摘要
目的动态观察Wnt诱导分泌蛋白3(Wnt2 inducible secreted protein 3,WISP3)突变体软骨细胞C20/A4的Ⅱ型胶原合成和分泌特点,观察其在细胞内的超微定位和超微结构变化。方法用脉冲追踪法,在14C标记的脯氨酸干预WISP3突变体软骨细胞C20/A4后的不同时间点(30,60,120,180,240,300min)收集细胞上清和细胞内蛋白,用液体闪烁系统检测胶原含量,用ELISA检测Ⅱ型胶原的量。用透视电镜观察突变型细胞的超微结构变化,用胶体金免疫电镜观察Ⅱ型胶原在突变型细胞内的超微定位。结果 (1)SW1353细胞及C20/A4细胞株在14C标记的脯氨酸干预后180min,分泌到细胞培养上清的胶原及细胞内合成的胶原同时达到高峰;(2)与SW1353细胞及C20/A4细胞株比较,14C标记的脯氨酸干预后,WISP3突变型C20/A4细胞内的Ⅱ型胶原合成高峰提前到120min,而WISP3过表达的C20/A4细胞胶原合成没有明显峰值,胶原分泌高峰均提前到120min,胶原合成和分泌不同步。(3)透视电镜发现WISP3突变型C20/A4细胞有线粒体的肿胀、粗面内质网扩张,核膜皱缩。Ⅱ型胶原主要定位于内质网和细胞膜,排列稀疏,分布不均匀。结论 WISP3突变型C20/A4细胞的胶原合成高峰提前,胶原合成和分泌的规律和节律改变可能是SEDT-PA患者发病的机制之一。WISP3突变型C20/A4细胞超微结构的改变和Ⅱ型胶原的分布改变可能是SEDT-PA发病的病理基础。
Objective To kinetically observe the production and secretion of collagen II in C20/A4 chondracytes with the mutant of Wnt2 inducible secreted protein 3 （WISP3） , and to observe the ultramicrolocalization and structure changes of collagen II in the cell. Methods 14 C-proline was incoporated into C20/A4 cells with or without WISP3 mutant. Intracellular and secretive proteins were collected at different culturing time （30,60,120,180,240, and 300 min）. Isotope liquid scintillation counter and ELISA were used to measure the content of collagen II. Cell ultrastructure was observed using transmissional electron microscope and the localization of type II collagen was determined using immunoelectronmicroscope. Results （1） In SW1353 and C20/A4 ceils, the peak rate of collagen synthesis and secretion was at 180 min after 14 C-proline incoporation. （2） The peak rate of collagen synthesis and secretion was advanced to 120 min after 14C-proline incoporation in C20/A4 cells with WISP3 mutant. No obvious peak rate was observed in C20/A4 cells with WISP3 overexpression. The peak secretion of the collagen was advanced to 120 min. The synthesis and secretion of the collagen were not simultaneous. （3） C20/A4 cells with WISP3 mutant displayed mitochondrion swelling, rough endoplasmic reticulum expanding, and karyotheca wrinkling under the transmissional electron microscope. Collagen II was mainly localized on the rough endoplasmic reticulum and the plasma membrane with sparse and asymmetric distribution. Conclusion The peak synthesis of the collagen is advanced in C20/A4 cells with WISP3 mutant. One of the mechanisms of the pathogenesis of SEDT-PA possibly related to the change in synthesis and secretion of collagen type II. The abnormal distribution and uhrastructure of collagen II in C20/A4 cells with WISP3 mutant could be the pathogenesis of SEDT-PA.
出处
《中国骨质疏松杂志》
CAS
CSCD
2010年第9期625-631,共7页
Chinese Journal of Osteoporosis
