悬滴培养法维持瘢痕疙瘩成纤维细胞表面标志CD105的表达及其功能

Maintenance of Cell Surface Marker CD105 and Its Function by Hanging-drop Culture Method

目的探讨悬滴培养法对维持体外培养的瘢痕疙瘩成纤维细胞表面标志的作用,并初步研究CD105调节细胞功能的作用。方法以耳部瘢痕疙瘩标本3例培养成纤维细胞,将第5代细胞分别常规贴壁培养和悬滴培养1周,对同一患者来源常规培养的第4代(P4)、第5代(P5)以及悬滴培养的第5代细胞(P5HD),采用多色流式细胞仪检测表面标志CD105、CD90、CD73和CD44的表达;并采用Real-time PCR的方法,检测上述表面标志,以及瘢痕疙瘩成纤维细胞相关功能基因CTGF、Col IA1和Col IA2的m RNA表达。结果流式检测显示CD105+和CD73+CD90+CD105+细胞比例在P4和P5HD组均高于P5组,统计学分析显示有显著性差异,但P4和P5HD组之间无差异;而CD73+、CD90+和CD44+各组细胞比例无差异。Real-time PCR结果显示,各组细胞CD105的m RNA表达与流式结果一致;且各组CTGF和Col IA1表达差异与CD105一致。结论悬滴培养法有助于维持体外瘢痕疙瘩成纤维细胞CD105,及其与纤维化和胶原合成相应功能基因的表达,从而保持细胞的生物学功能,但机制有待于进一步的研究。

Objective To investigate the maintenance of cell surface markers of keloid fibroblasts by hanging-drop culture method, and to preliminarily study the role of CD105 in regulating function of cells. Methods The fibroblasts were isolated from 3 keloid samples of ears, and the passage 5 cells were cultured using the adherent culture and the hangingdrop culture method for 1 week respectively. The passage 4 （P4） and 5 cells （P5） cultured by adherent culture method and the passage 5 cells cultured by hanging-drop cuhure method （P5HD）from the same patient were detected for the expression of cell surface markers including CD105, CD90, CD73 and CD44 by muhicolor flow cytometry analysis. The mRNA expressions of surface markers and CTGF, Col IA1, Col IA2 were detected using real-time PCR. Results The positive cell percent of CD105^＋ and CD73^＋CD90^＋CD105^＋ in P4 and P5HD group were significantly higher than in P5 group by flow cytometry analysis, but there was no difference between P4 and P5HD group. The positive cell percent of CD73^＋, CD90^＋ and CD44^＋ among three groups had no difference. The CD105 results of real-time PCR were in accordance with that of flow cytometry analysis. The mRNA expression tendency of CTGF and Col IA1 was similar to CD105. Conclusion The hanging- drop culture method is helpful to maintain the expression of CD105 surface marker and functional genes related to fibrosis and collagen production, then to keep the biological function of keloid fibroblasts, but the underlying mechanism needs to be discovered.