Toll样受体4基因敲除对心肌缺血的影响

Effect of Toll-like Receptor 4 Gene Knockout on Myocardial Ischemia

目的:研究干预Toll样受体4(Toll-Like receptor 4,TLR4)对心肌缺血小鼠脾脏中CD20^+细胞和CD68^+细胞的影响。方法:20只6周龄雄性C57BL/6野生型小鼠和20只6周龄雄性TLR4基因敲除(TLR4^-/-)小鼠分别随机设置空白对照组(NC组)、心肌缺血组(MI组)、TLR4^-/-对照组(TLR4^-/-NC组)、TLR4^-/-心肌缺血组(TLR4^-/-MI组),每组各10只。对照组给予腹腔注射20mg/kg生理盐水(NC)7天,心肌缺血组通过给予腹腔注射20mg/kg异丙肾上腺素(isoproterenol,ISO)7天建立心肌缺血模型。造模后使用心脏超声检查小鼠心脏射血分数(ejection fraction,EF)和左室短轴缩短率(left ventricular fractional shortening,LVFS),苏木精-伊红(hematoxylin-eosin,HE)染色和天狼猩红染色观察心肌病理学变化,原位末端标记法(terminal deoxynucleotidyl transferase dUTP nick end labeling,TUNEL)检测心肌组织中凋亡水平,以及流式细胞术(Flow cytometry, FCM)检测脾脏中CD20^+细胞、CD68^+细胞含量。结果:与NC组相比,MI组心肌纤维化加重,EF和LVFS降低,脾脏组织中CD20^+细胞、CD68^+细胞含量升高(均P<0.05);与MI组相比,TLR4^-/-MI组心肌组织损伤和纤维化程度减轻,EF和LVFS升高,脾脏组织中CD20^+细胞、CD68^+细胞含量下降(均P<0.05);结论:TLR4基因敲除对于小鼠心肌缺血有保护作用,心肌细胞损伤及炎症浸润程度减轻,脾脏中CD20^+细胞、CD68^+细胞含量减少,这可能和TLR4参与调节CD20^+细胞、CD68^+细胞在心肌缺血中的免疫反应有关。

Objective: To investigate the effects of intervening toll-like receptor 4(TLR4) on CD20^+ and CD68^+ cells in the spleen of myocardial ischemic mice. Methods:20 C57BL6 male mice and 20 TLR4 gene knockout male mice were randomly divided into the blank control group(group NC),myocardial ischemia group(group MI),TLR4^-/- control group(group TLR4^-/- NC)and TLR4^-/- myocardial ischemia group(group TLR4^-/- MI)with 10 mice in each group. The control group received intraperitoneal injection of 20mg/kg normal saline(NC)for 7 days. And the myocardial ischemia group also received intraperitoneal injection of 20mg/kg isoproterenol(ISO) for 7 days to establish the myocardial ischemia model. After the MI model builded, the ejection fraction (EF) and left ventricular fractional shortening(LVFS) were mearsured by the cardiac ultrasound. The myocardial pathological change was observed by Hematoxylin-eosin (HE)staining and Sirius red staining. The level of apoptosis in myocardial tissue was detected by the method of terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL). And the content of CD20^+ and CD68^+ cells in spleen were detected by the flow cytometry(FCM). Results: Compared with the NC group, myocardial fibrosis was aggravated in the MI group, EF and LVFS were decreased, and the contents of CD20^+ cells and CD68^+ cells in the spleen tissues were increased(<0.05). Compared with the MI group, the damage and fibrosis degree of the TLR4^-/-MI group was reduced, EF and LVFS were increased, and the contents of CD20^+ cells and CD68^+ cells in the spleen tissues were decreased(<0.05). Conclusions: TLR4 gene knockout has a protective effect on myocardial ischemia in mice. The degree of myocardial cell injury and inflammatory infiltration is reduced, and the content of CD20^+ cells and CD68^+ cells in the spleen is reduced, which may be related to the immune response of CD20^+ cells and CD68^+ cells involved in the regulation of TLR4 in myocardial ischemia.