两种消化时间对成纤维细胞体外培养的影响

Comparison between the influence of two kinds of digestion time on the in vitro culture of fibroblasts

目的:通过对比不同消化时间对成纤维细胞原代培养的影响,探索一种更为简便、可靠的实验方法,为原代心肌成纤维细胞的基础研究提供依据。方法:使用Ⅱ型胶原酶多次消化法,采用两种不同的消化时间对乳鼠心肌组织进行消化,利用差速贴壁法分离心肌细胞及心肌成纤维细胞,37℃孵育,使用台盼兰染色、免疫组化、3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT]等方法分析其形态、纯度、增殖能力。结果:消化时间8 min组的细胞总存活率为(94.7±0.6)%,10 min组为(93.9±0.8)%,二者比较差异无统计学意义(P>0.05);心肌成纤维细胞存活率8 min组为(55.3±9.0)%,10 min组为(91.8±5.5)%,两组比较差异有统计学意义(P<0.05);波形蛋白免疫组化,两组阳性细胞率均>95%,两组比较差异无统计学意义(P>0.05);增殖能力8min组为(5.33±0.80),10 min组为(61.15±5.20),两组比较差异有统计学意义(P<0.05)。结论:两种消化时间获得的心肌成纤维细胞在形态上、纯度上无明显差异,在增殖能力和心肌成纤维细胞数量上,10 min组优于8min组。

Objective： To explore a simpler and more reliable experimental method,providing the basis for basic research of primary cultured cardiac fibroblasts by comparing the influence of different digestion time on the primary culture of fibroblasts. Methods： Type II collagenase was used to digest for many times and two different kinds of digestion time were used to digest the cardiac tissues of neonatal rats. The cardiomyocytes and cardifibroblasts were isolated by differential adhesion and were incubated at 37 ℃. Trypan blue staining,immunohistochemistry,3-（ 4,5-dimethyl- 2- thiazolyl）- 2,5- diphenyl- 2- H- tetrazolium bromide（ MTT） etc. were used to analyze the morphology,purity and proliferation. Results： There was no significant difference between the survival rate of the two kinds of digestion time [8min（ 94. 7 ± 0. 6） %,10min（ 93. 9± 0. 8） % ]. The difference between the number of fibroblasts [（ 8min 55 ± 9. 0,10 min 91 ± 5. 5） ]was significant. In terms of vimentin immunohistochemical results,the positive rates of the two groups were both higher than 95 %. The proliferation of the 10 min group（ 61. 15 ± 5. 20） was significant,but there was no significant change in the 8 min group. Conclusion： There is no difference in the shape or in the purity of fibroblasts between the two groups. But in terms of the ability to proliferate,the 10 min group is better than that of the controlled group. Besides,the 10 min group can get more cardiac fibroblasts.