真菌毒素雪腐镰刀菌烯醇对培养软骨细胞影响的超微结构观察

采用软骨细胞体外单层培养的方法，观察真菌毒素ＮＩＶ对幼兔关节软骨细胞超微结构的作用。结果表明ＮＩＶ对软骨细胞膜系统和细胞浆具有明显的损伤作用，特别是对培养早期的软骨细胞具致命的损伤，以后其损伤程度随毒素浓度的升高而加重。ＮＩＶ与大骨节病的关系有待进一步探讨。     

雪腐镰刀菌烯醇对培养软骨细胞形态和细胞凋亡的影响

目的：研究雪腐镛刀菌烯醇（ＮＩＶ）对培养软骨细胞形态和细胞凋亡的影响。方法：分别用细胞爬片的电镜观察和姬母萨染色光学显微镜观察,以及体外细胞培养再建软骨组织切片ＨＥ染色观察等手段,以图发现其对软骨细胞的损伤。结果：ＮＩＶ低浓度组只引起细胞超微结构轻微改变,ＮＩＶ中、高浓度组引起细胞明显改变,且有凋亡小体的存在,结论：设定ＮＩＶ之低浓度可能是引起软骨细胞病理损伤的最小浓度;ＮＩＶ中、高浓度引起软骨细胞过度凋亡等明显病理损伤。     

脱氧雪腐镰刀菌烯醇（DON）致鸡胚软骨细胞损伤及硒的保…

本实验观察了脱氧雪腐镰刀菌烯醇（ＤＯＮ）对鸡胚关节软骨的作用。结果表明，ＤＯＮ对关节软骨细胞具有明显的毒性作用，其超微结构的改变主要为软骨细胞膜系统的损伤。软骨细胞质膜节段性缺损；线粒体嵴断裂溶解，膜结构模糊；粗面内质网断裂，核蛋白体脱粒；并出现大量变性空泡；软骨基质变疏松、崩解。在给予相同剂量的同时，补充５μｇ硒，软骨细胞损伤程度明显减轻，质膜、核膜均完整，软骨基质未见异常，仅见部分核蛋白体脱粒     

NIV毒素和硒对软骨细胞CD_（44）代谢的影响

目的进一步探讨大骨节病（KBD）的发病机制。方法取4月龄引产胎儿软骨细胞原代分离、培养并随机分为四组。对照组不处理,加硒组加入硒浓度为0.1 mg/L,雪腐镰刀菌烯醇毒素（NIV）组加入NIV毒素使终质量浓度为0.1 mg/L,联合组加硒和NIV毒素,浓度同上。RT-PCR法检测各组CD44 mRNA在软骨细胞中的表达;流式细胞仪检测各组软骨细胞表面CD44蛋白的表达;酶联免疫吸附法检测细胞培养液中可溶性CD44（sCD44）水平。结果 NIV组、联合组软骨细胞CD44 mRNA及蛋白表达水平均明显高于对照组和加硒组（P均〈0.05）。与NIV组比较,联合组CD44表达有所降低,但不明显。NIV组细胞培养液中溶解的sCD44水平最高,其次为联合组,对照组水平最低。结论 NIV毒素和硒能影响软骨细胞CD44的代谢,这可能是造成软骨细胞损伤的机制。     

FA促进培养软骨细胞及其质发生异常矿化

采用视酸（Ｒｅｔｉｎｏｉｃａｃｉｄ，ＲＡ）、抗坏血酸（ＶｉｔａｍｉｎＣ，Ｖｃ）比较研究大骨节病病区黄腐（Ｆｕｌｖｉｃａｃｉｄ，ＦＡ）对体外培养肥大软骨细胞及基质的作用，发现病区ＦＡ可促进贴壁单层培养的软骨细胞形成异常的胞外基质，促进异常矿化。病区ＦＡ刺软骨细胞产生的活性氧使近细胞膜的软骨囊内蛋白颗粒减少，细胞白纤维消失；粗长的胶原蛋白变细，胶原蛋白连接方式滨束状排列变炒杂乱的网状；造成蛋白多糖结构     

脱氧雪腐镰刀菌烯醇（DON）致鸡胚软骨细胞损伤及硒的保护效应

本实验观察了脱氧雪腐镰刀菌烯醇（ＤＯＮ）对鸡胚关节软骨的作用。结果表明，ＤＯＮ对关节软骨细胞具有明显的毒性作用，其超微结构的改变主要为软骨细胞膜系统的损伤。软骨细胞质膜节段性缺损；线粒体嵴断裂溶解，膜结构模糊；粗面内质网断裂，核蛋白体脱粒；并出现大量变性空泡；软骨基质变疏松、崩解。在给予相同剂量的同时，补充５μｇ硒，软骨细胞损伤程度明显减轻，质膜、核膜均完整，软骨基质未见异常，仅见部分核蛋白体脱粒。本实验证实ＤＯＮ引起骨细胞损伤及硒具有保护作用，可以初步认为在大骨节病的发病上，镰刀菌毒素具有不可忽视的作用。     

