共查询到20条相似文献,搜索用时 0 毫秒
1.
Pierre-andr Guerne Francisco Blanco Andr Kaelin Alain Desgeorges Martin Lotz 《Arthritis \u0026amp; Rheumatology》1995,38(7):960-968
Objective. To examine growth factor responses of human articular chondrocytes in aging and development. We have previously established a growth factor response profile for adult human articular chondrocytes and shown that transforming growth factor β (TGFβ) is the most potent mitogen among a variety of factors tested. Methods. Chondrocytes were isolated from human articular cartilage obtained from donors ages 11–83 years and tested in primary culture for proliferative responses to serum and recombinant preparations of the major chondrocyte growth factors. Proliferation was measured by 3H-thymidine incorporation and cell counting. Skeletal maturity of the young donors was determined by radiographic assessment of Risser's index. Results. Chondrocytes showed a continuous age-related decline in the proliferative response to serum. Analysis of recombinant growth factors showed that with increasing donor age, there was a decrease in the levels of DNA synthesis in response to all factors tested. In chondrocytes from adult donors, there was no change in the relative potencies of the different growth factors. The decrease in the levels of DNA synthesis as measured by 3H-thymidine incorporation corresponded to a reduced rate of in vitro cell replication with increasing donor age. In addition to the quantitative changes in the proliferative responses of chondrocytes with increasing age, there was a qualitative change in the pattern of growth factor responses during development. Cells from young donors (ages 10–20) responded better to platelet-derived growth factor, AA chain homodimer (PDGF-AA) than to TGFβ1, while the inverse pattern was seen in cells from adult donors. This decrease in the response to PDGF-AA was significantly correlated with increasing skeletal maturity. Conclusion. Chondrocyte growth factor responsiveness shows qualitative changes during development, and after skeletal maturity, there is a profound decline in the levels of DNA synthesis and cell replication in response to the known chondrocyte growth factors. 相似文献
2.
Objective. Transglutaminases (TGases) (E.G. 2.3.2.13) catalyze a posttranslational modification of proteins and are associated with biomineralization in growth plate cartilage. Type II TGase participates in the activation of latent transforming growth factor β (TGFß), a crucial factor for both normal cartilage mineralization and the pathologic mineralization that results in calcium pyrophosphate dihydrate (CPPD) crystal formation in aging articular cartilage. To explore a possible association between TGase levels and CPPD crystal formation in mature articular cartilage, TGase activity in articular chondrocytes from old and young pigs and in the articular cartilage vesicle (ACV) fraction of porcine articular cartilage was examined. In addition, the effects of TGase inhibitors on the production of inorganic pyrophosphate (PPi), a process necessary for CPPD crystallogenesis, were determined. Methods. TGase activity was measured with a radiometric assay in cultured articular chondrocytes from the knee joints of old (3–5 years old) and young (2–6 weeks old) pigs and in the ACVs. PPi levels were measured in chondrocyte-conditioned media in the presence of TGase inhibitors or control compounds. Results. Levels of TGase activity in the cytosolic fraction of old chondrocytes were 7-fold higher than those in identically cultured young chondrocytes. The mean ± SD activity level in the membrane fraction of lysed chondrocytes was 6.0 ± 0.6 units/mg protein in old articular chondrocytes and was undetectable in young chondrocytes. In ACVs, the mean ± SD TGase activity level was 1.23 ± 0.1 units/mg protein. Type II TGase protein was present in chondrocyte cytosol and in ACVs. TGase activity was increased by TGFß to 120% of control values (P < 0.01), and decreased by insulin-like growth factor 1 to 80% of control values (P < 0.01). TGase inhibitors blocked media accumulation of PPi, an essential precursor of CPPD crystal formation, and a sensitive marker of TGFß effect. Conclusion. These data suggest a potential link between TGase activity and processes of pathologic biomineralization that result in CPPD crystal formation in aging articular cartilage. 相似文献
3.
