人关节软骨细胞的体外分离、培养与鉴定

目的:研究人关节软骨细胞的体外分离、培养及鉴定方法,观察各代人关节软骨细胞的形态学特性。方法:取人创伤性截肢的无菌膝关节软骨,采用两步酶消化法分离培养人关节软骨细胞,并进行传代培养。通过倒置相差显微镜下观察细胞形态,绘制生长曲线,甲苯胺蓝染色及Ⅱ型胶原免疫组织化学染色对细胞进行鉴定。结果:两步酶消化法消化出的软骨细胞呈圆形,培养2-3天,细胞贴壁、变形,呈三角形或多角形,2周左右细胞融合成层,传代5次后出现去分化。软骨细胞增殖和生长缓慢。形态学、免疫组织化学染色显示细胞培养5代以内可以保持表型的稳定。结论:本研究采用胰蛋白酶及Ⅱ型胶原酶联合消化法获得大量高纯度、高活性的人关节软骨细胞。5代以内细胞生长良好,生物学特性明显,适合于实验研究,5代以后出现去分化现象。     

兔关节软骨细胞体外三维培养条件的优化

实验使用海藻酸钠水凝胶作为细胞支架.模拟软骨细胞体内生长的三维环境,研究了体外三维培养条件下,不同浓度的胎牛血清(FBS)和硒酸复合液(ITS)体系对兔透明软骨细胞(hyaline cartilage)的牛长、增殖和细胞外基质分泌活动的影响.结果 表明,三维模式培养21天,透明软骨细胞仍然具有较好的增殖活性.在硒酸复合液及低浓度血清时,细胞未去分化,保持分泌Ⅱ型胶原(Collagen Ⅱ)和软骨聚集蛋白聚糖(Aggrecan)的能力,与之比较,高浓度胎牛血清(10%)培养条件下,在21天开始细胞去分化,即硒酸复合液在一定的血清浓度下有助于维持软骨细胞生长、增殖,避免了软骨细胞受高浓度血清影响而去分化.     

Chondrogenesis of limb bud mesenchyme in vitro: stimulation by cations

To analyze the nature of cell-cell interactions in chondrogenesis, two cations that influence these interactions, calcium and poly-L-lysine (PL), were tested for their effects on chondrogenesis in vitro. High density cultures of chick limb bud mesenchyme (Hamilton-Hamburger stages 23/24), were exposed to culture media containing calcium (0.6-3.3 mM) or PL (1-10 micrograms/ml). Both cations stimulated chondrogenesis in a dose-dependent manner, and also promoted cartilage formation in normally non-chondrogenic, low cell density cultures. Chondrogenesis was assayed based on cartilage nodule number, [35S]sulfate incorporation, and expression of type II collagen as detected by immunohistochemistry. The calcium effect was not mimicked by other divalent cations (Cd, Co, Ni, Mg, Mn, and Sr). The effect of PL was dependent on its Mr (greater than or equal to 14K) and charge, and was mimicked by poly-D-lysine but not by lysine or other analogs of PL or lysine (epsilon-amino caproic acid, lysozyme, poly-L-arginine, and spermidine). Calcium and PL probably act by different mechanisms since their effects were additive, and required their presence on different days of culture: calcium acted on Day 1, and PL on Day 2. It is proposed that calcium may play a role in the cell aggregation phase of chondrogenesis whereas PL, or a naturally occurring polypeptide of similar nature, may promote chondrogenesis by crosslinking specific anionic components of the cell surface or extracellular matrix.     

Lysophosphatidic Acid Enhanced the Angiogenic Capability of Human Chondrocytes by Regulating Gi/NF-kB-Dependent Angiogenic Factor Expression

