诱导多能干细胞/原代心肌细胞的融合细胞表现出双向重建

Bidirectional reprogramming of fusion cells of pluripotent stem cells/primary cardiac myocytes

目的体外构建诱导多能干细胞(induced pluripotent stem cells,iPSc)与原代心肌细胞的融合细胞,初步探讨融合细胞体外生物学特性。方法绿色荧光蛋白(green fluorescent protein,GFP)转染八聚体结合转录因子4(octamer-binding transcription factor-4,Oct-4)小鼠来源的iPSc与小鼠乳鼠心肌细胞通过聚乙二醇(polyethylene glycol,PEG-4000)诱导融合,建立细胞融合模型。动态观察融合细胞生长形态变化情况,融合细胞碱性磷酸酶(alkali phosphatase,AKP)染色,免疫荧光鉴定融合细胞表达干细胞与心肌细胞特异性蛋白情况,以及染色体核型分析,确定融合细胞是否发生细胞核的融合及程度。结果聚乙二醇(PEG-4000)能够介导iPSc与心肌细胞融合,融合后第4天开始出现成集落状生长的细胞团;第2、3、4、5天的iPSc和融合细胞AKP阳性率分别为(0.935±0.039)、(0.939±0.022)、(0.954±0.017)、(0.944±0.027)和(0.761±0.044)、(0.740±0.023)、(0.681±0.034)、(0.748±0.045),同时间点的iPSc和融合细胞AKP阳性率比较有明显差异(P<0.05);融合细胞初期主要表现为iPSc的特点,Oct-4表达阳性,cTnT表达阴性,融合7 d后,逐渐表现出两种细胞的特点,Oct-4与cTnT均表达阳性;超过80%的杂交细胞染色体数目在76～80条之间。结论二倍体iPSc与二倍体心肌细胞的融合细胞,具有两亲本细胞的特性,表现出双向重建。

Objective To construct fusion cells with induced pluripotent stem cells（iPSc） and primary cardiac myocytes in vitro,and to investigate biological features of the fusion cells.Methods Polyethylene glycol（PEG-4000） was used to mediate the cell fusion of iPSc derived from green fluorescent protein（GFP） transgenes（octamer-binding transcription factor-4,Oct-4） mouse and cardiac myocytes from neonatal mouse.Morphological changes of the fusion cells were observed dynamically after alkaline phosphatase（AKP） staining.Specific proteins of stem cells and cardiac myocytes in fusion cells were detected by immunofluorescence.Chromosome karyotype analysis were performed to determine whether the occurrence of nuclear fusion and degree of integration.Results Fusion cells were constructed successfully by polyethylene glycol mediation.Colony-like cell clusters appeared in 4 d after fusion.The AKP positive rate of iPSc were 0.935±0.039,0.939±0.022,0.954±0.017,and 0.944±0.027 at the 2nd,3rd,4th and 5th days respectively,and that of fusion cells were 0.761±0.044,0.740±0.023,0.681±0.034,and 0.748±0.045 at the corresponding days respectively.At the same time points,there were significant differences between iPSc AKP-positive rates and those of fusion cells（P0.05）.In the initial stage,fusion cells mainly displayed iPSc characteristics,with Oct-4 positive while cTnT negative.Then the fusion cells began to display both characteristics of iPSc and cardiac myocytes in 7 d after fusion,with positive expression of Oct-4 and cTnT.More than 80% of fusion cells had 76 to 80 chromosomes.Conclusion Fusion cells from diploid iPSc and diploid myocardial cells display the characteristics of the two parental cells and show bidirectional reprogramming.