Ando K Yahata T Sato T Miyatake H Matsuzawa H Oki M Miyoshi H Tsuji T Kato S Hotta T 《Blood》2006,107(8):3371-3377
To characterize human hematopoietic stem cells (HSCs), xenotransplantation techniques such as the severe combined immunodeficiency (SCID) mouse repopulating cell (SRC) assay have proven the most reliable methods thus far. While SRC quantification by limiting dilution analysis (LDA) is the gold standard for measuring in vitro expansion of human HSCs, LDA is a statistical method and does not directly establish that a single HSC has self-renewed in vitro. This would require a direct clonal method and has not been done. By using lentiviral gene marking and direct intra-bone marrow injection of cultured CD34+ CB cells, we demonstrate here the first direct evidence for self-renewal of individual SRC clones in vitro. Of 74 clones analyzed, 20 clones (27%) divided and repopulated in more than 2 mice after serum-free and stroma-dependent culture. Some of the clones were secondary transplantable. This indicates symmetric self-renewal divisions in vitro. On the other hand, 54 clones (73%) present in only 1 mouse may result from asymmetric divisions in vitro. Our data demonstrate that current ex vivo expansion conditions result in reliable stem cell expansion and the clonal tracking we have employed is the only reliable method that can be used in the development of clinically appropriate expansion methods. 相似文献
4.
Virgil L. Woods Paul J. Schreck Dirk S. Gesink Hector O. Pacheco David Amiel Wayne H. Akeson Martin Lotz 《Arthritis \u0026amp; Rheumatology》1994,37(4):537-544
Objective. To perform a comprehensive analysis of the integrin forms expressed by normal human articular chondrocytes. Methods. Cartilage sections and collagenase-released chondrocytes were probed with a comprehensive panel of integrin isoform–specific monoclonal antibodies (MAb), using in situ immunohistochemistry techniques, indirect immunofluorescence and flow cytometry, and immunoprecipitation/sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Results. Chondrocytes in cartilage sections reacted with MAb specific for the α5, αv, and β1 integrin subunits and the αvβ3 and αvβ5 heterodimers. They also reacted with a polyclonal antibody specific for the intracytoplasmic portion of the α1 subunit. MAb specific for the αv subunit reacted more strongly with chondrocytes near the articular surface than with those in deeper layers of cartilage, and the αvβ3-specific MAb reacted exclusively with chondrocytes within the most superficial 30 μm of cartilage. Flow cytometric analysis and SDS-PAGE analysis of immunoprecipitates prepared from extracts of cell-surface radioiodinated chondrocytes confirmed the above observations, and additionally revealed the presence of the α3β1 integrin. Conclusion. Normal human articular chondrocytes prominently display substantial quantities of the α1β1, α5β1, and αvβ5 integrin heterodimers, as well as lesser quantities of the α3β1 and αvβ3 heterodimers, The αv subunit–containing integrins are detected more readily on the more superficial chondrocytes than on chondrocytes deep within cartilage. These observations provide the basis for analysis of the role of chondrocyte integrins in cartilage homeostasis and in the pathogenesis of joint diseases. 相似文献
5.
Hu DN Yang PY Ku MC Chu CH Lim AY Hwang MH 《The Kaohsiung journal of medical sciences》2002,18(3):113-120
Adult human articular chondrocytes were isolated from surgically excised articular cartilage from 15 patients suffering from trauma or disease of the knee. Cells were isolated with an enzymatic digestion method and cultured with F12 medium supplemented with serum. The effects of serum and basic fibroblast growth factor (bFGF) on the growth of cultured chondrocytes were studied. Serum stimulated the growth of chondrocytes at concentrations from 1-30%. bFGF significantly stimulated the growth of chondrocytes in a dose-dependent manner at concentrations from 1-100 ng/ml. Chondrocytes grew well in F12 medium supplemented with 10% fetal bovine serum (FBS). These cultured chondrocytes could be passaged for many generations and revealed an average of 2,702-fold increase of cell numbers during 2-6 months period (cumulative population doublings = 11 times). Cumulative population doublings increased to 24 times in cell cultured with medium supplemented with bFGF (10 ng/ml). Immunocytochemical study using anti-S-100 antibodies demonstrated that these cultures were pure cell cultures of chondrocytes. We have demonstrated that cell cultures of adult human articular chondrocytes can be established with these methods. Cultured chondrocytes provide an in vitro model system for studying the physiology and pathology of human articular chondrocytes and may be used for autologous transplantation of chondrocytes to treat articular cartilage defects. 相似文献
6.
