CAJ | 학술논문

目的：通过研究塑化剂邻苯二甲酸酯对软骨细胞凋亡因子bcl-2和Bax蛋白表达及超微结构的影响，探讨邻苯二甲酸酯在致软骨细胞凋亡中的作用机制。方法：用软骨细胞体外培养的方法，原代培养乳鼠关节软骨细胞，传第3代后加药，按染邻苯二甲酸酯剂量不同分为0／L （对照）、10－10／L、10－8／L、10－6／L组，染邻苯二甲酸酯培养7 d后，透射电镜下观察软骨细胞的超微结构改变，采用western-blot方法检测各实验组中软骨细胞bcl-2和Bax蛋白的表达。结果：透射电镜下对照组和10－10／L组的软骨细胞呈球形，粗面内质网发达，线粒体膜性结构完整；10－8／L 、10－6／L组的细胞内可见脂滴明显增多，胞质内出现大量空泡类物质，细胞内膜结构不清，部分细胞出现核固缩。10－8／L 、10－6／L组的软骨细胞bcl-2的蛋白表达（0．620±0．041和0．530±0．035）较对照组表达（0．805±0．032）显著降低（P＜0．01）；而Bax的表达（0．966±0．038和1．280±0．154）较对照组表达（0．602±0．045）显著增加（P＜0．01）。10－10／L组软骨细胞中bcl-2的表达（0．878±0．055）和Bax的表达（0．650±0．039）与对照组相比均无统计学差异（P＞0．05）。此外，10－8／L 、10－6／L组相比，bcl-2和Bax的表达也有显著差异（P＜0．01）。结论：10－8／L、10－6／L浓度的邻苯二甲酸酯可对软骨细胞超微结构造成损伤，其通过减少抑凋亡因子bcl-2的表达和增加促凋亡因子Bax的表达，从而产生促进软骨细胞凋亡的作用。
목적：통과연구소화제린분이갑산지대연골세포조망인자bcl-2화Bax단백표체급초미결구적영향，탐토린분이갑산지재치연골세포조망중적작용궤제。방법：용연골세포체외배양적방법，원대배양유서관절연골세포，전제3대후가약，안염린분이갑산지제량불동분위0／L （대조）、10－10／L、10－8／L、10－6／L조，염린분이갑산지배양7 d후，투사전경하관찰연골세포적초미결구개변，채용western-blot방법검측각실험조중연골세포bcl-2화Bax단백적표체。결과：투사전경하대조조화10－10／L조적연골세포정구형，조면내질망발체，선립체막성결구완정；10－8／L 、10－6／L조적세포내가견지적명현증다，포질내출현대량공포류물질，세포내막결구불청，부분세포출현핵고축。10－8／L 、10－6／L조적연골세포bcl-2적단백표체（0．620±0．041화0．530±0．035）교대조조표체（0．805±0．032）현저강저（P＜0．01）；이Bax적표체（0．966±0．038화1．280±0．154）교대조조표체（0．602±0．045）현저증가（P＜0．01）。10－10／L조연골세포중bcl-2적표체（0．878±0．055）화Bax적표체（0．650±0．039）여대조조상비균무통계학차이（P＞0．05）。차외，10－8／L 、10－6／L조상비，bcl-2화Bax적표체야유현저차이（P＜0．01）。결론：10－8／L、10－6／L농도적린분이갑산지가대연골세포초미결구조성손상，기통과감소억조망인자bcl-2적표체화증가촉조망인자Bax적표체，종이산생촉진연골세포조망적작용。
Objective:To in vitro investigate the effects phthalic acid esters(PAEs,a pasticizer) on the expression of Bcl-2 and Bax in chondrocytes and ultrastructure modification as well as the mechanisms in generating apoptosis of the chondrocytes .Methods: Primary articular chondrocytes of neonate rat were cultured with in vitro technique by the third generation.Then the chrondrocytes were infected with PAEs by concentration of 0/L,10 -10/L,10 -8/L and 10 -6/L,respectively,for 7 days,and observed under transmission electron microscope to examine the changes of their ultrastructure .Western blot was performed to determine the expression of Bcl-2 and Bax in chondrocytes in different dosage groups.Results: Microscopically,the chrondrocytes in dosage group of 10 -10/L were in round shape,with abundant rough endoplasmic reticulum(RERs) and complete structure of mitochondria membrane,while in dosage groups of 10 -8/L and 10 -6/L,displayed intensified intracellular lipid droplets ,with a number of vacuoles being seen in the cytoplasm ,incomplete structure of the intracellular membranes and partial pyknosis of the chromatine .Bcl-2 expression was significantly down-regulated in dosage group of 10 -8/L(0.620 ±0.041) and 10 -6/L(0.530 ±0.035) compared to the control group(0.805 ±0.032)(P <0.01),whereas Bax significantly increased(0. 966 ±0.038 for 10 -8/L,and 1.280 ±0.154 for 10 -6/L) compared to the control group (Bcl-2:0.602 ±0.045;Bax:0.602 ±0.045) (P<0.01). There was no statistical difference regarding the expression of Bcl-2 or Bax between 10 -10/L group and control group(P>0.05),yet the difference was significant by expression of Bcl-2 and Bax in dosage group of 10 -8/L and 10 -6/L(P<0.01).Conclusion: PAE at concentration of either 10 -8/L or 10 -6/L may lead to damage to the ultrastructure of chondrocyte .The potential mechanism may be involved in inactivation of the anti-apoptotic Bcl-2 and up-regulated Bax,eventually result in apoptosis of the chondrocytes.