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目的探讨microRNA-1(miR-1)对他莫昔芬(tamoxifen,TMX)诱导的心肌特异性Dicer基因缺失小鼠心脏结构重构及心功能的影响。方法采用Myh6-Cre/Loxp重组系统,通过Dicerloxp/loxp小鼠和Myh6-cre ERT小鼠杂交,获得Myh6-cre ERT/Dicerloxp/loxp小鼠。将18只8周龄雄性Myh6-cre ERT/Dicerloxp/loxp小鼠按随机数字表法分为对照组、模型组、miR-1治疗组。采用腹腔注射20 mg/mL TMX溶液(0.1 mL),连续注射5 d,建立心肌特异性Dicer基因缺失心力衰竭小鼠模型,对照组腹腔注射玉米油;模型建立后,miR-1治疗组通过尾静脉注射miR-1类似物(miR-1 mimic)10 nmol/只,对照组给予同等剂量生理盐水,模型组给予miRNA阴性对照试剂,2次/周。1周后,经胸二维超声心动图检测各组小鼠舒张末期左室内径(LVIDd)、收缩末期左室内径(LVIDs)、舒张末期左室容积(EDV)、收缩末期左室容积(ESV)、射血分数(EF)及左室短轴缩短率(FS),然后收集心肌标本,通过HE染色、Masson三色染色及天狼猩红染色观察小鼠心肌结构重构程度;利用原位缺口末端标记法(terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling,TUNEL)免疫荧光染色检测心肌组织细胞凋亡情况;通过细胞增殖指标Ki67免疫组化染色评估细胞增殖水平。结果建模后1周,模型组小鼠心功能明显下降,LVIDs及ESV均高于对照组(P<0.05),EF及FS则显著低于对照组(P<0.05),miR-1治疗后上述指标均较模型组明显改善(P<0.05)。组织病理学检测结果显示,与对照组相比,模型组小鼠心肌细胞排列紊乱,细胞肥大,炎症细胞浸润明显,心肌纤维化显著,而治疗组小鼠心肌细胞形态正常,无明显肥大和炎性浸润,未见明显的间质纤维化;TUNEL免疫荧光染色及Ki67免疫组化染色结果显示,miR-1治疗组小鼠较模型组心肌组织凋亡比例增加(P<0.05),且存在较明显的细胞增殖(P<0.05)。结论 miR-1可抑制TMX诱导的心肌特异性Dicer基因缺失心力衰竭模型中心肌细胞的固有改变和间质的改变,抑制心脏结构重构,维持心功能。
Objective To investigate the effect of microRNA-1 (miR-1) on cardiac remodeling and cardiac function in cardiac-specific Dicer-deficient mice induced by tamoxifen (TMX). Methods Myh6-cre ERT / Dicerloxp / loxp mice were obtained by hybridization of Dicerloxp / loxp mice and Myh6-cre ERT mice using the Myh6-Cre / Loxp recombination system. Eighteen 8-week-old male Myh6-cre ERT / Dicerloxp / loxp mice were randomly divided into control group, model group and miR-1 treatment group. The mice were injected intraperitoneally with 20 mg / mL TMX solution (0.1 mL) for 5 days. The mice model of heart-specific Dicer gene-deficient heart failure was established. The control group was intraperitoneally injected with corn oil. After the model was established, 10 nmol / mouse of miR-1 mimic was injected intravenously, the control group was given the same dose of saline, and the model group was given miRNA negative control reagent twice a week. One week later, transthoracic echocardiography was used to detect the left ventricular end-diastolic diameter (LVIDd), end-systolic left ventricular diameter (LVIDs), end-diastolic left ventricular volume (EDV), end-systolic left ventricular volume ), Ejection fraction (EF) and left ventricular fractional shortening (FS). Then myocardial samples were collected and the myocardial remodeling was observed by HE staining, Masson trichrome staining and Sirius red staining. Cell apoptosis was detected by immunofluorescence staining with terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling (TUNEL). Cell proliferation was assessed by Ki67 immunohistochemical staining. Results After 1 week of modeling, the cardiac function of model group was significantly decreased, LVIDs and ESV were significantly higher than those of control group (P <0.05), EF and FS were significantly lower than those of control group (P <0.05) The above indicators were significantly improved than the model group (P <0.05). Histopathological examination showed that compared with the control group, the model group of mice were disarranged cardiomyocytes, hypertrophy of cells, infiltration of inflammatory cells, significant myocardial fibrosis, while the treatment group, normal myocardial cells, no obvious hypertrophy and inflammation There was no significant interstitial fibrosis in TUNEL. The results of TUNEL immunofluorescence staining and Ki67 immunohistochemistry showed that the percentage of apoptotic myocardium in miR-1 treated group was higher than that in untreated group (P <0.05) Significant cell proliferation (P <0.05). Conclusion miR-1 can inhibit the intrinsic changes of cardiac myocytes and interstitial changes induced by TMX in cardiac-specific Dicer gene-deficient heart failure model, restrain cardiac remodeling and maintain cardiac function.