脾脏pDCs活化对小鼠心肌缺血再灌注损伤的影响

Effect of activation of splenic plasmacytoid dendritic cells on myocardial ischemia-reperfusion injury in mice

目的评价脾脏浆细胞样树突状细胞(pDCs)活化对小鼠心肌缺血再灌注损伤的影响。方法动物实验:SPF级健康雄性C57BL/6J小鼠36只,10周龄,体质量22~27 g,按照随机数字表法分为3组(n=12):假手术组(Sham组)、心肌缺血组(MI组)和心肌缺血再灌注组(MI/R组)。MI组阻断小鼠左冠状动脉前降支(LAD)40 min,MI/R组阻断LAD 40 min再灌注1 h,Sham组仅穿线不结扎。造模成功后,随机选取各组3只摘取心脏采用TTC-亚甲蓝双染色法测定心肌梗死面积,随机选取各组3只摘取心脏采用HE染色法观察心肌组织病理学结果,其余各组6只先采集腹主动脉血样,采用ELISA法检测血浆干扰素α(IFN-α)浓度,再摘取心脏后收集小鼠心脏灌洗液(CP)。细胞实验:SPF级健康雄性C57BL/6J小鼠12只,10周龄,体质量22~27 g,采用免疫磁珠法分离脾脏pDCs(阳性细胞比例>85%),采用随机数字表法分为Sham组CP刺激pDCs组(pDCs+CP-Sham组)、MI组CP刺激pDCs组(pDCs+CP-MI组)、MI/R组CP刺激pDCs组(pDCs+CP-MI/R组)和PBS刺激pDCs组(pDCs+PBS组),分别采用Sham组、MI组、MI/R组小鼠CP和PBS刺激8 h。采用流式细胞术检测各组pDCs表面CD45、共刺激分子CD80、CD86和主要组织相容性复合物Ⅱ(MHCⅡ)分子的表达,采用ELISA法检测各组细胞培养上清液IFN-α的浓度。结果动物实验:与Sham组和MI组比较,MI/R组小鼠心肌梗死体积百分比升高,血浆IFN-α浓度增加(P<0.05),心肌细胞出现明显空泡变性,心肌纤维断裂严重,大量炎症细胞浸润;Sham组各指标与MI组比较差异无统计学意义(P>0.05)。细胞实验:与pDCs+CP-Sham组比较,pDCs+CP-MI组CD80、CD86和MHCⅡ分子表达上调(P<0.05),pDCs+CP-MI/R组上述指标差异无统计学意义(P>0.05);与pDCs+CP-MI/R组比较,pDCs+CP-MI组上述指标表达上调(P<0.05);与pDCs+CP-Sham组和pDCs+CP-MI/R组比较,pDCs+CP-MI组细胞培养上清液IFN-α浓度增加(P<0.05),pDCs+CP-Sham组与pDCs+CP-MI/R组IFN-α浓度比较差异无统计学意义(P>0.05)。结论小鼠心肌缺血再灌注损伤的机制可能与心肌缺血后脾脏pDCs受刺激活化产生IFN-α有关。

Objective To evaluate the effect of activation of splenic plasmacytoid dendritic cells(pDCs)on myocardial ischemia-reperfusion(I/R)injury in mice.Methods The experiment was performed in two parts.Animal experiment Thirty-six SPF healthy male C57BL/6J mice,aged 10 weeks,weighing 22-27 g,were assigned to 3 groups(n=12 each)using a random number table method:sham operation group(Sham group),myocardial ischemia group(MI group)and myocardial I/R group(MI/R group).The myocardial ischemia was induced by occluding the left anterior descending coronary artery for 40 min in MI group,while the model of myocardial I/R was established by occlusion of the left anterior descending coronary artery for 40 min followed by 1-h reperfusion in MI/R group.Following successful preparation of the model,3 animals from each group were randomly selected,and their hearts were removed for determination of myocardial infarct size through a combination of TTC and methylene blue double staining.Another 3 animals from each group were randomly selected,and their hearts were removed for examination of pathological changes of myocardial tissues using HE staining.Blood samples were collected from the abdominal aorta of 6 mice left in each group for determination of plasma interferon alpha(IFN-α)concentrations by enzyme-linked immunosorbent assay.Then the animals were sacrificed and hearts were harvested for collection of cardiac perfusate(CP).Cell experiment Twelve SPF healthy male C57BL/6J mice,aged 10 weeks,weighing 22-27 g,were selected and the splenic pDCs were isolated using anti-mPDCA-1 MicroBeads according to the manufacturer′s instructions(with a positivity rate of>85%for the isolated cells).The cells were divided into 4 groups:group pDCs stimulated by CP in Sham group(pDCs+CP-Sham group),group pDCs stimulated by CP in MI group(pDCs+CP-MI group),group pDCs stimulated by CP in MI/R group(pDCs+CP-MI/R group)and pDCs stimulated by PBS group(pDCs+PBS group).The CP in Sham,MI and MI/R groups and PBS were used to induce and culture pDCs for 8 h.Flow cytometry was employed to detect the expression of CD45 and co-stimulatory molecules CD80,CD86 and Major Histocompatibility ComplexⅡ(MHCⅡ)on the surface of pDCs.The levels of IFN-αin the cell culture supernatant were determined using enzyme-linked immunosorbent assay.Results Animal experiments Compared with Sham group and MI group,the percentage of myocardial infarct size was significantly increased,the concentrations of plasma IFN-αwere increased(P<0.05),and cardiomyocytes displayed evident vacuolar degeneration,severe myocardial fiber rupture,and infiltration of a substantial number of inflammatory cells in MI/R group.There was no significant difference in each parameter between Sham group and MI group(P>0.05).Cell experiment Compared with pDCs+CP-Sham group,the expression of CD80,CD86 and MHCⅡwas significantly up-regulated in pDCs+CP-MI group(P<0.05),and no significant change was found in the aforementioned parameters in pDCs+CP-MI/R group(P>0.05).The expression of aforementioned parameters was significantly up-regulated in pDCs+CP-MI group as compared with pDCs+CP-MI/R group(P<0.05).Compared with pDCs+CP-Sham group and pDCs+CP-MI/R group,the concentrations of IFN-αin the cell culture supernatant were significantly increased in pDCs+CP-MI group(P<0.05).There was no statistically significant difference in the concentrations of IFN-αbetween pDCs+CP-MI/R group and pDCs+CP-Sham group(P>0.05).Conclusions The mechanism underlying myocardial I/R injury may be related to activation of splenic pDCs leading to the production of IFN-αfollowing myocardial ischemia in mice.