干扰SEDL基因表达对软骨细胞生理活性的影响

The influence of interferential SEDL gene in the physiological activity of human chondrocytes

目的初步研究干扰SEDL基因表达后对人软骨细胞中II型胶原蛋白表达水平、内质网位置分布及超微结构和软骨细胞活性的影响。方法将靶向干扰SEDL基因的病毒转染入人软骨细胞中,QPCR及Western Blot分别检测SEDL基因及Sedlin蛋白表达水平,评价干扰SEDL基因的软骨细胞模型是否建立成功;免疫荧光及Western Blot检测II型胶原蛋白表达情况,荧光探针检测内质网位置分布改变,电镜检测内质网超微结构改变,MTT法检测软骨细胞增殖活性情况。结果 (1)转染干扰SEDL基因病毒的软骨细胞SEDL基因表达明显降低,具有统计学差异(P<0.05)。Sedlin蛋白表达降低,表明干扰SEDL基因的细胞模型构建成功。(2)随着Sedlin蛋白的减少,II型胶原蛋白表达量降低,具有统计学差异(P<0.05)。(3)细胞模型中内质网分布发生改变,其形态变圆,触角变少,颜色变淡,细胞与细胞间内质网荧光排列不规则,紊乱。(4)电镜下观察,细胞模型中内质网超微结构出现水肿扩张。(5)转染干扰SEDL基因病毒的软骨细胞活性在48 h达峰,之后细胞活性下降,且具有统计学差异(P<0.05)。结论 Sedlin降低影响软骨细胞II型胶原蛋白表达,软骨细胞中内质网形态位置分布及超微结构发生改变,致使软骨细胞活性降低。

Objective To preliminarily study theinfluenceof interferential SEDL genein theexpression of collagen II,distribution and ultrastructureof endoplasmic reticulum,and theactivity of human chondrocytes. Methods Lentivirus which can target interferenceSEDL genewas transfected into human cartilagecells（ HC-a）. Theexpression of SEDL geneand Sedlin protein weredetected using QPCR and Western blotting. Theestablishment of cartilagecell model with SEDT genewas evaluated. Theexpression of collagen II was detected using Western blotting and immunofluorescence. Theultrastructural changes of endoplasmic reticulum weretested using electron microscope. MTT method was used to detect theproliferation of thecartilagecells. Results（ 1） Theexpression of SEDL genein thecartilagecells was significantly reduced after lentivirus was transfected（ P〈0. 05）,and theexpression of Sedlin protein was also decreased,indicating that theSEDT cell model was successfully established.（ 2） With thedecreaseof expression of Sedlin protein,theexpression of collagen II was observably reduced（ P〈0. 05）.（ 3） Thedistribution of endoplasmic reticulum was changed in thecell model. Thecells becameround in shape,less protrusion,fadein color,and theendoplasmic reticulum arranged irregularly.（ 4） Theedematous endoplasmic reticulum was observed under transmission electron microscope.（ 5） Activity of thecells reached peak within 48 h after lentivirus transfection. Then thecell activity significantly declined（ P〈0. 05）. Conclusion Thereduced expression of Sedlin results in thedeclineof collagen II expression. Thechanged distribution and ultra microstructureof endoplasmic reticulum result in a decreaseof cartilagecell activity.