摘要目的 明确人β防御素2(human β-defensin-2,hBD2)基因氧化型低密度脂蛋白(oxidized low-density lipoprotein,OX-LDL)诱导的人单核巨噬细胞(human leukemic monocyte ,THP-1)泡沫化的作用和机制.方法 用OX-LDL诱导THP-1细胞建立单核细胞泡沫化模型并通过油红O染色进行模型鉴定.通过慢病毒系统在THP-1细胞中过表达hBD2基因,并检测hBD2过表达对OX-LDL诱导的THP-1细胞泡沫化的影响.酶联免疫吸附试验测定各组细胞上清液炎症因子肿瘤坏死因子( tumor necrosis factor, TNF)-α、白细胞介素(interleukin, IL)-1β和IL-6含量.组间差异比较采用t检验.结果 重组病毒感染后72 h,细胞的基因转染效率接近100%.细胞对照组hBD2蛋白水平为0.122 ±0.024,对照病毒感染组为0.123 ±0.022,hBD-2感染组为0.981 ±0.183,细胞对照与hBD-2感染组比较差异有统计学意义(t＝-3.175,P＝0.007);病毒感染后72 h,细胞对照组hBD2基因mRNA相对表达量为0.131 ± 0.021,对照病毒感染组为0.128 ±0.022, hBD-2感染组为1.001 ±0.105,细胞对照组与hBD-2感染组比较差异有统计学意义(t＝-7.213,P＝0.003).油红O染色结果表明,OX-LDL诱导THP-1细胞72 h,能够明显引起细胞内脂质堆积,hBD2过表达则可以有效抑制OX-LDL诱导引起的THP-1细胞内脂质堆积. THP-1组和THP-1+Lv-hBD2+OX-LDL诱导组hBD2 mRNA相对表达量均显著高于THP-1+OX-LDL诱导组,差异有统计学意义(t值分别为3.237和-6.021,P值分别为0.004和0.003),THP-1组和THP-1+Lv-hBD2+OX-LDL诱导组hBD2蛋白水平均显著高于THP-1+OX-LDL诱导组,差异均有统计学意义(t值分别为0.314和-4.061,P值分别为0.006和0.007),THP-1细胞经OX-LDL诱导72 h,细胞上清液中TNF-α、IL-1β和IL-6 含量明显上升,与THP-1 组比较差异均有统计学意义( t 值分别为-3.825、-2.017和-3.551, P值分别为0.007、 0.004和0.005);THP-1+Lv-hBD2+OX-LDL组细胞中TNF-α、 IL-1β和IL-6含量明显降低,与THP-1+OX-LDL组比较差异均有统计学意义(t值分别为4.132、 3.681和2.991,P值分别为0.003、 0.002和0.007).结论 hBD2能够有效抑制OX-LDL诱导的THP-1泡沫化,且可能与其抑制OX-LDL诱导的THP-1细胞炎性反应有关.

abstractsObjective To clarify the role of human β-defensin2 ( hBD2) on preventing oxidized low-density lipoprotein (OX-LDL) induced human leukemic monocyte (THP-1) foaming.Methods The monocyte foaming model was established using THP-1 cell induced by OX-LDL and the model was identified by oil red staining.The hBD2 was overexpressed on THP-1 cells by using lentivirus system and the effect of hBD 2 overexpression on THP-1 cell foaming induced by OX-LDL was detected.The levels of inflammatory factors including tumor necrosis factor (TNF)-α, interleukin (IL)-1βand IL-6 in cell supernatant of each group were detected by enzyme-linked immunosorbent assay ( ELISA).Differences between the groups were compared by using the t test.Results The gene transfection efficiency of the cells was close to 100%at 72 h after infection. The hBD2 protein levels were 0.122 ±0.024 in the control group, 0.123 ±0.022 in Lv-control infection group and 0.981 ±0.183 in Lv-hBD2 infection group; and the level in control group was statistically higher than that in hBD-2 infection group (t＝-3.175, P＝0.007).The relative levels of hBD2 mRNA at 72 h after virus infection were 0.131 ±0.021 in control group, 0.128 ±0.022 in Lv-control group and 1.001 ±0.105 in Lv-hBD2 infection group; and the level in control group was statistically higher than that in hBD-2 infection group (t＝-7.213, P＝0.003).The results of oil red staining showed that OX-LDL inducing THP-1 cells for 72 h could significantly induce lipid accumulation in cells.Overexpression of hBD2 could effectively inhibit lipid accumulation in THP-1 cells induced by OX-LDL.The expression of hBD2 mRNA in THP-1 group was significantly higher than that in THP-1+OX-LDL group (t＝3.237, P＝0.004); and the difference was also significant when comparing THP-1+Lv-hBD2+OX-LDL group with THP-1+OX-LDL group (t＝-6.021, P＝0.003).The level of hBD2 protein in THP-1 group was significantly higher than that in THP-1+OX-LDL group (t＝0.314, P＝0.006); and the difference was also significant when comparing THP-1+Lv-hBD2+OX-LDL group with THP-1+OX-LDL group (t＝-4.061,P＝0.007).The levels of TNF-α, IL-1βand IL-6 in the supernatant of THP-1 cells induced by OX-LDL for 72 h were significantly increased compared with those in THP-1group (t＝-3.825,-2.017 and -3.551, respectively; P＝0.007, 0.004 and 0.005, respectively). The levels of TNF-α, IL-1βand IL-6 in THP-1+Lv-hBD2+OX-LDL group were significantly lower than those in THP-1+OX-LDL group ( t＝4.132, 3.681, and 2.991, respectively; P＝0.003, 0.002, and 0.007, respectively).Conclusions hBD2 can effectively inhibit THP-1 foaming induced by OX-LDL, which may be related to its inhibition of inflammatory response.