氯化钆诱导大鼠腹腔巨噬细胞内质网应激及凋亡探究.docVIP

氯化钆诱导大鼠腹腔巨噬细胞内质网应激及凋亡探究【摘要】 目的 探讨氯化钆对大鼠腹腔巨噬细胞内质网应激和凋亡的作用。方法分离和培养大鼠腹腔巨噬细胞(PMΦ),分别经氯化钆(Gd)和衣霉素(Tunicamycin)处理后,采用细胞 免疫组化S-P法检测ARMET的表达,免疫荧光双标染色检测ARMET和CHOP的表达,PI染色观察对细胞凋亡的影响。结果 氯化钆处理组细胞数目减少,胞体变小变圆;ARMET在氯化钆组(5×10-6mol/L)的阳性细胞百分数为(42.1±6.6)%,在正常对照组的阳性率为(6.1±1.6)%,差异有统计学意义(P0.05);免疫荧光双标染色提示氯化钆组ARMET和CHOP均高表达;PI染色可见氯化钆组细胞被深染、胞核断裂等凋亡特征。结论 氯化钆可诱导大鼠腹腔巨噬细胞内质网应激和凋亡,其作用机制可能与胞内钙超载引起的非折叠蛋白反应有关。 【关键词】 氯化钆;巨噬细胞;内质网应激;凋亡 Study of endoplasmic reticulum stress and apoptosis of peritoneal macrophage in ratinduced by Gadolinium chloride HUANG Xue-gui, ZHAO Jun.Hefei Women and Children Health Center, Hefei230000, China 【Abstract】 Objective To investigate the effect of endoplasmic reticulum stress and apoptosis of peritoneal macrophage in rat induced by Gadolinium chloride. Methods Rat peritoneal macrophage(PMΦ) were isolated and cultured. After PMΦ treated with Gadolinium chloride and Tunicamycin, the expression of ARMET was determined by immunohistochemical method, immunoflurorescent double staining were employed to detect the expression of CHOP and ARMET, PI stain was used to observe PMΦs apoptosis. Results The amount of cells were greatly reduced and the cell body were shrinked to round in GdCl3group. The percentage of cell ARMET positive-expressed were (42.1±6.6)% in GdCl3group(5×10-6 mol/L), while the value of control group were(6.1±1.6)%, the difference between the two groups was significant(P 1.1.2 PMΦ给药方案 将PMΦ培养至第7 d,细胞融合达到80%时进行给药处理。试验分为正常对照组、GdCl3组、Tunicamycin组。①正常对照组:分为两组,每组细胞分别加入与② 、③组等量的生理盐水与DMSO溶媒。②GdCl3组:精密称取GdCl3.6H2O晶体7.9 mg溶于生理盐水,充分溶解后过滤除菌,得5×10-3M的母液,取母液4 μl加入4 ml DMEM中,混匀后换液,每孔加1 ml,培养24 h后处理。③Tunicamycin组:取用DMSO配制的2.5 mg/ml的母液用DMEM按1:1000稀释,每孔加1 ml,培养4 h后处理。以上各组均设四个复孔。 1.2.3 细胞免疫化学 PBS摇洗10 min×2次,用预冷的4%多聚甲醛4℃固定30 min, 再用含0.1% BSA+0.1%皂角素的PBS,4℃封闭30 min。每孔滴加一抗(ARMET,1:20,用含0.1% BSA+0.1%皂角素的PBS稀释)100 μl,4℃孵育过夜。次日PBS洗后滴加带HRP标记的猪抗兔二抗(1:100)37℃孵育1 h,DAB显色,苏木素复染,盐酸酒精分色,蓝化,滴加DAPI染核, PBS洗10 min×2次后, 荧光显微镜下观察,摄像。 1.2.4 免疫化学阳性细胞计数 在DAB显色的细胞组织化学