K＋、Cl-离子通道阻滞对体外培养兔关节软骨细胞基质合成代谢的影响  

Effects of potassium and chloride channel blockers on the matrix anabolism of rabbit articular chondrocytes in vitro

作　　者：任立新[1] 王琦[1] 丁娟[1] 杨朝晖[1] 段王平[1] 卫小春[1] 

机构地区：[1]山西医科大学第二医院骨科骨与软组织损伤修复山西省重点实验室,太原030001

出　　处：《中华实验外科杂志》2011年第4期608-610,共3页Chinese Journal of Experimental Surgery

基　　金：基金项目：国家重点基础研究发展计划资助项目（2009CB526514）;国家自然科学基金资助项目（30872616）;山西省基础研究资助项目（2008021045-1、2008012011-2）

摘　　要：目的观察阻断体外培养兔关节软骨细胞膜上的K＋、cl-离子通道对其糖胺多糖（GAG）、II型胶原基质合成代谢的影响。方法2个月龄新西兰白兔，无菌条件下切取双膝关节软骨，酶解法分离软骨细胞，以5×10^4／孔接种于24孔板中。正常培养2d细胞贴壁后换液，并以12个孔为1组随机分为3组，对照组：用DMEM／F12正常培养；K＋离子通道阻滞组（简称4-AP组）：利用含1mmol／L4-氨基吡啶（4一AP）的DMEM／F12培养，特异性阻断电压门控型K＋离子通道；c1一离子通道阻滞组（简称SITS组）：利用含1mmol／L4-乙酰氨基4＋-异硫氰基_2，2＋．乙拌磷酸均二苯乙烯（SITS）的DMEM／F12培养，阻断阴离子通道，主要是Cl-离子通道。分别在换液后第3、6、9天留取各孔上清液并分为2份，1份以阿尔新蓝法检测其GAG的含量，同时另1份以酶联免疫吸附试验（ELISA）法检测Ⅱ型胶原的含量（n=12）。结果4-AP组在第3天时与对照组比较GAG含量明显下降（P〈0．05），但第6天和第9天差异无统计学意义（P〉0．05），同时在第3天和第6天Ⅱ型胶原含量显著增加（P〈0．05），第9天时有下降趋势，但差异无统计学意义（4-AP组GAG含量分别为17．23±1．47、20．98±2．61、33．13±8．13；11型胶原含量分别为0．793±0．214、0．789±0．084、0．715±0．388）；SITS组在第3、6、9天与对照组比较GAG含量都显著增加（P〈0．05），同时在第3天和6天Ⅱ型胶原含量显著增加（P〈0．05），第9天仍然有增加的趋势，但差异无统计学意义（SITS组GAG含量分别为54．30±4．43、56．47±3．14、58．71±2．50；II型胶原含量分别为0．793±0．125、0．853±0．060、0．925±0．053）。结论阻断K＋、cl-离子通道后显著促进软骨细胞GAG、Ⅱ型胶原的合成，尤其是阻断cl-离子通道后，GAG的合成增加尤为明显。Objective To observe the effects of potassium and chloride channel blockers on the glycosaminoglycan （GAG） and collagen type Ⅱ synthesis of rabbit articular chondrocytes in vitro. Meth- ods Two-month-old New Zealand rabbits were killed and their knee joints were taken out under the aseptic condition. The chondrocytes were isolated by enzymolysis method and cuhivated into a 24-well plate （ see- ded 5 ± 104 cells per well）. Then the chondrocytes were divided into three groups randomly after culture for 2 days when the cells were adherent. In the control group the cells were cultured by DMEM/F12, in the potassium channel blocking group [ 4-aminopyridine （4-AP） group ] the cells were cultured in the medium containing 1 mmol/L 4-AP, and in the chloride channel blocking group [ 4-acetamido-4-isothiocyano-2,2- disulfonic acid stilbene （SITS） group] the cells were cultured in the medium containing 1 mmol/L SITS. The GAG synthesis was measured by Alician Blue method and collagen type Ⅱ synthesis was estimated by the enzyme linked immunosorbent assay （ELISA） in the media at the 3rd, 6th and 9th day after medium change （ n = 12）. Results As compared with the control group, the GAG content in 4-AP group was sig- nificantly decreased at the 3rd day （ P 〈 0. 05 ）, but showed no significant difference at 6th and 9th day （ P 〉 O. 05 ） ; the collagen type Ⅱ content was significantly increased at 3rd and 6th day （ P 〈 0. 05 ）, and showed a decrease tendency at 9th day without significant difference （ GAG ： 17.23 ± 1.47, 20.98 ± 2. 61, 33. 13 ±8.13; collagen type Ⅱ ： 0. 793 ±0. 214, 0. 789 ±0. 084, 0. 715 ±0. 388）. The GAG content in the SITS group was significantly increased at the 3rd, 6th and 9th day （P 〈 0.05 ） , while the collagen type Ⅱ content was significantly increased at 3rd and 6th day （P 〈 0. 05）, and showed an increase tendency at 9th day without significant difference （GAG： 54. 30 ±4. 43, 56.47 ± 3. 14, 58.71 ± 2. 50; collagen type �

关 键 词：软骨细胞 离子通道 糖胺多糖 Ⅱ型胶原 

分 类 号：R68[医药卫生—骨科学]