细胞骨架破坏对体外培养兔软骨细胞基质代谢分泌的影响

Effects of cytoskeleton destruction on secretion and matrix metabolism of rabbit cartilage Cells in vitro

目的观察微丝、微管和中间纤维3种细胞骨架（CSK）成分破坏对体外培养关节软骨细胞基质代谢的影响。方法取2个月龄新西兰大白兔10只，取双膝关节软骨行软骨细胞培养，随机分为4组，即正常对照组、微丝破坏组（加入0．01mg／L细胞松弛素一D破坏肌动蛋白）、微管破坏组（加入0．4mg／L秋水仙素破坏微管蛋白）、中间纤维破坏组（加入175mg／L丙烯酰胺破坏波形蛋白）。培养3d后，各组取部分细胞消化并爬片，行免疫荧光染色及共聚焦显微镜荧光值半定量检测，观察细胞微丝蛋白、微管蛋白和波形蛋白的形态及含量变化。并于培养第3、6、9天取细胞上清液，用酶联免疫吸附试验（ELISA）法和阿尔新蓝法检测各组上清液中Ⅱ型胶原和糖胺多糖（GAG）的含量。结果微丝、微管、中间纤维破坏组与对照组比较，荧光度值分别从83．70、82．82、77．93降至60．45、57．86、55．56（P〈0．05，n=30）。微管破坏组和中间纤维破坏组在第3、6、9天上清液中的Ⅱ型胶原和GAG含量均明显低于对照组（P〈0．05），而微丝破坏组与对照组在第3、6、9天上清液中的Ⅱ型胶原和GAG含量差异均无统计学意义（P〉0．05）。结论预设浓度的CSK破坏剂可以破坏软骨细胞相应的CSK成分，且微管和中间纤维破坏组软骨细胞基质代谢水平显著下降，而微丝破坏组对软骨细胞基质代谢的水平无明显影响。

Objective Rearch the changes of secretion and matrix metabolism in cartilage cells when the three protein component filaments of chondrocyte cytoskeleton （CSK） were destroyed. Methods 10 New Zealand white rabbits aged 2 months, We took the knee cartilage, obtain the cells using two-step enzymatic digestion and elimination method for cell culture . To be adherent cells, were randomly divided into four groups, namely normal control group, microfilament damage group （adding 0.01 mg/L of cytoch- alasin-D destroy actin）, microtubule damage group （adding 0. 4 mg/L colchicines destroy tubulin）, inter- mediate filament damage group （ adding 175 mg/L of acrylamide destroy vimentin）. Cultured for 3 days, some cells from each group were digested and climbing films, line by immunofluorescence staining and con- focal fluorescence microscope, semi-quantitative detection. The cells were observed actin protein tubulin and vimentin form and content. On the 3rd, 6th and 9th day, obtained the cultured supernatant of each group detected type II collagen and proteoglycan （GAG） content by ELISA method and Aleian blue. Re- sults The composition of three cytoskeletal proteins, the average value of the corresponding analysis of fluorescence, showing microfilaments, microtubules, intermediate filaments destruction group compared with the control group fluorescence, fluorescence values were reduced from 83.70,82. 82,77.93 to 60.45, 57.86,55.56 （P 〈 0. 05, n = 30）. Meanwhile, the destruction of microtubules and intermediate fiber group on the 3rd, 6th and 9th day, type II collagen and GAG were significantly lower than the control group （P 〈0.05）, while the destruction of microfilaments group on the 3rd, 6th and 9th day collagen II and GAG content were not statistically different with control group （P 〉 0.05）. Conclusion The concen- tration of CSK default agents can destroy the CSK components. Chondrocyte matrix metabolism of microtu- bules and intermediate filament groups were significantly decreased, while microfilament destruction had no effect on cartilage matrix metabolism.