摘要
目的探讨在聚己内酯薄膜上将DiI标记的骨髓间充质干细胞(BMSCs)与乳鼠心肌细胞接触共培养制作心肌补片的可能机制。方法选取5~6周龄SD大鼠2只,分离培养获得SD大鼠BMSCs,流式细胞术鉴定表面抗原。选取乳鼠15只,分离培养获得乳鼠心肌细胞。BMSCs培养至3代,使用DiI染料标记BMSCs。在聚己内酯薄膜上将DiI标记的BMSCs与心肌细胞接触共培养并设为实验组,对照组中将心肌细胞替换为等量未标记的BMSCs。共培养后24 h在荧光显微镜下观察细胞生长情况,并用扫描电镜观察共培养情况。共培养后7 d对细胞进行免疫荧光染色检测心肌标志物表达,在荧光显微镜下观察聚己内酯薄膜上BMSCs表达心肌标志物的情况。共培养的第1、7天使用流式细胞术分析BMSCs的干细胞分化率。使用钙黄绿素单独对心肌细胞染色,再在聚己内酯薄膜上将其与DiI标记的BMSCs接触共培养,观察细胞间染料转移,并对细胞进行免疫荧光染色,检测缝隙连接蛋白43的表达,观察缝隙连接与接触共培养之间的关系。结果流式细胞术鉴定示BMSCs表面CD90、CD44H强阳性,CD11b/c、CD45阴性。共培养后24 h,荧光显微镜下观察到细胞依附于聚己内酯薄膜上,DiI标记的BMSCs发红光,未标记的心肌细胞不发光;扫描电镜下观察到聚己内酯薄膜上细胞数量多,细胞状态正常。共培养第7天,部分DiI标记的BMSCs表达心肌肌钙蛋白T、α-心肌肌动蛋白。流式细胞术分析示第7天实验组的干细胞分化率高于对照组[(20.12±0.15)%比(3.49±0.20)%,P<0.05]。共培养后第2天,缝隙连接蛋白43免疫荧光染色结果示部分BMSCs可见明显绿色点状荧光;共培养后第3天,染料转移试验示部分BMSCs发出明显绿色荧光。结论DiI标记的BMSCs与心肌细胞在聚己内酯薄膜上接触共培养可以制作心肌补片,并且接触共培养促进分化形成心肌补片的机制可能与缝隙连接以及缝隙连接介导�
Objective To investigate the possible mechanism of DiI labeled bone marrow mesenchymal stem cells(BMSCs)in contact co-cultured with neonatal rat cardiomyocytes(CMs)on polycaprolactone(PCL)film to make myocardial patch.Methods BMSCs from Sprague Dawley rats(aged 5-6 weeks)were isolated,cultured,and characterized for surface marker expression using flow cytometry.CMs from 15 neonatal rats were isolated and cultured.After cultured for 3 generations,BMSCs were labeled with DiI dye.On PCL film,DiI labeled BMSCs were co-cultured with CMs as the experimental group,and CMs were replaced with the same amount of unlabeled BMSCs in the control group.After 24 h of co-culture,the cell growth was observed under fluorescence microscope and the co-culture was observed under scanning electron microscope.Immunofluorescence staining was performed after 7 days to detect myocardial markers,including cardiac troponin T(cTnT)andα-actinin.BMSC differentiation on the PCL film was observed under a fluorescence microscope.The differentiation efficiency of BMSCs into cardiomyoid cells was analyzed by flow cytometry on days 1 and 7 of co-culture.Intercellular dye transfer was observed by staining CMs with calcein and co-culturing them with DiI-labeled BMSCs on PCL film.The cells were stained with immunofluorescence to detect the expression of connexin 43(Cx43)and observe the relationship between gap junction and contact co-culture.Results Flow cytometry showed strong positivity for CD90 and CD44 and negativity for CD11b/c and CD45 on BMSCs.After 24 h of co-culture,DiI labeled BMSCs glowed red on the PCL film,while unlabeled CMs did not;the number of cells on PCL film was large and cell morphology appeared normal under scanning electron microscope.On the 7th day of co-culture,some DiI labeled BMSCs expressed cTnT andα-actinin.Flow cytometry showed a higher differentiation rate of stem cells in the experimental group on day 7 compared to the control group((20.12±0.15)%vs.(3.49±0.20)%,P<0.05).From the second day of co-culture,some BMSCs exh
作者
张子畅
穆军升
周帆
伯平
尤斌
Zhang Zichang;Mu Junsheng;Zhou Fan;Bo Ping;You Bin(Department of Cardiac Surgery,Xuanwu Hospital Capital Medical University,Beijing 100053,China;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 People's Liberation Army General Hospital,Beijing 100039,China)
出处
《中华心血管病杂志》
CAS
CSCD
北大核心
2024年第5期525-531,共7页
Chinese Journal of Cardiology
基金
国家自然科学基金(81870181,82270255)。
