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目的:建立一种高效分离成年SD大鼠背部毛乳头细胞(DPCs)的方法,考察DPCs在体外培养条件下的生长特性。方法:采用改良二步酶消化法分离大鼠背部DPCs,应用相差显微镜进行形态学观察,流式细胞技术检测细胞周期,细胞计数法绘制生长曲线,流式细胞技术和免疫细胞化学法检测细胞表面分子标志。结果:分离培养的DPCs多呈短梭形,融合前细胞呈现凝集性生长趋势;传代细胞呈多角形或短梭形,传代后第3天开始呈对数生长,第9天达增殖高峰。第1、3、5代的细胞增殖时间分别为68、52和36h。流式细胞仪检测第1、3、5代细胞周期,处于G0/G1期分别为(90.21±5.13)%、(81.23±1.85)%和(75.16±5.32)%,随着传代次数的增加,G0/G1期细胞比例逐渐下降,但仍大部分处于静止期;经免疫化学SABC法染色显示,α-SMA抗体表达阳性,CK抗体表达阴性,细胞表面分子表达CD44、CD90,但不表达CD34。结论:改良二步酶消化法能成功分离培养成年SD大鼠背部DPCs,体外培养的DPCs与干细胞的生物学特性相吻合,有望成为一种新的细胞替代疗法的种细胞来源。
OBJECTIVE: To establish a method for efficient isolation of DPCs from adult SD rats and to investigate the growth characteristics of DPCs under in vitro culture conditions. Methods: DPCs were isolated from rats by modified two-step enzymatic digestion. Morphology was observed by phase contrast microscopy. Cell cycle was measured by flow cytometry. Cell growth curve was drawn by cell counting. Flow cytometry and immunocytochemistry were used to detect the cell surface Molecular sign. Results: The DPCs isolated mostly showed a short fusiform shape, and the cells showed the tendency of agglutination growth before fusion. The subcultured cells were polygonal or short fusiform, and began to logarithmic growth on the third day after passage and peaked on the ninth day. The cell proliferation times of the 1st, 3rd and 5th generation were 68, 52 and 36h, respectively. Flow cytometry was used to detect the cell cycle of the 1st, 3rd, 5th passage, which was (90.21 ± 5.13)%, (81.23 ± 1.85)% and (75.16 ± 5.32)% at G0 / The percentage of cells in G0 / G1 phase decreased gradually, but most of them remained in quiescent phase. Immunohistochemical SABC staining showed that α-SMA antibody was positive and CK antibody was negative. Cell surface molecules expressed CD44 and CD90, but did not express CD34. CONCLUSION: DPCs can be successfully isolated and cultured from the back of adult SD rats by the improved two-step enzyme digestion method. The DPCs cultured in vitro are consistent with the biological characteristics of stem cells, which is expected to become a new kind of cell source for cell replacement therapy.