小鼠出生后早期心脏成纤维细胞数量及性状变化研究

Changes of number and characteristics of heart fibroblasts in early postnatal mice

目的心脏成纤维细胞占心脏细胞数量的50%以上,在心脏生理病理过程中发挥重要作用。小鼠出生后7 d是心脏失去再生能力的时间转折点,本研究着眼于研究心脏成纤维细胞在小鼠出生后早期数量及性状的变化情况,为心肌再生机制研究提供基础性实验数据。方法出生1 d和7 d的C57小鼠进行心尖切除手术(Apex resection,AR),体视镜拍照及HE染色观察心尖处切口,21 d后观察再生情况;分离出生后1 d和7 d的C57小鼠心脏组织,称取心脏湿重比较重量差异,病理切片进行HE染色观察两个时间点的心脏病理结构,免疫荧光染色观察1 d和7 d心脏组织中成纤维细胞数量变化;为了更加准确定量心脏成纤维细胞的数量变化,我们利用成纤维细胞表面标志物胸腺细胞分化抗原1(Thymocyte differentiation antigen 1,Thy1)通过流式分选技术对心脏组织细胞悬液进行心肌成纤维细胞的分选,比较1 d和7 d小鼠心脏组织中心肌成纤维细胞比例;为了验证心脏成纤维细胞性状是否发生变化,我们分离1 d和7 d小鼠心脏成纤维细胞,分析成纤维细胞的特异性标志物Thy1、成纤维细胞特异性蛋白(Fibroblast-specific protein 1,Fsp-1)、骨膜蛋白(Periostin)、血小板衍生生长因子受体α(plateletderived growth factor receptorα,Pdgfrα)、Ⅰ型胶原α1(Collagen alpha-1,Col1a1)、转录因子21(Transcription factor 21,Tcf21)在m RNA水平的表达变化情况。结果出生1 d的小鼠心尖切除手术后21 d可以完全再生(再生率为86.67%),而7 d小鼠手术后不能完全再生(再生率为0);小鼠出生后7 d心脏组织平均比1 d心脏湿重重13.13mg(3.11倍)(1 d:(6.22±0.19),7 d:(19.35±0.56),P<0.0001;n=6)。心脏成纤维细胞免疫荧光染色证明,7 d心脏成纤维细胞数量明显高于1 d心脏;流式细胞分选证明小鼠出生后7 d心脏组织中心肌成纤维细胞占总细胞比例比出生后1 d时高2.9%(1.38倍)(1 d:(7.7±0.74),7 d:(10.6±0.95),P=0.029;n=3);在成纤维细胞转录水平,成纤维细胞标志蛋白Thy1、Fsp-1、Periostin在小鼠出生后7 d表达高于出生后1 d的成纤维细胞(Thy1:1 d:(1.01±0.12),7 d:(2.71±0.27),P=0.0288;Fsp-1:1 d:(1.04±0.27),7 d:(5.28±0.10),P=0.0046;Periostin:1d:(0.91±0.01),7 d:(1.13±0.05),P=0.0119;n=3),而Pdgfrα、Col1a1、Tcf21三种成纤维细胞标志蛋白表达则低于出生后1 d的小鼠心脏中的成纤维细胞(Pdgfrα:1 d L:(1.09±0.04),7 d:(0.62±0.01),P=0.0068;Col1a1:1d:(1.00±0.09),7 d:(0.57±0.02),P=0.0433;Tcf21:1 d:(1.00±0.03),7 d:(0.54±0.02),P=0.0054;n=3),证明出生后1 d到7 d的过程中心脏成纤维细胞性状发生了变化。结论小鼠出生后早期(1 d和7 d)心脏成纤维细胞的比例和性状均发生了明显变化,为研究哺乳动物心肌再生能力的丢失的机制研究提供一定的线索。

Objective Cardiac fibroblasts account for more than 50%of heart cells and play an important role in the physiological and pathological processes of the heart.Seven days of age is an important turning point in mice when the heart loses its ability to regenerate.This study analyzed the changes in the number and characteristics of cardiac fibroblasts in the early postnatal period of mice,and provides basic experimental data for the study of the myocardial regeneration mechanism.Methods Apical resection(AR)was performed on 1-day-old and 7-day-old C57 mice.The apex incision was photographed with a stereoscope and stained with hematoxylin and eosin(HE),and regeneration was observed at 21 days post resection.Heart tissues of C57 mice at postnatal day 1(P1)and P7 were isolated and weighed to compare the wet weight.Cardiac pathological structures at the two time points were observed by HE staining in pathological sections,and fibroblasts were observed by immunofluorescence staining.To accurately quantify the number of cardiac fibroblasts,we used fibroblast surface marker thymocyte differentiation antigen 1(Thy1)to sort cardiac tissue cell suspensions by flow sorting technology.To investigate changes in cardiac fibroblast characteristics,we isolated mouse cardiac fibroblasts on P1 and P7 and analyzed the m RNA expression of fibroblast marker proteins Thy1,fibroblast-specific protein 1(Fsp-1),Periostin,platelet-derived growth factor receptorα(Pdgfrα),collagen alpha-1(Col1 a1),and transcription factor 21(Tcf21).Results Heart tissue of 1-day-old mice could regenerate(regeneration rate was 86.67%)21 days after apical resection,whereas that of 7-day-old mice could not regenerate(regeneration rate was 0).The average wet weight of heart tissue in mice at P7 was 13.13 mg(3.11 times)heavier than that at P1(P1:6.22±0.19,P7:19.35±0.56,P<0.0001;n=6).Immunofluorescence staining indicated that the number of cardiac fibroblasts at P7 was significantly higher than that at P1.Flow cytometry showed that the proportion of cardiac fibroblasts to total cells in heart tissue at P7 was 2.9%(1.38 times)higher than that at P1(P1:7.7±0.74,P7:10.6±0.95,P=0.029;n=3).At the level of fibroblast transcription,Thy1,Fsp-1,and Periostin expression was higher at P7 than at P1(Thy1:P1:1.01±0.12,P7:2.71±0.27,P=0.0288;Fsp-1:P1:1.04±0.27,P7:5.28±0.10,P=0.0046;Periodin:P1:0.91±0.01,P7:1.13±0.05,P=0.0119;n=3),and Pdgfrα,Col1 a1,and Tcf21 expression was lower at P7 than at P1(Pdgfrα:P1:1.09±0.04,P7:0.62±0.01,P=0.0068;Col1 a1:P1:1.00±0.09,P7:0.57±0.02,P=0.0433;Tcf21:P1:1.00±0.03,P7:0.54±0.02,P=0.0054;n=3),which indicated that cardiac fibroblast traits changed during the period from1 to 7 days after birth.Conclusions The proportion and characteristics of cardiac fibroblasts in mice changed significantly during the early postnatal period(1 to 7 days),which provides some clues to the mechanism of the loss of mammalian myocardial regeneration.