Dil体外标记的大鼠骨髓间充质干细胞与乳鼠心肌细胞接触共培养诱导分化为心肌样细胞的实验研究  

Experimental Study on Co-culture of Dil-labeled Rat Bone Marrow Mesenchymal Stem Cells and Neonatal Rat Cardiomyocytes to Induce Differentiation into Cardiomyocyte-like Cells

作　　者：张子畅 周帆 郑建伟 穆军升 伯平 尤斌 ZHANG Zi-chang;ZHOU Fan;ZHENG Jian-wei;MU Jun-sheng;BO Ping;YOU Bin(Department of Cardiac Surgery,Beijing Anzhen Hospital,Capital Medical University,Beijing Institute of Heart Lung and Blood Vessel Diseases,Beijing 100029,China;Department of Ultrasound,The Third Medical Center of Chinese PLA General Hospital,Beijing 100039,China;Heart Center of Sunshine Union Hospital Weifang 261205,China)

机构地区：[1]首都医科大学附属北京安贞医院,北京市心肺血管疾病研究所心脏外科,北京市100029 [2]解放军总医院第三医学中心超声科,北京市100039 [3]阳光融和医院心脏中心,潍坊市261205

出　　处：《中国分子心脏病学杂志》2021年第6期4344-4350,共7页Molecular Cardiology of China

基　　金：2018年国家自然科学基金(81870181);中国科协学会2021年度公共服务能力提升项目立项(2021GGFW003)。

摘　　要：目的对Dil标记的大鼠骨髓间充质干细胞(BMSCs)与乳鼠心肌细胞(CMs)进行接触共培养,观察接触共培养对Dil标记的干细胞向CMs分化的影响,对其内在机制进行初步探究。在细胞标记下,为干细胞治疗心肌梗死寻找一种合适的诱导分化方法.方法分离培养获得SD大鼠BMSCs,流式细胞鉴定表面标志物分离获得乳鼠CMs.BMSCs培养至3代后,使用Dil染料对BMSCs进行标记.将Dil标记的BMSCs与CMS接触共培养,并设仅有Dil标记的BMSCs为对照组,24 h后在显微镜下观察细胞生长情况.在共培养7日后,使用心肌标志物(cTnT、α-actin)对细胞进行免疫荧光染色,在荧光显微镜下观察干细胞的分化情况.在共培养的第1、7日使用流式分析BMSCs向CMs的定向分化效率。使用钙黄绿素对CMS染色,观察接触共培养细胞间染料转移;使用缝隙连接蛋白43(Cx43)对细胞进行免疫荧光染色,观察缝隙连接与接触共培养之间存在的关系.结果BMSCsM式细胞术鉴定:CD90、CD44H强阳性,CD11b/c、CD45阴性。共培养24h后显微镜下观察可见:细胞贴壁,荧光激发时Dil标记的BMSCs发红光,未标记的CMs不发光第7日,部分Dil标记的BMSCs表达cTnT、α-actin.流式分析显示:实验组第7日干细胞分化率高于对照组(20.06%:2.45%,P<0.05).另外,共培养第2日起,部分BMSCs免疫荧光Cx43染色阳性;并且共培养第1日钙黄绿素在CMs中显示绿色荧光,共培养第3日起部分BMSCs显示绿色荧光:结论Dil标记的BMSCs在与CMS接触共培养下可被诱导分化为心肌样细胞,并且接触共培养促进其分化为心肌样细胞,可能与通过缝隙连接传递相关信号通路有关.Objective To study the contact co-culture of Dil-labeled bone marrow mesenchymal stem cells(BMSCs)and neonatal cardiomyocytes(CMs),observe the effect of contact co-culture on differentiation of Dil-labeled BMSCs into CMs,and explore the underlying mechanism to find a suitable method of inducing differentiation of CMs from stem cells for the treatment of myocardial infarction under conditions of cell-labeled.Methods BMSCs from Sprague Dawley rats were isolated,cultured,and characterized for expression of surface markers by flow cytometry.CMs of suckling mice were also isolated.BMSCs cultured for three generations were labeled with Dil dye before contact co-culture with CMs,and Dil-labeled BMSCs were used as a control group.After 24h,cell growth was observed by microscopy.After 7d of contact co-culture,cells were immunofluorescent stained for myocardial markers including cardiac troponin T(cTnT)and α-actin,and observe the differentiation of stem cells by fluorescence microscopy.The differentiation efficiency of BMSCs into CMs was quantitatively analyzed by flow cytometry on the 1st and 7th days of co-culture.Calcein-stained CMs were used to observe intercellular dye transfer in co-cultures,and immunofluorescence staining of the gap junction protein connexin 43(Cx43)was used to examine the relationship.Results BMSCs were identified by strongly positive expression of CD90 and CD44H,and negative expression of CD1 lb/c and CD45.After co-culture for 24h,microscopy revealed that cells adhered to the plate.During fluorescence excitation,Dil-labeled BMSCs emitted red light,while unlabeled CMs did not emit light.On the 7th day,some Dil-labeled BMSCs expressed cTnT and α-actin.Flow cytometry revealed an increased rate of BMSC differentiation in the experimental group compared with the control group on the 7th day(20.06%vs 2.45%,P<0.05).In addition,from the 2nd day of contact co-culture,some BMSCs exhibited positive immunofluorescence for Cx43.Green fluorescence indicated the presence of calcein in CMs on the 1st day and so

关 键 词：干细胞治疗 骨髓间充质干细胞 心肌细胞 接触共培养 Dil标记 

分 类 号：R542.22[医药卫生—心血管疾病]