大鼠气管上皮细胞培养：3种方法的比较

Culture of tracheal epithelial cells in rats:Comparison of three methods

背景:目前常用分离气管上皮细胞的方法有蛋白酶法和胰酶法,但各有不足,控制不好会影响细胞的增殖和活力。目的:改进气管上皮细胞培养技术,比较链酶蛋白酶、胰酶和联合消化3种不同酶消化法在培养气管上皮细胞中的区别。设计、时间及地点:对比观察,实验于2007-09/2008-07在同济大学分子生物开放实验室和解放军第二军医大学动物实验室完成。材料:SD雄性大鼠12只,体质量200～250g。链酶蛋白酶、胰酶为Gibco公司产品。方法:将SD大鼠分3组,每组4只。链酶蛋白酶组使用1g/L链酶蛋白酶在4℃消化18h;胰酶冷消化组使用2.5g/L胰酶4℃消化18h;联合消化组使用链酶蛋白酶4℃消化18h后用2.5g/L胰酶37℃消化5min,用以上3种方法分离、培养SD大鼠气管上皮细胞。主要观察指标:用细胞计数板,激光共聚焦显微镜测定细胞分离的数量以及纯度、成活率。结果:链酶蛋白酶、胰酶冷消化和联合消化法所得细胞数量分别为(1.21±0.17)×106;(1.35±0.13)×106和(1.36±0.16)×106。联合消化法所得细胞状态最好,贴壁能力和增殖能力最好,其次为链酶蛋白酶法,胰酶冷消化法较差。结论:链酶蛋白酶与胰酶的联合法是一种有效的培养气管上皮细胞的方法。

BACKGROUND： So far, pronase digestion and trypsin digestion methods are the common methods for isolating tracheal epithelial cells, but they have differences. They can affect the proliferation and activities of cells if not well controlled. OBJECTIVE： To improve culture technology of tracheal epithelial calls, and compare pronase digestion, trypsin digestion and combination digestion methods for culture of tracheal epithelial cells. DESIGN, TIME AND SETTING： A controlled observational experiment was performed at Open Laboratory of Molecular Biology in Tongji University and Animal Laboratory in the Second Military Medical University, from September 2007 to July 2008. MATERIALS： Twelve male SD rats weighing 200-250 g were included. Pronase and trypsin （Gibco Company） were also used for this study. METHODS： All SD rats were divided into three groups, with four in each： pronase, trypsin and combination. Under anesthesia, the tracheas were separated from all rats. In the pronase group, 1 g/L pronase were injected into the tracheals 18 hours at 4 ℃ for digestion. In the trypsin group, 2.5 g/L trypsin were injected into the tracheals 18 hours at 4 ℃ for digestion. In the combination group, 1 g/L pronase were injected into the tracheals 18 hours at 4 ℃ for digestion, and then 2.5 g/L trypsin were injected into the tracheals 5 minutes at 37 ℃. The three methods mentioned above were used to isolate and culture the tracheal epithelial cells. MAIN OUTCOME MEASURES： The quantity, survival rate and purity of the tracheal epithelial cells were determined by cell counting plate and laser confocal microscopy. RESULTS： The number of cells in the pronase, trypsin and combination groups was （1.21 ±0.17）×10^6, （1.35±0.13）×10^6, （1.36±0.16）×10^6, respectively. The cells harvested in the combination group had the best adherent and proliferative abilities, second for the cells in the pronese, and the cells in the trypsin had the worst. CONCLUSION： The combination of pronase and trypsin is an effective method for tracheal epithelial cells culture.