脱氧雪腐镰刀菌烯醇对培养软骨细胞影响的超微结构观察

采用体外软骨细胞培养方法，观察脱氧雪腐镰刀菌烯醇（ＤＯＮ）对幼兔关节软骨细胞超微结构的作用，结果表明ＤＯＮ对软骨细胞膜系统和细胞器具有明显的损伤作用，特别是对培养早期的软骨细胞具致命损伤。ＤＯＮ与大骨节病关系待进一步探讨。     

交链孢霉甲基醚（AME）对软骨细胞代谢的影响

本文应用ＡＭＥ作用于培养的兔关节软骨细胞，动态观察其对软骨细胞生长代谢的影响。结果表明ＡＭＥ对软骨细胞ＤＮＡ含量无明显影响，而能使软骨基质中葡萄糖醛酸（ＧｌｃＵＡ）含量降低，在培养第９日ＡＭＥ与葡萄糖醛酸含量呈显著负相关关系。提示ＡＭＥ对软骨组织的毒性作用，在于抑制成熟期软骨细胞合成和分泌蛋白多糖。     

镰刀菌毒素对软骨细胞的损伤及硒的保护作用

单端孢霉烯族化合物Ｔ－２毒素引起鸡胚关节软骨产生变性改变，其超微结构的变化主要为软骨细胞膜系统的损伤。具体表现为软骨细胞质膜节段性缺损；线粒体肿胀，嵴断裂、变短、溶解，部分线粒体空泡化；粗面内质网呈池状扩张增多；细胞核电子密度增高核膜扩张形成核周池，并出现节段性破损；胞浆疏松，胞浆内出现髓样小体和变性空泡；软骨细胞边缘软骨基质变疏松。硒可以保护Ｔ－２毒素对软骨细胞的损伤。Ｔ－２毒素对软骨细胞的损…     

慢性丙型肝炎患者外周血单个核细胞中HCV RNA和NS5抗原的检测

目的 研究丙型肝炎病毒（ＨＣＶ）感染外周血个核细胞（ＰＢＭＣ）的情况及其意义。方法 运用非核素原位杂交法（ＮＩＳＨ）和抗生蛋白链菌素－生物素法（ＳＡＢＣ）分别检测２０例慢性丙型肝炎患者ＰＢＭＣ中的ＨＣＶ ＲＮＡ和ＮＳ５抗原。结果 ２０例患者中有８例（４０％）,ＰＢＭＣ中ＨＣＶ ＲＮＡ呈阳性,其中６例（３０％）ＰＢＭＣ中ＮＳ５抗原同时呈阳性。ＨＣＶＲＮＡ主要分布于胞浆中,而ＮＳ５抗原还可以出现在胞膜     

榆林大骨节病区水中黄腐酸对培养软骨细胞作用的观察

目的；本实验观察黄腐酸（ＦＡ）对软细胞，结构，生长，代谢和功能的影响，方法：用大骨节病病区水中ＦＡ，以不同浓度（５ｍｇ／Ｌ，１０ｍｇ／Ｌ，２０ｍｇ／Ｌ）作用于体外培养的兔关节软骨细胞，检测结果作Ｆ检验进行统计学处理，结果：显示实验组织细胞膜功能，蛋白质，ＤＮＡ，蛋白聚糖等不低于对照组或高于对照组，结论：ＦＡ对培养软骨细胞的ＤＮＡ合成，分裂增殖和细胞结构等没有影响。     