Ex vivo expansion of hematopoietic stem cells (HSCs) is important for many clinical applications, and knowledge of the surface phenotype of ex vivo-expanded HSCs will be critical to their purification and analysis. Here, we developed a simple culture system for bone marrow (BM) HSCs using low levels of stem cell factor (SCF), thrombopoietin (TPO), insulin-like growth factor 2 (IGF-2), and fibroblast growth factor-1 (FGF-1) in serum-free medium. As measured by competitive repopulation analyses, there was a more than 20-fold increase in numbers of long-term (LT)-HSCs after a 10-day culture of total BM cells. Culture of BM "side population" (SP) cells, a highly enriched stem cell population, for 10 days resulted in an approximate 8-fold expansion of repopulating HSCs. Similar to freshly isolated HSCs, repopulating HSCs after culture were positive for the stem cell markers Sca-1, Kit, and CD31 and receptors for IGF-2. Surprisingly, prion protein and Tie-2, which are present on freshly isolated HSCs, were not on cultured HSCs. Two other HSC markers, Endoglin and Mpl, were expressed only on a portion of cultured HSCs. Therefore, the surface phenotype of ex vivo-expanded HSCs is different from that of freshly isolated HSCs, but this plasticity of surface phenotype does not significantly alter their repopulation capability. 相似文献
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8.
Apoptosis and megakaryocytic differentiation during ex vivo expansion of human cord blood CD34+ cells using thrombopoietin 总被引:1,自引:0,他引:1
Ryu KH Chun S Carbonierre S Im SA Kim HL Shin MH Shin HY Ahn HS Woo SY Seoh JY Fraser JK 《British journal of haematology》2001,113(2):470-478
Thrombopoietin (TPO), the primary regulator of megakaryocytopoiesis, plays important roles in early haematopoiesis. Previously, we have demonstrated that TPO induces a characteristic pattern of apoptosis during ex vivo expansion of cord blood (CB) CD34+ cells. In this study, we have demonstrated that the TPO-induced apoptotic cells belong to the megakaryocytic (MK) lineage and that initially expanding MK progenitors declined along with the appearance of TPO-induced apoptosis. Human CB CD34+ cells were expanded in serum-free conditions with TPO. Multidimensional flow cytometry using simultaneous measurement of apoptosis and immunophenotyping showed that the TPO-induced apoptotic cells appeared in CD61+ fractions. Immunocytochemical analysis of the fluorescent activated cell-sorted fractions showed that the apoptosis-associated CD44low fraction expressed CD61. Clonogenic assay revealed 7.4 +/- 0.50-fold increase of total megakaryocyte colony-forming units (CFU-MKs) during the initial 9 d. Thereafter, the number of CFU-MKs decreased in parallel with the increase of apoptosis. When the MK colonies were subdivided according to size, the proportion of large colonies progressively decreased, while that of medium and small colonies increased. In particular, from d 6 small colonies became predominant. These results suggested that the MK progenitors matured as they expanded during ex vivo expansion with TPO and then proceeded to apoptosis. 相似文献
9.
Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. 总被引:155,自引:12,他引:155
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It is here reported that mesenchymal stem cells known to give rise to limb-bud mesoderm can, at the single-cell level, also differentiate into cells of visceral mesoderm and can be expanded extensively by means of clinically applicable methods. These cells were named mesodermal progenitor cells (MPCs). MPCs were selected by depleting bone marrow mononuclear cells from more than 30 healthy human donors of CD45(+)/glycophorin-A (GlyA)(+) cells. Cells were cultured on fibronectin with epidermal growth factor and platelet-derived growth factor BB and 2% or less fetal calf serum. It was found that 1/5 x 10(3) CD45(-)GlyA(-) cells, or 1/10(6) bone marrow mononuclear cells, gave rise to clusters of small adherent cells. Cell-doubling time was 48 to 72 hours, and cells have been expanded in culture for more than 60 cell doublings. MPCs are CD34(-), CD44(low), CD45(-), CD117 (cKit)(-), class I-HLA(-), and HLA-DR(-). MPCs differentiated into cells of limb-bud mesoderm (osteoblasts, chondrocytes, adipocytes, stroma cells, and skeletal myoblasts) as well as visceral mesoderm (endothelial cells). Retroviral marking was used to definitively prove that single MPCs can differentiate into cells of limb bud and visceral mesoderm. Thus, MPCs that proliferate without obvious senescence under clinically applicable conditions and differentiate at the single-cell level not only into mesenchymal cells but also cells of visceral mesoderm may be an ideal source of stem cells for treatment of genetic or degenerative disorders affecting cells of mesodermal origin. 相似文献
10.
Long-term ex vivo maintenance and expansion of transplantable human hematopoietic stem cells. 总被引:11,自引:4,他引:7
We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34(+) thy-1(+) cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34(+) thy-1(+) cells on AC6.21 stroma, a murine bone marrow-derived stromal cell line, caused expansion of cells with CD34(+) thy-1(+) phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34(+) thy-1(+) phenotype. The ex vivo-expanded CD34(+) thy-1(+) cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34(+) thy-1(+) cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34(+) CD38(-) cells, shows a similar pattern of proliferative response. This suggests that ex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34(+) thy-1(+) cells. 相似文献
11.
