摘要
目的 :探讨cyclinD2 、p16蛋白表达与新生大鼠心肌细胞体外培养早期增殖、分化的关系及意义。方法 :培养出生后 1d大鼠心肌细胞 ,予生长曲线、流式细胞计数检测培养过程中增殖改变 ,透射电镜观察超微结构变化 ,免疫细胞化学结合图像分析检测心肌细胞内cyclinD2 、p16蛋白变化。结果 :①生长曲线、流式细胞计数显示 :心肌细胞在培养的前 3d内具有一定增殖能力 ,但呈下降趋势同时逐步分化 ;3d后增殖能力丧失。超微结构显示细胞内有大量肌丝和线粒体。②心肌细胞在培养的 5d中 ,cyclinD2 表达量在第 3、4、5d分别是第 1d的 89 99% (P <0 0 5 )、83 6 1% (P <0 0 5 )和 6 9 6 5 % (P <0 0 1) ;p16表达量在 2、3、4、5d分别是第 1d的 1 6 3倍、1 72倍、1 99倍和2 84倍 (均P <0 0 1)。结论 :新生大鼠心肌细胞出生后早期体外培养前 3d有一定增殖能力 ,但呈下降趋势同时逐步分化 ;cyclinD2 表达下调和p16表达上升可能参与心肌细胞早期分化。
AIM: To investigate the protein expression of cyclin D_2 and p16 in proliferation and differentiation of cultured cardiac myocytes.METHODS: One-day-old Sparague-Dawley rats were used. Cardiac myocytes(CM) were collected by a trypsin-dispersal method and cultured. Cell growth line and fluorescence activated cell sorting (FACS) were used to investigate the proliferation of CM. Ultra-thin sections were made to observe the ultrastructure of CM under transmission electron microscope. The expression of cyclin D_2 and p16 in CM were measured using immunocytochemistry and image analysis.RESULTS: ①Results of cell growth line and FACS analysis showed that cultured CM could proliferate in the first 3 cultured days, but the ability decreased quickly, concomitant with differentiation. CM was obseved quiescent in cell cycle three days later. The ultrastructure of CM showed the large amount of myofilaments and mitochondrion. ②The protein expression of cyclin D_2 in 3,4,5 day CM group was 0.89 times( P <0 05),0.80 times ( P <0.05) and 0.56 times ( P <0.01) of that in 1 day group, respectively. The expression of p16 in CM was increased during the culture process, 2,3,4,5 day group were 1.63 times, 1.72 times, 1.99 times and 2.84 times ( P <0.01) of that in 1 day group, respectively.CONCLUSION: Cultured neonatal rat cardiac myocytes could proliferate during the first 3 days after incubation, but the ability of proliferation decreased, from the fourth day, concomitant with differentiation. Cyclin D_2 and p16 play the key roles in CM postnatal development. Downregulation of cyclin D_2 and upregulation of p16 may induce CM differentiation.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2002年第1期32-35,I005,共5页
Chinese Journal of Pathophysiology