Lysophosphatidic acid (LPA) has been found to mediate myeloid differentiation, stimulate osteogenesis, alter cell proliferation and migration, and inhibit apoptosis in chondrocytes. The effect of LPA on the angiogenic capability of chondrocytes is not clear. This study aimed to investigate its effect on the angiogenic capability of human chondrocytes and the underlying mechanism of these effects. Human chondrocyte cell line, CHON-001, commercialized human chondrocytes (HC) derived from normal human articular cartilage, and human vascular endothelial cells (HUVECs) were used as cell models in this study. The angiogenic capability of chondrocytes was determined by capillary tube formation, monolayer permeability, cell migration, and cell proliferation. An angiogenesis protein array kit was used to evaluate the secretion of angiogenic factors in conditioned medium. Angiogenin, insulin-like growth factor-binding protein 1 (IGFBP-1), interleukin (IL)-8, monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase (MMP)-9, and vascular endothelial growth factor (VEGF) mRNA and protein expressions were evaluated by Q-RT-PCR and EIA, respectively. LPA receptor (LPAR) expression was determined by RT-PCR. Signaling pathways were clarified using inhibitors, Western blot analysis, and reporter assays. The LPA treatment promoted the angiogenic capability of CHON-001 cells and HC, resulting in enhanced HUVEC capillary tube formation, monolayer permeability, migration, and cell growth. Angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9, and VEGF mRNA and protein expressions were significantly enhanced in LPA-treated chondrocytes. LPA2, 3, 4 and 6 were expressed in CHON-001 and HC cells. Pretreatment with the Gi/o type G protein inhibitor, pertussis toxin (PTX), and the NF-kB inhibitor, PDTC, significantly inhibited LPA-induced angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9, and VEGF expressions in chondrocytes. The PTX pretreatment also inhibited LPA-mediated NF-kB activation, suggesting the presence of active Gi/NF-kB signaling in CHON-001 and HC cells. The effect of LPA on the angiogenesis-inducing capacity of chondrocytes may be due to the increased angiogenesis factor expression via the Gi/NF-kB signaling pathway.     

Chondrogenesis in mouse limb buds in vitro: Effects of dibutyryl cyclic AMP treatment

Abstract. We studied the effects of dibutyryl cyclic AMP (dbcAMP) on mouse limb-bud chondrogenesis at three stages of embryonic development. After 24 h of culture, limb buds with or without a covering of ectoderm were treated with 1 mM dbcAMP for 48 h and were then compared with untreated cultured limb buds. Treatment with dbcAMP enhanced cartilaginous differentiation in organ cultures of stage-17 and -19 (according to Theiler's) limb buds, although the presence of ectoderm reduced the level of dbcAMP stimulation. By stage 20, treatment with dbcAMP irreversibly inhibited cartilaginous differentiation. These results suggest that the responsiveness of mesenchymal limb-bud cells to dbcAMP is stage related. The results of histological studies as well as of analyses of DNA content and sulphated glycosaminoglycan accumulation supported the hypothesis that dbcAMP treatment induces recruitment of initially non-chondrogenic cells whose commitment explains the enhancement of cartilaginous differentiation. Limb-bud competence for chondrogenesis throughout the three developmental stages studied is also discussed.     

The Emergence of Culture: The Evolution of a Uniquely Human Way of Life

The Emergence of Culture: The Evolution of. Uniquely Human Way of Life . Philip G. Chase. New York: Springer Press, 2006. 227 pp.     

Chondrogenesis in mouse limb buds in vitro: Effects of dibutyryl cyclic AMP treatment

We studied the effects of dibutyryl cyclic AMP (dbcAMP) on mouse limb-bud chondrogenesis at three stages of embryonic development. After 24 h of culture, limb buds with or without a covering of ectoderm were treated with 1 mM dbcAMP for 48 h and were then compared with untreated cultured limb buds. Treatment with dbcAMP enhanced cartilaginous differentiation in organ cultures of stage-17 and -19 (according to Theiler's) limb buds, although the presence of ectoderm reduced the level of dbcAMP stimulation. By stage 20, treatment with dbcAMP irreversibly inhibited cartilaginous differentiation. These results suggest that the responsiveness of mesenchymal limb-bud cells to dbcAMP is stage related. The results of histological studies as well as of analyses of DNA content and sulphated glycosaminoglycan accumulation supported the hypothesis that dbcAMP treatment induces recruitment of initially non-chondrogenic cells whose commitment explains the enhancement of cartilaginous differentiation. Limb-bud competence for chondrogenesis throughout the three developmental stages studied is also discussed.     

Synergistic Action of Transforming Growth Factor-β and Insulin-like Growth Factor-I Induces Expression of Type II Collagen and Aggrecan Genes in Adult Human Articular Chondrocytes

Reexpression of aggrecan and type II collagen genes in dedifferentiated adult human articular chondrocytes (AHAC) in suspension culture varied widely depending on the specific lot of bovine serum used to supplement the culture medium. Some lots of serum provided strong induction of aggrecan and type II collagen expression by AHAC while others did not stimulate significant production of these hyaline cartilage extracellular matrix molecules even following several weeks in culture. Addition of 50 ng/ml insulin-like growth factor-I (IGF-I) to a deficient serum lot significantly enhanced its ability to induce aggrecan and type II collagen mRNA. Given this observation, IGF-I and other growth factors were tested in defined serum-free media for their effects on the expression of these genes. Neither IGF-I nor insulin nor transforming growth factor β (TGF-β) alone stimulated induction of aggrecan or type II collagen production by dedifferentiated AHAC. However, TGF-β1 or TGF-β2 combined with IGF-I or insulin provided a strong induction as demonstrated by RNase protection and immunohistochemical assays. Interestingly, type I collagen, previously shown to be downregulated in serum supplemented suspension cultures of articular chondrocytes, persisted for up to 12 weeks in AHAC cultured in defined medium supplemented with TGF-β and IGF-I.     