12.
OBJECTIVE: To determine 1) the kinetics and strength of adhesion of human articular chondrocytes to a cut cartilage surface, and 2) the role of specific integrins in mediating such adhesion, using an in vitro model. METHODS: Human articular chondrocytes isolated from cadaveric donors (mean +/- SD age 38 +/- 13 years) were cultured in high-density or low-density monolayer. Following release from culture with trypsin and a 2-2.5-hour recovery period, chondrocytes were analyzed either for adhesion to cartilage or for integrin expression by flow cytometry. RESULTS: Following culture in monolayer, adhesion of chondrocytes to cartilage increased with time, from 6-16% at 10 minutes to a maximum of 59-82% at 80-320 minutes. After 80 minutes of adhesion, the resistance of cells to flow-induced shear stress (50% detachment) was approximately 21 Pa. Chondrocyte adhesion to cartilage decreased with pretreatment of cells with monoclonal antibodies that bound to and blocked certain integrins. After an 80-minute incubation time, adhesion of chondrocytes cultured in high-density monolayer decreased from the value of IgG1-treated controls (55%) with blocking of the beta1 integrin subunit (to 23%) or with blocking of alpha 5 beta 1 (to 36%). Following expansion of chondrocytes in low-density monolayer, the mechanisms of adhesion to cartilage were generally similar. After an 80-minute incubation time, adhesion of chondrocytes cultured in low-density monolayer decreased from the value of IgG1-treated controls (62%) with blocking of the beta1 integrin subunit (to 30%) or with blocking of alpha 5 beta 1 (to 44%). Additionally, adhesion of these cells decreased to 46% by blocking of alpha v beta 5, with a similar trend in effect for chondrocytes cultured in high-density monolayer. Blocking of the alpha 1 or alpha 3 integrin subunits or alpha v beta 3 had no detectable effect on adhesion, even though these receptors were detected by flow cytometry. CONCLUSION: Under the culture and seeding conditions studied, beta1, alpha 5 beta 1, and alpha v beta 5 integrins mediate human chondrocyte adhesion to cartilage. These chondrocyte integrins have a potential role in the initial adhesion and retention of chondrocytes at a cartilage defect site following clinical procedures of chondrocyte transplantation. 相似文献
13.
人胚胎关节软骨细胞库的建立 总被引:2,自引:0,他引:2
目的:体外建立人胚胎关节软骨细胞冻存、复苏的稳定技术,为软骨组织工程提供大量的、具有软骨细胞生物活性的种子细胞。方法:将传代培养的人胚胎关节软骨细胞用含10%DMSO、50%胎牛血清的IMDM培养液悬浮后,置于-196℃的液氮中长期保存,建立人胚胎关节软骨细胞库。然后将细胞库中的冻存细胞进行复苏培养,观察其生长状况,并测定软骨细胞中DNA及糖醛酸的含量。结果:冻存软骨细胞复苏后仍保持旺盛的增殖与分泌细胞外基质的功能。结论:关节软骨细胞库的建立,可以长期保存培养的软骨细胞。 相似文献
14.
Increased matrix metalloproteinase-13 production with aging by human articular chondrocytes in response to catabolic stimuli 总被引:1,自引:0,他引:1
Forsyth CB Cole A Murphy G Bienias JL Im HJ Loeser RF 《The journals of gerontology. Series A, Biological sciences and medical sciences》2005,60(9):1118-1124
Chondrocyte anabolic activity has been shown to decline with aging, but catabolic activity has received little attention. In this study, the effect of aging on the chondrocyte catabolic response was determined by stimulating isolated human chondrocytes with fibronectin fragments (FN-f) or interleukin-1beta and measuring matrix metalloproteinase-13 (MMP-13) production as a catabolic response. A significant age-related increase in chondrocyte MMP-13 production was noted. FN-f stimulation of MMP-13 expression was blocked using a nuclear factor kappa-B (NFkappaB) inhibitor suggesting a role for NFkappaB in this chondrocyte catabolic response. Chondrocyte production of the NFkappaB-regulated cytokine interleukin-1beta was also found to increase with donor age in unstimulated cells. These results demonstrate a significant age-related increase in chondrocyte catabolic responsiveness which could contribute to the development of osteoarthritis in older adults. 相似文献
15.
VLA-4 expression and cell cycling status during ex vivo expansion of human cord blood CD34+ cells 总被引:1,自引:0,他引:1
Seoh JY 《Acta haematologica》2001,105(2):111-115
16.
