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目的:建立基于显微形态标记及流式分选分离大鼠心脏Telocytes(CTs)的方法。方法:采用抗体c-Kit免疫磁珠法获得原代心脏Telocytes,显微注射Di I标记具“Telopode”典型形态的细胞,使用流式分离及回收单个Di I+细胞;使用免疫荧光技术和RT-PCR方法对经回收的单个细胞来源的细胞进行表型鉴定。结果:显微注射Di I能较好地标记具Telocytes典型形态的细胞,结合流式分选及单细胞回收,能有效回收经标记的Di I+细胞,经回收的Di I标记阳性Telocytes的贴壁率为14.9%,增殖率为5.6%,呈克隆样生长率为2.4%,该呈克隆样生长的细胞能通过消化传代。免疫荧光染色证明,该回收Telocytes表达其相对特异性表面标记物c-Kit和CD34,RT-PCR的结果也证明:经回收Telocytes表达其相对特异基因c-Kit、CD34、Vimentin和PDGFR-β。结论:研究所建立的方法能有效分离及单细胞回收高纯度的心脏Telocytes,经回收的心脏Telocytes具有增殖及传代能力,且能维持其特异表型。
OBJECTIVE: To establish a method for the isolation of rat heart Telocytes (CTs) based on microscopic morphology and flow sorting. Methods: The primary cardiac Telocytes were obtained by immunomagnetic beads method and the cells labeled with “Telopode ” were labeled with Di I by microinjection. The cells were isolated and recovered by flow cytometry. The immunofluorescence technique and RT-PCR was used to phenotype cells recovered from single cells. RESULTS: Microinjection of Di I could label well the typical cells with Telocytes. Combining with flow sorting and single cell recovery, the labeled Di I + cells could be effectively recovered. The labeled Di I -positive Telocytes adhered well The rate was 14.9%, the rate of proliferation was 5.6%, and the rate of clonal growth was 2.4%. The clonally-grown cells passed the passage of digestion. Immunofluorescent staining confirmed that the recovered Telocytes expressed the relative specific surface markers c-Kit and CD34, and the results of RT-PCR also proved that Telocytes expressed the relative specific genes c-Kit, CD34, Vimentin and PDGFR-β. CONCLUSION: The established method can effectively separate and recover high-purity cardiac Telocytes from single cells. The recovered cardiac Telocytes have the ability of proliferation and passage, and can maintain their specific phenotype.