体外转染Cyclin A2基因对原代大鼠心肌细胞增殖的影响

目的探讨体外转染细胞周期素A2(Cyclin A2)基因对原代大鼠心肌细胞增殖的影响,为心脏再生提供细胞学依据。方法 SD乳鼠12只,分离、培养、鉴定原代乳鼠心肌细胞并分为3组,实验组:转染带有强化绿色荧光蛋白(GFP)和Cyclin A2基因的重组腺病毒(Ad-Cyclin A2-GFP);空病毒组:转染不含目的基因带有GFP的重组腺病毒(Ad-Null-GFP);阴性对照组:未做转染处理,仅加入等量的培养基。利用GFP示踪技术,评估原代心肌细胞转染效率;转染后的细胞继续体外培养3~5d,利用免疫荧光技术分别检测Cyclin A2、磷酸化组蛋白H3(H3P)、心肌肌钙蛋白-T(c Tn T)。结果 1.荧光GFP示踪表明原代心肌细胞的转染效率高达(97±0.74)%;2.免疫荧光标记显示,空病毒组和对照组结果相似;心肌细胞特异性标记蛋白c Tn T分布于细胞质,原代心肌细胞纯度高达(95±0.62)%;心肌细胞Cyclin A2主要在胞核内聚集,少数分布于细胞质。H3P为核蛋白在细胞核内分布。3.转染Cyclin A2后,实验组H3P阳性率明显高于空病毒组和对照组,差异具有统计学意义(P0.05);实验组可见大量多核细胞,以双核为主,伴少量3核细胞。结论腺病毒作为转染载体对原代心肌细胞有很好的侵染效率;Cyclin A2超表达促进原代心肌细胞形成双核。

Effect of Cyclin A2 on the proliferation of rat primary cardiomyocytes

Objective To investigate the effect of Cyclin A2 on the proliferation of cardiomyocytes. Methods Twelve SD neonatal rats were used, cardiomyocytes were isolated from the neonatal rat heart, cultured in dish and identified by immunofluorescence. The primary cultured cardiomyocytes were divided into three groups: group 1 which cells were transduced with adenovirus expressing Cyclin A2 and eGFP, group 2 which cells were transduced with andenovirus expressing only eGFP and group 3 in which, untransduced cells were used as negative control. eGFP was employed to determine transduction efficiency. The expression of Cyclin A2, phosphorylated histone H3 (H3P), cardiac troponin -T (cTnT) were detected by immunofluorescence at day 3 after cardiomyocytes were transduced with andenovirus. Results 1. Analysis of eGFP expression showed that the transduction efficiency was (97±0.74)%. 2. cTnT, a specific marker protein for myocardial cells, was mainly found in the cytoplasm in eGFP-expressing and untransduced cells, suggesting that adenovirus/eGFP had no toxicity in the cultured cardiomyocytes. Immuno-staining results indicated that the percentage of isolated cTnT positive cells was (95±0.62)%. CyclinA2 was expressed mainly in the nucleus. H3P was also localized to the nucleus.3. In the CyclinA2 -overexpressing cells, the percentage of H3P positive cells were significantly increased compared to those untransduced or only eGFP-expressing cells (P<0.05). The experimental group showed a large number of multinucleated cells, in which the dual-core cell-based, followed by three nuclear cells. Conclusion Adenovirus vector transfection of primary cardiomyocytes has a good infection efficiency. Cyclin A2-overexpression in primary cardiomyocytes promotes to form a dual-core.