逆转录病毒载体介导人胰岛素样生长因子-Ⅰ在软骨细胞中的表达

目的 探讨逆转录病毒载体介导人胰岛索样生长因子-Ⅰ(human insulin-like growth factor-I,hIGF-I)体外转染兔软骨细胞后软骨细胞hIGF-I的表达情况及其生物学行为变化.方法 将构建的含有目的 基因的逆转录病毒载体pLNc-IGF-GFP体外转染兔关节软骨细胞,经G418筛选阳性克隆,采用RT-PCR及免疫化学染色检测转染软骨细胞中hIGF-I的表达情况,并在荧光显微镜下观察标记基因GFP的表达情况.采用MTT法、流式细胞仪、免疫细胞化学染色、二甲基驱甲蓝法及ELISA法对转染hIGF-I后的软骨细胞的增殖能力及表型进行检测.结果 G418筛选后获得的软骨细胞阳性克隆,经RT-PCR和免疫细胞化学检测表明hIGF-1基因在mRNA和蛋白质水平得到稳定表达,荧光显微镜下观察转染的软骨细胞可激发出绿色荧光,证明转染成功.转染软骨细胞株的增殖能力、Ⅱ型胶原和蛋白多糖的分泌水平,在转染后6 w内均高于同时间点未转染的软骨细胞,差异具有显著性(P<0.05).结论 逆转录病毒载体能有效地将hIGF-I基因转染至兔软骨细胞并获得稳定表达,同时转染后的软骨细胞增生活跃,能够在较长时间维持软骨细胞表型,为进一步研究软骨缺损的组织工程修复和基因治疗打下基础.     

Repair of articular cartilage defect by autologous transplantation of basic fibroblast growth factor gene-transduced chondrocytes with adeno-associated virus vector

OBJECTIVE: To examine the effects of basic fibroblast growth factor (bFGF) gene-transduced chondrocytes on the repair of articular cartilage defects. METHODS: LacZ gene or bFGF gene was transduced into primary isolated rabbit chondrocytes with the use of a recombinant adeno-associated virus (AAV) vector. These gene-transduced chondrocytes were embedded in collagen gel and transplanted into a full-thickness defect in the articular cartilage of the patellar groove of a rabbit. The efficiency of gene transduction was assessed according to the percentage of LacZ-positive cells among the total number of living cells. The concentration of bFGF in the culture supernatant was measured by enzyme-linked immunosorbent assay to confirm the production by bFGF gene-transduced chondrocytes. At 4, 8, and 12 weeks after transplantation, cartilage repair was evaluated histologically and graded semiquantitatively using a histologic scoring system ranging from 0 (complete regeneration) to 14 (no regeneration) points. RESULTS: LacZ gene expression by chondrocytes was maintained until 8 weeks in >85% of the in vitro population. LacZ-positive cells were found at the transplant sites for at least 4 weeks after surgery. The mean concentration of bFGF was significantly increased in bFGF gene-transduced cells compared with control cells (P < 0.01). Semiquantitative histologic scoring indicated that the total score was significantly lower in the bFGF-transduced group than in the control group throughout the observation period. CONCLUSION: These results demonstrated that gene transfer to chondrocytes by an ex vivo method was established with the AAV vector, and transplantation of bFGF gene-transduced chondrocytes had a clear beneficial effect on the repair of rabbit articular cartilage defects.     

A citrus flavonoid, nobiletin, suppresses production and gene expression of matrix metalloproteinase 9/gelatinase B in rabbit synovial fibroblasts

OBJECTIVE: Flavonoids including nobiletin are known to exert many biological actions in vitro. We investigated the chondroprotective effect of citrus flavonoids, especially nobiletin, using cultured rabbit synovial fibroblasts and articular chondrocytes. METHODS: We examined the effects of citrus flavonoids on the production and gene expression of matrix metalloproteinases (MMP) and prostaglandin E2 (PGE2)production in rabbit synovial fibroblasts. RESULTS: Six flavonoids isolated from Citrus depressa Rutaceae including tangeretin, 6-demethoxytangeretin, nobiletin, 5-demethylnobiletin, 6-demethoxynobiletin, and sinensetin suppressed the interleukin 1 (IL-1) induced production of proMMP-9/progelatinase B in rabbit synovial cells in a dose dependent manner (<64 microM); nobiletin most effectively suppressed proMMP-9 production along with the decrease in its mRNA. Nobiletin also reduced IL-1 induced production of PGE2 in the synovial cells, but did not modify the synthesis of total protein. These suppressive effects of nobiletin were also observed in rabbit articular chondrocytes. Nobiletin inhibited proliferation of rabbit synovial fibroblasts in the growth phase. CONCLUSION: These results suggest nobiletin is a novel antiinflammatory candidate that has the potential to inhibit PGE2 production, matrix degradation of the articular cartilage, and pannus formation in osteoarthritis and rheumatoid arthritis.     

