【作者】 任立新；

【导师】 卫小春；

【作者基本信息】 山西医科大学 ， 骨外科学， 2010， 硕士

【摘要】 目的：探讨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(glycosaminoglycan, GAG)、Ⅱ型胶原基质合成代谢的影响。方法：2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5×104/孔接种于24孔板中。正常培养2天细胞贴壁后换液,并以12个孔为一组随机分为三组,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lmmol/L4-氨基吡啶(4-aminopyridine,4-AP)的DMEM/F12培养,特异性阻断电压门控型K离子通道；CL离子通道阻滞组(简称SITS组)：利用含lmmol/L 4-乙酰氨基-4‘-异硫氰基-2,2’-乙拌磷酸均二苯乙烯(4-acetamido-4’-isothiocyano-2,2’-disulfonic acid stilbene, 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两份,一份以阿尔新蓝法检测其GAG的含量,同时另一份以ELISA法检测Ⅱ型胶原的含量(n=12)。结果：与对照组比较,4-AP组在第3天时GAG含量明显下降(P<0.05),但第6天和9天并无显著差别(p>0.05),同时Ⅱ型胶原在第3天和6天含量显著增加(P<0.05),第9天时有下降趋势,但统计学检验无明显差异；SITS组在第3、6、9天GAG含量都显著增加(P<0.05),同时Ⅱ型胶原在第3天和6天含量显著增加(P<0.05),第9天仍然有增加的趋势,但统计学检验无明显差异。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。更多还原

【Abstract】 Objective:To investgate the effects of potassium and chloride channel blockers on The glycosaminoglycan(GAG) and collagen type II synthesis of rabbit articular chondrocytes in vitro.Methods:2 months New Zealand rabbits were killed and their knee joint were taken out under aseptic condition. The chondrocytes were isolated by enzymolysis method and cultivated in 24-well plates (seeded 5×104 cells per well).Then the chondrocytes were divided into three groups randomly after cultured 2 days when the cells were adherent. The control group was cultured by DMEM/F12 while the potassium channel blockon group(4-AP group)was cultured in the media contained 1mmol/L 4-aminopyridine (4-AP), which can lead to the blockon of the potassium channel,and the chloride channel blockon group(SITS group)was cultured in the media contained 1mmol/L 4-acetamido-4-isothiocyano-2,2-disulfonic acid stilbene(SITS), which can lead to the blockon of the chloride channel.The GAG synthesis were measured by Alician Blue method and collagen typeⅡsynthesis were estimated by the ELISA in the media when medium was change after3-,6-and 9-day (n=12)Result:Compared with the control group.the GAG content of 4-AP group has significant decrease at 3th day(P<0.05),but has no significant difference at 6th and 9th day (p>0.05),while the collagen typeⅡcontent has significant increased at 3th and 6th day (P<0.05), and has decrease tendency at 9th day. but have no significant difference.The GAG content of SITS group has significant increased at 3th,6th and 9th day (P<0.05).while the collagen typeⅡcontent has significant increased at 3th and 6th day (P<0.05),and has increased tendency at 9th day, but have no significant difference.Conclusion:The blockon of potassium and chloride channel can increase the GAG and collagen typeⅡsynthesis of chondrocytes in vitro, especially when block chloride channel,the GAG synthesis was significant.更多还原