高内涵筛选与流式细胞分析技术在心肌成纤维细胞增殖研究中的应用

Application of high-content screening and flow cytometry analysis techniques to evaluation of myocardial fibroblasts proliferation

心肌成纤维细胞(cardiac fibroblasts,CFs)增殖参与了心脏疾病的病理生理过程并发挥重要作用。本文旨在应用高内涵筛选(high-content screening,HCS)和流式细胞术(flow cytometry,FCM)分析评价CFs增殖,以期建立精确定量评价细胞增殖和细胞周期的方法。采用酶消化法分离C57BL/6J新生乳小鼠(出生24～48 h)CFs,盘状结构域受体(discoidin domain receptor 2,DDR2)染色显示分离后培养的CFs纯度>95%。CFs经血清饥饿12 h后给予10%胎牛血清(fetal bovine serum,FBS)处理24 h,进行溴脱氧尿苷(5-bromodeoxyuridine,BrdU)和二脒基苯基吲哚(diamidino-phenylindole,DAPI)原位染色或BrdU和7-氨基放线菌素D(7-Aminoactinomycin D,7-AAD)单细胞悬液染色,分别利用HCS或FCM检测CFs增殖和细胞周期。结果表明:(1)HCS分析显示,10%FBS诱导组BrdU阳性细胞比例与无血清对照组比较有显著性差异[(12.96±0.67)%vs(2.77±0.33)%;P<0.05];细胞周期分析显示,细胞处于G0/G1期DNA为二倍体(2N),进入S期DNA复制,G2期DNA倍增(4N),到M期DNA平分到两个子细胞核中(2N)。(2)FCM分析显示,10%FBS诱导组BrdU阳性细胞比例比无血清对照组明显增大,差异显著[(11.10±0.42)%vs(2.22±0.31)%;P<0.05];DNA含量直方图细胞周期分析显示,10%FBS诱导组S期平台比对照组明显抬高。(3)对比分析HCS和FCM检测结果,BrdU阳性比例以及G0/G1、S、G2/M期细胞百分比等各项指标均无统计学差异。证实HCS和FCM两种技术能够应用于定量分析CFs增殖和细胞周期,方法实用可靠,结果一致。

The proliferation of cardiac fibroblasts （CFs） is a key pathological process in the cardiac remodeling. To establish an objec- tive, quantitative method for the analysis of cell proliferation and cell cycle, we applied the high-content screening （HCS） and flow cytometry （FCM） techniques. CFs, isolated by enzyme digestion from newborn C57BL/6J mice, were serum starved for 12 h and then given 10% fetal bovine serum （FBS） for 24 h. Followed by BrdU and DAPI （or 7-AAD） staining, CFs proliferation and cell cycle were analyzed by HCS and FCM, respectively. Discoidin domain receptor 2 （DDR2） staining indicated that the purity of isolated CFs was over 95%. （1） HCS analysis showed that the ratio of BrdU-positive cells was significantly increased in 10% FBS treated group compared with that in serum-free control group [（12.96± 0.67）% vs （2.77 ±0.33）%; P 〈 0.05]. Cell cycle analysis showed that CFs in G0/G1 phase were diploid, and CFs in S phase were companied with proliferation, DNA replication and enlarged nuclei; CFs in G2 phase were tetraploid, and CFs in M phase produced two identical cells （2N）. （2） FCM analysis showed that the ratio of BrdU-positivecells was increased in 10% FBS treated group compared with that in the control group [（11.10 i 0.42）% vs （2.22 ±0.31）%; P 〈 0.05]; DNA content histogram of cell cycle analysis indicated that the platform of S phase elevated in 10% FBS group compared with con- trol group. （3） There were no differences between the two methods in the results of proliferation and cell cycle analysis. In conclusion, HCS and FCM methods are reliable, stable and consistent in assessment of the proliferation and cell cycle in CFs.