Mutant Fusion Proteins with Enhanced Fusion Activity Promote Measles Virus Spread in Human Neuronal Cells and Brains of Suckling Hamsters

Subacute sclerosing panencephalitis (SSPE) is a fatal degenerative disease caused by persistent measles virus (MV) infection in the central nervous system (CNS). From the genetic study of MV isolates obtained from SSPE patients, it is thought that defects of the matrix (M) protein play a crucial role in MV pathogenicity in the CNS. In this study, we report several notable mutations in the extracellular domain of the MV fusion (F) protein, including those found in multiple SSPE strains. The F proteins with these mutations induced syncytium formation in cells lacking SLAM and nectin 4 (receptors used by wild-type MV), including human neuronal cell lines, when expressed together with the attachment protein hemagglutinin. Moreover, recombinant viruses with these mutations exhibited neurovirulence in suckling hamsters, unlike the parental wild-type MV, and the mortality correlated with their fusion activity. In contrast, the recombinant MV lacking the M protein did not induce syncytia in cells lacking SLAM and nectin 4, although it formed larger syncytia in cells with either of the receptors. Since human neuronal cells are mainly SLAM and nectin 4 negative, fusion-enhancing mutations in the extracellular domain of the F protein may greatly contribute to MV spread via cell-to-cell fusion in the CNS, regardless of defects of the M protein.     

Chondrogenesis in mouse limb bud in vitro. I. Effect of the ectoderm

The role of the ectoderm in the chondrogenesis of mouse limb bud mesoderm was investigated in vitro at several developmental stages by analysis of the evolution of DNA content, the accumulation of sulfated glycosaminoglycans and histochemical procedures. Young limb buds or the undifferentiated apex of older buds (stages 17 and 19 of Theiler's table) from which the ectoderm had been removed with trypsin treatment initiated a large chondrogenesis but not morphogenesis. When the ectoderm was present, these limb buds showed a polarized proximal to distal outgrowth and differentiated skeletal primordia. Mesodermal cells of stage 20 limb bud apex were able to differentiate autopodial skeletons with or without the presence of the ectoderm: cartilaginous areas of the limb skeleton seem determined at this developmental stage. These results, which show the importance of the ectoderm in limb bud morphogenesis, are compared with results obtained using other methods with mouse or bird buds.     

Characterization of the Chondrocyte Actin Cytoskeleton in Living Three-Dimensional Culture: Response to Anabolic and Catabolic Stimuli

The actin cytoskeleton is a dynamic network required for intracellular transport, signal transduction, movement, attachment to the extracellular matrix, cellular stiffness and cell shape. Cell shape and the actin cytoskeletal configuration are linked to chondrocyte phenotype with regard to gene expression and matrix synthesis. Historically, the chondrocyte actin cytoskeleton has been studied after formaldehyde fixation - precluding real-time measurements of actin dynamics, or in monolayer cultured cells. Here we characterize the actin cytoskeleton of living low-passage human chondrocytes grown in three-dimensional culture using a stably expressed actin-GFP construct. GFP-actin expression does not substantially alter the production of endogenous actin at the protein level. GFP-actin incorporates into all actin structures stained by fluorescent phalloidin, and does not affect the actin cytoskeleton as seen by fluorescence microscopy. GFP-actin expression does not significantly change the chondrocyte cytosolic stiffness. GFP-actin does not alter the gene expression response to cytokines and growth factors such as IL-1band TGF-b. Finally, GFP-actin does not alter production of extracellular matrix as measured by radiosulfate incorporation. Having established that GFP-actin does not measurably affect the chondrocyte phenotype, we tested the hypothesis that IL-1band TGF-bdifferentially alter the actin cytoskeleton using time-lapse microscopy. TGF-bincreases actin extensions and lamellar ruffling indicative of Rac/CDC42 activation, while IL-1bcauses cellular contraction indicative of RhoA activation. The ability to visualize GFP-actin in living chondrocytes in 3D culture without disrupting the organization or function of the cytoskeleton is an advance in chondrocyte cell biology and provides a powerful tool for future studies in actin-dependent chondrocyte differentiation and mechanotransduction pathways.     