Regulation of YKL-40 production by human articular chondrocytes 总被引:6,自引:0,他引:6
OBJECTIVE: YKL-40 (human cartilage glycoprotein 39) is one of the most abundant proteins secreted by cultured chondrocytes. The objectives of the present study were to identify regulators of YKL-40 production in cartilage and chondrocytes and to map the localization of YKL-40 in chondrocytes. METHODS: Human articular chondrocytes and cartilage explants (obtained from subjects at autopsy, from a tissue bank, and from osteoarthritis [OA] patients undergoing total joint replacement surgery) were stimulated with cytokines, growth factors, and other agents. YKL-40 expression was analyzed by Northern blot and polymerase chain reaction. YKL-40 secretion into the media was determined by enzyme-linked immunosorbent assay. RESULTS: YKL-40 production increased to very high levels during the early phase of chondrocyte monolayer culture and in normal cartilage explant cultures as a response to tissue injury. Spontaneous YKL-40 release was higher in OA than in normal cartilage explant cultures. In chondrocyte monolayer cultures, interleukin-1beta (IL-1beta) and transforming growth factor beta (TGFbeta) decreased the levels of secreted YKL-40, and this was associated with a reduction in YKL-40 messenger RNA levels. IL-1beta, but not TGFbeta, reduced YKL-40 production in cartilage explant cultures. Media from explants treated with cycloheximide had no detectable YKL-40, suggesting that the released protein was newly synthesized. Immunofluorescence microscopy showed YKL-40 staining in the Golgi system of the chondrocytes, but YKL-40 could not be detected in the extracellular matrix. CONCLUSION: The spontaneous increase in the production of YKL-40 in the early phase of culture appears to represent a cellular response to changes in the extracellular matrix environment. This, coupled with the profound suppressive effects of IL-1beta and TGFbeta on YKL-40 production, identifies a novel regulatory pattern for this major chondrocyte-derived protein. 相似文献
17.
S C Gilman 《The Journal of rheumatology》1987,14(5):1002-1007
Treatment of rabbit articular chondrocytes with 0.5-5 units/ml of recombinant human interleukin-1 (IL-1) induced phospholipase A2 (PLA2) activation, prostaglandin E2 (PGE2) biosynthesis and latent neutral protease secretion by these cells. When normalized on the basis of thymocyte costimulatory activity, the beta (pI 7) form of recombinant IL-1 was about 5-fold more potent than the alpha (pI 5) species, although the maximum response induced by either IL-1 form was similar. Recombinant murine IL-1 was also a potent dose dependent activator of chondrocyte arachidonate metabolism and protease secretion. In contrast to IL-1, neither IL-2 nor tumor necrosis factor alpha (TNF alpha) activated PLA2, PGE2, or neutral protease secretion in these cells and neither of these cytokines inhibited the chondrocyte metabolic response to IL-1. These results provide unequivocal evidence that IL-1 is a potent activator of chondrocyte arachidonate metabolism and hydrolytic protease secretion. That all 3 recombinant IL-1 molecules stimulate chondrocytes and yet share limited sequence homology suggests that an amino acid sequence common to all 3 species is required for chondrocyte activation. 相似文献
18.
V M Srivastava C J Malemud A J Hough J H Bland L Sokoloff 《Arthritis and rheumatism》1974,17(2):165-169
19.
S Alsalameh B Jahn A Krause J R Kalden G R Burmester 《The Journal of rheumatology》1991,18(3):414-421
It is postulated that chondrocytes may be actively involved in the pathogenesis of inflammatory joint diseases, presumably by providing tissue specific antigens that may initiate or sustain autoimmune reactions. To investigate whether chondrocytes may also function as accessory cells in ongoing immune processes, mixed leukocyte-chondrocyte cultures and antigen presentation assays were studied. Freshly isolated and short term cultured HLA class II antigen (Ia) negative as well as gamma-interferon treated Ia positive chondrocytes were weakly or not stimulatory to allogeneic or autologous resting lymphocytes derived from either normal donors or patients with rheumatoid arthritis. In an antigen presenting system using tetanus toxoid, the majority of chondrocyte preparations tested induced an antigen driven response in HLA matched allogeneic or autologous resting T cells which, however, was much less when compared to blood monocytes. In contrast, using activated T cells derived from tetanus toxoid specific T cell lines, an efficient antigen presenting capacity could be demonstrated in both Ia positive and initially Ia negative chondrocytes. Interestingly, the latter population had acquired Ia antigens upon incubation with the T cell line. 相似文献
20.