Effect of the disruption of three cytoskeleton components on chondrocyte metabolism in rabbit knee cartilage
Effect of the disruption of three cytoskeleton components on chondrocyte metabolism in rabbit knee cartilage
复制标题
三种细胞骨架成分破坏对兔膝关节软骨细胞代谢的影响
DOI：
10.3760/cma.j.issn.0366-6999.20140337
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发表时间：
2014-11-05
影响因子：
6.1
通讯作者：
Wei Xiaochun
中科院分区：
文献类型：
--
作者：
Duan Wangping;Wei Lei;Wei Xiaochun
背景软骨细胞的表型和基质的生物合成依赖于具有完整的细胞骨架结构。微丝、微管和中间丝是软骨细胞骨架结构的三个重要组成部分。本研究的目的是确定和比较这三种细胞骨架元素的破坏对兔膝关节软骨细胞的凋亡和基质合成的影响，在vitro.Methods软骨细胞分离全层膝关节软骨的两个月大的兔使用酶的方法（n=24）。分离的细胞稳定三天，然后暴露于低，中，高剂量的化学试剂，破坏三个主要的细胞骨架元素的利益：秋水仙素的微管，丙烯酰胺的中间丝，和细胞松弛素D的肌动蛋白微丝。一组对照细胞用载体处理。在暴露于破坏性化学试剂后的第1天和第2天，通过流式细胞术使用膜联蛋白-FITC结合测定评估早期细胞凋亡。在第3天通过激光扫描共聚焦显微镜（LSCM）结合免疫荧光染色分析细胞内细胞骨架的组成和分布。采用实时荧光定量PCR和酶联免疫吸附试验（ELISA）检测细胞培养第3、6、9天细胞聚集蛋白聚糖（AGG）和II型胶原（Col-2）mRNA水平及培养液中AGG和Col-2 mRNA水平。结果在初始药物剂量反应研究中，0.1 μ mol/L秋水仙碱、2.5 mmol/L丙烯酰胺、和10 μ g/L细胞松弛素D处理2天。上述选择的秋水仙素和丙烯酰胺处理浓度在9天培养中显著降低了活细胞的数量，并破坏了显著更多的细胞核。实时荧光定量PCR和ELISA结果表明，AGG和Col-2的mRNA水平和介质浓度显着降低与秋水仙素和丙烯酰胺处理的培养物相比，未经处理的细胞在3，6，和9天，这种抑制与较高的基质金属蛋白酶-13在这些细胞中的表达。细胞增殖，单层形态，基质代谢不受影响，在细胞松弛素D处理的细胞相比，在9天的culture.Conclusions的微管蛋白和中间丝的破坏诱导软骨细胞凋亡，增加基质金属蛋白酶的表达，并降低AGG和Col-2的表达在兔膝关节软骨细胞培养。我们的研究结果表明，微管蛋白和中间丝在兔膝关节软骨细胞合成软骨基质中起着关键作用。
Background Chondrocytes' phenotype and biosynthesis of matrix are dependent on having an intact cytoskeletal structure. Microfilaments, microtubules, and intermediate filaments are three important components of the cytoskeletal structure of chondrocytes. The aims of this study were to determine and compare the effects of the disruption of these three cytoskeletal elements on the apoptosis and matrix synthesis by rabbit knee chondrocytes in vitro.Methods Chondrocytes were isolated from full-thickness knee cartilage of two-month-old rabbits using enzymatic methods (n=24). The isolated cells were stabilized for three days and then exposed to low, medium, and high doses of chemical agents that disrupt the three principal cytoskeletal elements of interest: colchicine for microtubules, acrylamide for intermediate filaments, and cytochalasin D for actin microfilaments. A group of control cells were treated with carrier. Early apoptosis was assessed using the Annexin-FITC binding assay by flow cytometry on days 1 and 2 after exposure to the disrupting chemical agents. The components and distribution of the cytoskeleton within the cells were analyzed by laser scanning confocal microscopy (LSCM) with immunofluorescence staining on day 3. The mRNA levels of aggrecan (AGG) and type II collagen (Col-2) and their levels in culture medium were analyzed using real-time PCR and enzyme-linked immunosorbent serologic assay (ELISA) on days 3, 6, and 9.Results In the initial drug-dose response study, there was no significant difference in the vitality of cells treated with 0.1 mu mol/L colchicine, 2.5 mmol/L acrylamide, and 10 mu g/L cytochalasin D for two days when compared with the control group of cells. The concentrations of colchicine and acrylamide treatment selected above significantly decreased the number of viable cells over the nine-day culture and disrupted significantly more cell nuclei. Real-time PCR and ELISA results showed that the mRNA levels and medium concentrations of AGG and Col-2 were significantly decreased for cultures treated with colchicine and acrylamide when compared with untreated cells at three, six, and nine days, and this inhibition was correlated with higher matrix metalloprotease-13 expression in these cells. Cellular proliferation, monolayer morphology, and matrix metabolism were unaffected in cytochalasin D-treated cells when compared with control cells over the nine-day culture period.Conclusions The disruption of the microtubulin and intermediate filaments induced chondrocyte apoptosis, increased matrix metalloprotease expression, and decreased AGG and Col-2 expression in rabbit knee chondrocyte cultures. Our findings suggest that microtubulin and intermediate filaments play a critical role in the synthesis of cartilage matrix by rabbit knee chondrocytes.