的形象。就像《爱,是不能忘记的》中的钟雨和《波希米亚花瓶》中的梧桐,她们一个爱着与自己相处不足24小时,连手也不曾握过的“老干部”,一个爱着不能过年轻夫妇那种生活的简,都在享受精神上的爱。她们从旧式的婚姻中解脱出来,被“老干部”和简强大的“精神力量”所吸引,爱的是“成熟而坚定的政治头脑”、“在动荡中的革命年代里出生入死的经历,他活跃的思想,工作上的魄力,文学艺术上的修养”……这是80年代新女性心目中的男性标准。她们开始自主地摆脱对有金钱、有地位的男性的依附关系,在婚恋问题上有了崭新的价值观念,这是女性在追求自我历程中的一个进步。张洁大胆突破了传统观念的束缚,敢于“冒天下之大不韪”为自己也为女性喊出了“爱”的心声。
Pierce? Co-Immunoprecipitation (Co-IP) Kit（26149） 中文说明书 介绍： Thermo 公司的Pierce?免疫共沉淀试剂盒，可通过将铆钉抗体固定在琼脂 糖支撑物上，从裂解液中或其他复杂混合物中，分离出天然蛋白复合物。Co-IP 是一种研究蛋白与蛋白相互作用通用的方法，该方法使用一种诱饵蛋白与抗原 进行免疫沉淀反应，然后可通过免疫共沉淀任何与诱饵蛋白具有相互作用的猎 物蛋白。传统的Co-IP方法使用蛋白A或G共同洗脱抗体的重链和轻链，这很 可能导致将相关的蛋白一起洗脱下来，掩盖一些重要的结果。Pierce?免疫共沉 淀试剂盒通过将共价结合抗体固定在一个胺类活性反应树脂上解决了这一问题。该试剂盒包含足够的用于蛋白结合和恢复的缓冲液，完成对照试验的高校离心 柱和收集管，这些产品进一步缩短了操作实验的时间。 重要产品信息： 略 Co-IP实验步骤： A.抗体固定 注意：以下试验步骤是针对用无胺和其他载体蛋白稀释的10-75μg亲和纯 化抗体（参考重要产品信息一节）。根据实际使用比例参考这一协议步骤。参 考重要产品信息节表1中的建议抗体用量和树脂体积用量。 1.室温平衡胺连接耦合树脂（AminoLink?Plus Coupling Resin）和试剂； 2.为每个Co-IP反应准备2ml 1×Coupling Buffer（超纯水稀释20×Coupling Buffer）；
3.轻轻涡旋混匀装有AminoLink?Plus Coupling Resin的瓶子，使其处于悬浮状态。使用大口径（或剪掉一段枪头端），添加50μl树脂悬液到Pierce提供的离心柱中，将离心柱放入微量离心管中，1000g离心1min，弃滤液； 4.添加200μl 1×Coupling Buffer 清洗树脂2次，离心弃滤液； 5.将离心柱放于纸巾上，轻巧离心柱底部，去除剩余的液体，插上底塞； 6.准备10-75μg亲和纯化抗体用于结合蛋白，调整体积至200μl，使用足够的超纯水和20×Coupling Buffer来制备1×Coupling Buffer。例如：添加10μl 20×Coupling Buffer，180μl超纯水和10μl浓度为1μg/μl。可直接添加含有超纯水、20×Coupling Buffer、亲和纯化抗体的树脂在离心柱中。 7.在通风厨中，每200μl反应体系，添加3μl氰基硼氢化钠溶液； 注：氰基硼氢化钠属剧毒物质，操作时要小心并穿戴防护服。 8.拧紧离心柱上螺帽，室温涡旋孵育90-120min，确保浆体在孵育过程中处于悬浮状态； 9.握紧底塞，拧开并拿走螺帽，将离心柱置于收集管中离心，保存滤液以便验证抗体耦合； 10.打开螺帽，添加200μl 1×Coupling Buffer，离心弃滤液，重复此步骤1次； 11. 向离心柱中添加200μl Quenching Buffer，离心弃滤液； 12. 将离心柱放于纸巾上，轻巧离心柱底部，去除残留液体，插上底塞。在树脂上添加200μl Quenching Buffer； 13. 在通风厨中，添加3μl氰基硼氢化钠溶液，拧紧螺帽；轻轻摇动并孵育15min； 14.取出底塞，拧开螺帽，将离心柱置于一收集管中，离心弃滤液； 15.打开螺帽，采用200μl 1×Coupling Buffer洗脱树脂，离心。再次重复此步骤； 16.用150μl Wash Solution洗脱树脂6次，每次洗脱后离心； 17.不管是进行细胞裂解、Co-IP，还是储存树脂，都需要继续进行下列步骤； 18.用200μl 1×Coupling Buffer洗脱树脂2次，每次需离心；
张洁作品中的女性意识 摘要张洁笔下的主要女性是当时社会中的“另类”，而又是“普遍类”，在此，我将从这几个方面解析在张洁小说作品中的女性意识：她们强烈 的事业心，要求独立和解放，争做强者。以及易被忽视的“女性自身 价值”。 关键词张洁的小说女性意识 “男人的雌化和女人的雄化，将是一个不可避免的、世界性的问题”，张洁，她是这样的定义着当时的社会趋向，预言着后来的世界，女人必将强大起来。她作品中的女人们很多都不愿意雄化，却不得不被迫雄化。 在张洁的作品中体现出来的女性意识主要有女性的反抗精神，强烈的事业心，要求独立、解放。 首先，我们可以很明显的看到，张洁的作品中的女性，都是离了婚或是不愿意结婚的女性，而且离了婚的她们也不愿意再婚。她们在一起承受着各种的磨难，追求着自己的事业，无论她们的事业之路有多么的艰辛。如《方舟》中的荆华、柳泉、梁倩，三个离婚后的女人自小学毕业分开后，再次聚到一块儿。她们有着同样地痛苦婚姻，她们同样地离了婚，她们同样的在工作上有着重重困难，但又同样的不懈地为事业奋斗。 在小说《方舟》中，我们可以看到两位热忠于事业的女性：柳泉、梁倩。 柳泉，在工作上一丝不苟，能够把所有的工作都安排的井井有条，在短时间内精准的记住那些外宾的住房，她可以轻松自若地翻译。她是那么的用心的将自己的工作做好，在工作中，她会自然地流露出那种自信，在工作之后的酒宴上，她表现出了那种“知识妇女，在意识到自己的聪明才智时才有的微笑”，可以使“每一个正直的男人肃然起敬的微笑”。这充分的透露着她在强烈的事业心的驱使下，工作上得到肯定后的满足与自信。 梁倩，在电影的拍摄上，精益求精，最求高的艺术造诣，竭尽能力的要想做出高的成就，摆脱父亲的名望的荫庇，她要通过自己独立的奋斗，让世人都看见她的成就，认可她的工作，她的付出，她的工作成绩，她要实现她的信仰：电影