ox-LDL诱导泡沫细胞形成中AnnexinⅡ的表达改变及意义

Role of AnnexinⅡ in the Formation of Foam Cells Induced by Ox-LDL

目的观察AnnexinⅡ在氧化型低密度脂蛋白(ox-LDL)诱导泡沫细胞形成中表达的改变及其与细胞内胆固醇酯(CE)/总胆固醇(TC)比值(CE/TC)改变的相关性,从而探讨AnnexinⅡ在ox-LDL诱导泡沫细胞形成中的意义。方法常规培养RAW264.7单核细胞株,待细胞长至90%汇合度时,分别加入不同浓度的ox-LDL(50、100、200 mg/L)孵育24 h,以未经氧化修饰的天然低密度脂蛋白(n-LDL,200 mg/L)为阴性对照,测定细胞内TC和CE含量,油红O染色鉴定泡沫细胞;比色法测定乳酸脱氢酶(LDH)释出量和MTT法检测细胞存活率来观察oxLDL对细胞的毒性作用;RT-PCR和Western blot检测AnnexinⅡmRNA和蛋白的表达水平。结果不同浓度的oxLDL(50、100、200 mg/L)孵育24 h,仅200 mg/L ox-LDL组细胞活力显著降低,LDH释出量显著升高,与n-LDL(200mg/L)组比较差异具有显著性(P<0.01),50 mg/L和100 mg/L ox-LDL对细胞活力和LDH释出量无显著影响(P>0.05);CE/TC比值分别是55.0%±6.7%、61.8%±4.8%和59.6%±5.2%,与n-LDL(200 mg/L)组比较差异均具有显著性(P<0.05),其中浓度为100 mg/L的ox-LDL作用最显著;ox-LDL呈浓度依赖性抑制AnnexinⅡmRNA和蛋白的表达,AnnexinⅡmRNA和蛋白的表达改变与CE/TC比值呈负相关(r_1=-0.9374,P<0.05;r_2=-0.9548,P<0.05)。结论 ox-LDL呈浓度依赖性抑制AnnexinⅡ表达,提示其可能影响细胞内胆固醇自平衡从而诱导泡沫细胞形成。

Aim The purpose of the study was to observe the expression change of Annexin II and the correla- tionship between the expression levels of Annexin I1 and the ratio of intracellular total cholesterol （TC） to cholesterol ester （CE） and to explore the significance of Annexin lI in the formation of foam cells induced by ox-LDL. Methods RAW264.7 monocyte cell lines with a 90% confluency were exposed to ox-LDL （50, 100 and 200 mg,/L） and native LDL （n-LDL, 200 rag/L） for 24 h, respectively, and the following parameters such as TC, CE, the release of LDH, cell via- bihty and expression levels of Annexin I1 were detected by colorimetry,Western blot and RT-PCR etc. Results Our data showed that the release of LDH and cell viability in exposure to ox-LDL （200 mg/L） for 24 h were increased and decreased significantly and there were statistically significant differences （P〈0. 01） compared with the n-LDL （200 mg/L） group. However, the release of LDH and cell viability in exposure to 50 mg/L and 100 mg/L of ox-LDL had no remarkable chan- ges （P〉0.05）. Moreover, the ratio of CE/TC were 55.0%±6.7%, 61.8%±4.8% and 59.6%±5.2%, respectively exposed to ox-LDL （50, 100 and 200 mg/L） and there were significant differences compared with n-LDL （200 rag/L） （P〈0. 05） , and peaked at the 100 mg/L of ox-LDL. In addition, further results showed that ox-LDL inhibited the expression of Annexin II mRNA and protein in a concentration-dependence manner and the expression levels of Annexin 11 mRNA and protein were negatively correlated with the ratio of CE/TC （rI =-0.9374, P〈0. 05; r2 =-0.9548, P〈0. 05）. Conclusion Ox-LDL inhibited the expression of Annexin lI mRNA and protein in a concentration-dependent manner, which suggested that it may affect the intracellular cholesterol self balance and induce the formation of foam cells.