过氧化氢对大鼠离体肺组织精细切片活性的影响

Effect of Hydrogen Peroxide on Viability of Precision-Cut Lung Slices in Rats

目的研究过氧化氢(H2O2)对离体肺组织精细切片活性的影响,建立肺组织氧化损伤的简易模型。方法制备离体肺组织精细切片,以0.5mLKrebsHenseleit缓冲液作为孵育液,在37℃培养箱内进行孵育。在孵育液中加入H2O2,使其浓度为2mmol/L,分别孵育0(对照组)、5、10、15和20min;然后在不含H2O2的孵育液中进行恢复孵育1h。测定肺片的四甲基偶氮唑盐(MTT)染色、孵育液中乳酸脱氢酶(LDH)和肺片组织ATP含量。结果随H2O2损伤时间延长,MTT染色变浅,其吸光度逐渐减小,损伤时间大于10min组与对照组比较有显著差异(P<0.01)。H2O2损伤10min组肺片的LDH释放多于对照组(P<0.05),损伤时间更长者增加更明显(P<0.01)。H2O2损伤5min组的肺片组织中ATP含量明显低于对照组(P<0.05),损伤时间更长者下降更明显(P<0.01)。经偏相关分析发现,肺片MTT染色与LDH释放呈负相关(r=-0.866,P<0.01),与ATP含量呈正相关(r=0.869,P<0.01)。结论离体肺组织切片在含H2O2的孵育液中进行孵育时,随孵育时间延长其活性降低。此模型简单可靠,可用于肺组织氧化损伤的研究。

Objective To evaluate the viability changes of rat precision-cut lung slices after hydrogen peroxide （H2O2） injury and to establish a simple model for the research on lung oxidative injury. Methods The precisioncut lung slices in rats were prepared and incubated in 37℃ with Krebs-Henseleit（K-H） buffer as incubation liquid. Lung slices were incubated with 2 mmol/L H2O2 for 0 （control）, 5, 10, 15 and 20 min, respectively. Quantitative measurement of the 3-（4,5-dimethyhhiazol-2-yl）-2,5-diphenyhetrazolium bromide （MTT） was assayed by colorimetry, and the contents of ATP in the incubated lung slices and the lactic dehydrogenase （LDH） in the incubated supernate of lung slices were detected. Results With incubation time prolonged for more than 10 min, the lung slices were stained less by MTT, and absorbance was decreased compared with the control （P 〈0.01）. LDH detected in the supernate increased significantly in the lung slices injured for 10 min （P 〈0.05） and for more than 10 min （ P 〈 0.01 ）. The ATP level in the lung slices decreased significantly when injured for 5 min （ P 〈 0.05 ） and for more than 5 min （P 〈0.01）. MTT staining was found to have negative correlation with LDH release （r = -0.866, P 〈 0.01 ） and positive correlation with ATP content （ r = 0. 869, P 〈 0. 01 ） by partial correlation analysis. Conclusion When incubated with H2O2, the viability of precision-cut lung slices could be decreased as incubation time prolonged. This simple and effective in vitro model could be useful for the research on lung oxidative injury.