Biologic effects of an interleukin-1 receptor antagonist protein on interleukin-1-stimulated cartilage erosion and chondrocyte responsiveness

Recombinant human interleukin-1 alpha (IL-1 alpha) and recombinant human IL-1 beta stimulate matrix proteoglycan degradation and inhibit glycosaminoglycan synthesis in bovine nasal cartilage explants. A 17-kd human recombinant IL-1 receptor antagonist protein (IRAP) caused a concentration-dependent (0.2-200 ng/ml) suppression of the effects of IL-1 alpha and IL-1 beta in cartilage organ cultures. IRAP inhibited the binding of radiolabeled IL-1 alpha to rabbit articular chondrocytes. Matrix metalloproteinase (collagenase, gelatinase, and stromelysin) and prostanoid production by IL-1-activated rabbit articular chondrocytes was also suppressed by IRAP. These results could have potential significance in the development of a new antiarthritis therapy based on an IRAP.     

Effects of transforming growth factor beta s and basic fibroblast growth factor on articular chondrocytes obtained from immobilised rabbit knees.

OBJECTIVE: To clarify the effects of transforming growth factor beta 1 (TGF beta 1), TGF beta 2, and basic fibroblast growth factor (bFGF) on cell proliferation and proteoglycan (PG) synthesis in articular chondrocytes obtained from immobilised rabbit knees. METHODS: The right knees of rabbits were immobilised in full extension for up to 42 days using fiberglass casts. Specimens for histology were stained with safranin O. Chondrocytes were isolated from the weight bearing regions of the femur and tibia of the immobilised knees and cultured with combinations of growth factors. Cell proliferation and PG synthesis were determined by 3H-thymidine and 35S-sulphate incorporations. RESULTS: Histological study revealed loss of metachromasia in the articular cartilage at seven days, fissuring and cell clusters at 28 days, and loss of cartilage layers 42 days after immobilisation. Radioisotope assay of the chondrocytes revealed no remarkable change in DNA synthesis in the presence of either TGF beta 1 or TGF beta 2 alone. bFGF markedly stimulated cell proliferation in specimens obtained 0 to seven days after immobilisation. The combination of either TGF beta 1 or TGF beta 2 with bFGF had a synergistic effect, inducing significant increases in DNA synthesis four, seven, and 14 days after immobilisation. PG synthesis by chondrocytes from immobilised joints was not significantly altered by these agents. CONCLUSION: TGF beta 1 or TGF beta 2 in combination with bFGF exert synergistic effects on cell proliferation in articular chondrocytes obtained from the rabbit knee during the early days after immobilisation by a cast. These results suggest a critical role of cytokine combinations in the development of articular cartilage degeneration after immobilisation.     

Effect of platelet lysate on growth and sulfated glycosaminoglycan synthesis in articular chondrocyte cultures

Human platelet lysate (PL) has a strong growth promoting action on rabbit articular chondrocytes in monolayer culture. The responsible factor is heat stable (56°C, 30 minutes) and above 10,000 MW. PL (80 μg protein/ml) reduces cell protein content and sulfated glycosaminoglycan synthesis. Synthesis of DNA is stimulated within the first 12 hours of culture but the decline in radiosulfact incorporation lags. PL acts to a slight extent on chondrocytes in serum-free media, but its effect is potentiated by “platelet-poor human serum” or fetal bovine serum. PL is one of several agents having such effects on chondrocytes cultured in serum-containing media. Stimulation of growth in this cell type thus reduces nonreplicative biosynthetic activity nonspecifically. In the epiphyseal growth plate and in pathologic alterations of permeability of the martix of articular cartilage, platelet-derived factors, together with somatomedin or other cofactors in serum, may be the principal mediator of growth of chondrocytes in vivo.     

Action of multiplication-stimulating activity on [3H]thymidine incorporation in rabbit and human fetal chondrocytes in vitro

The mitogenic action of multiplication-stimulating activity (MSA) on normal mammalian chondrocytes has been examined. Addition of MSA (NIH, PkII-MSA, 2.5-500 ng/ml or Collaborative Research, CR-MSA, 50-250 ng/ml) to primary suspensions of chondrocytes prepared by enzymic digestion of costal and articular cartilage of rabbits (356-481 g body wt) resulted in a dose-dependent increase in [3H]thymidine incorporation into the trichloroacetic acid-precipitated cell contents. CR-MSA (50-250 ng/ml) also had a significant stimulatory effect on [3H]thymidine incorporation into human fetal chondrocytes (22 weeks of gestation) prepared by enzymic digestion. When PkII-MSA was added in the presence of 1.25% of a standard adult or cord plasma to either rabbit or human fetal (18 weeks) chondrocytes, the increase in [3H]thymidine incorporation appeared to be synergistic. The mitogenic action of MSA can thus be demonstrated on primary suspensions of mammalian chondrocytes. The action of MSA on human chondrocytes has not previously been reported.