基于c-Kit^+显微形态标记及流式分选分离心脏Telocytes

c-Kit^+ Combining with DiI Micro-label to Sort the Cardiac Telocytes

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 methodology for specific sorting of cardiac telocytes using c-Kit＋Combining with Di I Micro-label and flow cytometry. Methods： The cardiac telocytes were isolated via a c-Kit antibody conjugated magnetic bead method. The cells which have unique telocyte morphology, with long＂Telopode＂,were labeled with Di I microinjection. The labeling Di I＋cells were sorted and recovered in 96-well plate by single cell flow cytometry technique. The immunofluorescence and RT-PCR were applied to confirm the phenotype of collected cells. Results： The cardiac telocytes（ c-Kit＋） which have unique telocyte morphology were able to be labeled by Di I microinjection. The Di I labeled cells were able to be sorted and recovered in single cell level. The adherent cells of collected Di I＋cells were 14. 9% under culture. The 5. 6% of cultured Di I＋cells was able to proliferate and the 2. 4% of cultured Di I＋cells was able to form cell-clone. In addition,the formed cell-clone was able to subculture. The immunofluorescence staining confirmed that the recovered Di I＋cells were c-Kit-and CD34-positive. RT-PCR results also showed that the recovered Di I＋cells were positive for cKit,CD34,Vimentin and PDGFR-β,which are the markers for telocytes. Conclusion： The methodology which was established in present study was able to isolate and sort cardiac telocytes in single cell level. The sorted cardiac telocytes were able to proliferate and maintain the unique phenotype.