CAJ | 학술논문

目的：探讨Toll样受体4（ TLR4）对氧化型低密度脂蛋白（ oxidized low density lipopro-tein，ox-LDL）诱导的巨噬细胞凋亡的影响及其机制研究。方法体外培养THP-1细胞，用PMA（丙二醇甲醚醋酸酯）诱导成巨噬细胞，分别设立正常对照组、ox-LDL组、ox-LDL＋LPS组、衣霉素组，用MTT和流式细胞术检测细胞存活率及凋亡情况、油红O染色观察细胞吞噬脂质情况，q-RT-PCR、Western blot检测内质网应激相关基因及葡萄糖调节蛋白78（glucose-regulated protein78， GRP78）和CCAAT／增强子结合蛋白同源蛋白（ CCAAT／enhancer-binding protein homologous protein ， CHOP ）的表达情况，并且用TLR4-siRNA抑制TLR4活性观察其对通路的影响。结果流式及MTT结果显示ox-LDL＋LPS组的凋亡细胞数较ox-LDL组明显增加（P＜0．01）， ox-LDL＋LPS组的内质网应激相关基因和蛋白GRP78、CHOP均较ox-LDL组增加，且两组都明显大于正常对照组（ P＜0．01），抑制TLR4活性后内质网应激程度明显减轻（ P＜0．05）。结论 TLR4加重ox-LDL诱导的巨噬细胞凋亡，其机制是加重内质网应激程度，增加CHOP表达，起到促凋亡作用。
목적：탐토Toll양수체4（ TLR4）대양화형저밀도지단백（ oxidized low density lipopro-tein，ox-LDL）유도적거서세포조망적영향급기궤제연구。방법체외배양THP-1세포，용PMA（병이순갑미작산지）유도성거서세포，분별설립정상대조조、ox-LDL조、ox-LDL＋LPS조、의매소조，용MTT화류식세포술검측세포존활솔급조망정황、유홍O염색관찰세포탄서지질정황，q-RT-PCR、Western blot검측내질망응격상관기인급포도당조절단백78（glucose-regulated protein78， GRP78）화CCAAT／증강자결합단백동원단백（ CCAAT／enhancer-binding protein homologous protein ， CHOP ）적표체정황，병차용TLR4-siRNA억제TLR4활성관찰기대통로적영향。결과류식급MTT결과현시ox-LDL＋LPS조적조망세포수교ox-LDL조명현증가（P＜0．01）， ox-LDL＋LPS조적내질망응격상관기인화단백GRP78、CHOP균교ox-LDL조증가，차량조도명현대우정상대조조（ P＜0．01），억제TLR4활성후내질망응격정도명현감경（ P＜0．05）。결론 TLR4가중ox-LDL유도적거서세포조망，기궤제시가중내질망응격정도，증가CHOP표체，기도촉조망작용。
Objective To study the effects of Toll-like receptor 4(TLR4) on oxidized low density lipoprotein ( ox-LDL) induced macrophage apoptosis and its possible mechanism .Methods THP-1 derived macrophages were divided into four groups including untreated control group , ox-LDL treated group , ox-LDL+LPS treated group and tunicamycin treated group .MTT assay and flow cytometry analysis were performed to measure cell vitality and cell apoptosis , respectively .Oil red O staining was used to observe the phagocytosis of lipids by macrophages .The persistent and intense endoplasmic reticulum ( ER) stress markers were de-tected by analyzing the expression of glucose-regulated protein 78 ( GRP78 ) and CCAAT/enhancer-binding protein homologous protein ( CHOP) at mRNA and protein levels by q-RT-PCR and Western blot .Small in-terfering RNA ( siRNA) was used to silence the expression of TLR 4 to further elucidate its possible mecha-nism.Results Flow cytomotry and MTT assay showed that the number of apoptotic cells in ox-LDL+LPS treated group were increased more significantly than that in ox-LDL treated group (P<0.01), and cell apop-tosis in both two groups were greater than that in control group (P<0.01).Compared with control group, the expression of GRP78 and CHOP at mRNA and protein levels were up-regulated in ox-LDL+LPS treated group and ox-LDL treated group (P<0.01), and the expression of GRP78 and CHOP in ox-LDL+LPS treated group was significantly higher than that in ox-LDL treated group (P<0.01).Silenced expression of TLR4 al-leviated the endoplasmic reticulum stress (P<0.05).Conclusion Increased expression of CHOP contribu-ted to cell apoptosis .TLR4 might promote ox-LDL induced macrophage apoptosis through accelerating endo-plasmic reticulum stress .