WARP Interacts with Collagen VI-Containing Microfibrils in the Pericellular Matrix of Human Chondrocytes

Collagen VI and WARP are extracellular structural macromolecules present in cartilage and associated with BM suprastructures in non-skeletal tissues. We have previously shown that in WARP-deficient mice, collagen VI is specifically reduced in regions of the peripheral nerve ECM where WARP is expressed, suggesting that both macromolecules are part of the same suprastructure. The object of this study was to conduct a detailed analysis of WARP-collagen VI interactions in vitro in cartilage, a tissue rich in WARP and collagen VI. Immunohistochemical analysis of mouse and human articular cartilage showed that WARP and collagen VI co-localize in the pericellular matrix of superficial zone articular chondrocytes. EM analysis on extracts of human articular cartilage showed that WARP associates closely with collagen VI-containing suprastructures. Additional evidence of an interaction is provided by immunogold EM and immunoblot analysis showing that WARP was present in collagen VI-containing networks isolated from cartilage. Further characterization were done by solid phase binding studies and reconstitution experiments using purified recombinant WARP and isolated collagen VI. Collagen VI binds to WARP with an apparent K_d of approximately 22 nM and the binding site(s) for WARP resides within the triple helical domain since WARP binds to both intact collagen VI tetramers and pepsinized collagen VI. Together, these data confirm and extend our previous findings by demonstrating that WARP and collagen VI form high affinity associations in vivo in cartilage. We conclude that WARP is ideally placed to function as an adapter protein in the cartilage pericellular matrix.     

人软骨细胞压力实验模型的建立与评估

目的:人承重关节内受到的多种机械应力(剪切力、张力、静水压力等)在调节关节软骨细胞的生理功能方面起着重要作用。建立对人膝关节软骨细胞施加不同强度周期性静水压的压力模型,观察不同压力强度下软骨细胞的生长形态、增殖和凋亡情况。方法:采用酶消化法分离培养正常成人膝关节软骨细胞,将培养的第3代软骨细胞分为6组:对照组、0.5 MPa组、1.0 MPa组、3.0MPa组、5.0 MPa组、8.0 MPa组,应用高压恒温静水压加载系统分别给予各组不同强度压力作用5 d,每日1 h。甲苯胺蓝染色法和Ⅱ型胶原免疫组织化学染色法鉴定软骨细胞,倒置相差显微镜观察细胞形态和生长状况,流式细胞术检测细胞凋亡,四甲基偶氮唑蓝(MTT)法绘制细胞生长曲线。结果:与对照组相比,0.5 MPa、3.0 MPa组无明显差异(P0.05);1.0 MPa组能促进软骨细胞增殖,抑制凋亡(P0.05);5.0 MPa组出现细胞增殖能力下降,细胞活力降低,凋亡率增加(P0.05);8.0 MPa组则表现出明显的细胞增殖的抑制和细胞凋亡趋势(P0.05),以及细胞形态学的改变。结论:不同强度的周期性压力对人软骨细胞的新陈代谢产生了不同影响,尤其在软骨细胞的增殖和凋亡水平方面。利用本压力实验模型能体外模拟人负重关节软骨细胞的受压情况,初步确定了人软骨细胞压力实验中压力梯度的选择。为软骨细胞的压力损伤研究提供了实验数据,为进一步探寻压力作用与骨关节炎的关系提供了实验平台。     

Reversion of the Malignant Phenotype of Human Breast Cells in Three-Dimensional Culture and In Vivo by Integrin Blocking Antibodies

In a recently developed human breast cancer model, treatment of tumor cells in a 3-dimensional culture with inhibitory β1-integrin antibody or its Fab fragments led to a striking morphological and functional reversion to a normal phenotype. A stimulatory β1-integrin antibody proved to be ineffective. The newly formed reverted acini re-assembled a basement membrane and re-established E-cadherin–catenin complexes, and re-organized their cytoskeletons. At the same time they downregulated cyclin D1, upregulated p21^cip,waf-1, and stopped growing. Tumor cells treated with the same antibody and injected into nude mice had significantly reduced number and size of tumors in nude mice. The tissue distribution of other integrins was also normalized, suggesting the existence of intimate interactions between the different integrin pathways as well as adherens junctions. On the other hand, nonmalignant cells when treated with either α6 or β4 function altering antibodies continued to grow, and had disorganized colony morphologies resembling the untreated tumor colonies. This shows a significant role of the α6/β4 heterodimer in directing polarity and tissue structure. The observed phenotypes were reversible when the cells were disassociated and the antibodies removed. Our results illustrate that the extracellular matrix and its receptors dictate the phenotype of mammary epithelial cells, and thus in this model system the tissue phenotype is dominant over the cellular genotype.