小鼠肺组织羟脯氨酸含量测定的实验方法优化

Optimized experimental method for determination of hydroxyproline in lung tissue of mice

目的羟脯氨酸(HYP)的检测手段众多,但鲜有精确适用于小鼠肺组织的方法。文章拟建立一种准确、可靠的基于小鼠肺组织的HYP水平测定方法,以更为有效地评估其纤维化发展程度。方法以碱水解法为基础,比较碱水解液浓度及水解时间对小鼠肺组织HYP水平测定结果的影响;比较实验相关条件改变对HYP标准品测定结果的影响;以上述实验结果为基础,比较干燥和湿润的小鼠肺组织HYP水平测定结果。结果通过优化得出,碱水解液最佳浓度为2 mol/L,最佳水解时间为20 min;柠檬酸缓冲液最适pH值为6.0~6.5,适用于氯胺T最佳溶剂为甲醇,氯胺T溶液的最佳反应时间为15 min,高氯酸溶液的最佳反应时间为5 min,4-(二甲氨基)苯甲醛溶液的最佳显色条件为85℃水浴3 min,用干燥肺组织测得的HYP含量[(212.26±8.83)mg/g肺组织]较用湿润肺组织[(162.70±15.04)mg/g肺组织]高(P<0.01)。用改进后方案测得的肺组织HYP水平([(375.20±6.83)mg/g肺组织])明显高于使用优化前[(345.99±8.57)mg/g肺组织]。以HYP标准品浓度为横坐标,对应的OD值为纵坐标绘制标准曲线,其回归方程为y=0.014x+0.0725,相关系数为R 2=0.9991。回收率分别为118.68%、117.38%和117.88%,均在80%~120%之间,说明本法较为可靠。结论建立了一种准确、可靠的基于小鼠肺组织的HYP水平测定方法。

Objective There are many methods for the detection of hydroxyproline(HYP),but few of them are suitable for the detection of lung tissue in mice.We intend to establish an accurate and reliable method for measuring HYP levels based on mouse lung tissues to assess the degree of fibrosis development more effectively.Methods Based on the alkali hydrolysis method,the effects of the concentration of alkali hydrolysate and hydrolysis time on the determination results of HYP level in mice lung tissue were compared;the effects of the changes of experimental conditions on the determination results of HYP standard were compared;and the results of the determination of HYP level in mice lung tissue under dry and wet conditions were compared on the basis of the above experimental results.Results The optimum concentration of alkali hydrolysate is 2 mol/L and the optimum hydrolysis time is 20 min.The optimum pH value of citric acid buffer is 6.0-6.5.The optimum solvent for chloramine T is methanol,the optimum reaction time for chloramine T solution is 15 min,the optimum reaction time for perchloric acid solution is 5 min,and the optimum reaction time for 4-(dimethylamino)benzoyl toluene is 5 min.The optimum condition of aldehyde solution color development is that it is bathed in water at 85 for 3 minutes.Some related reagents are stored in suitable environment after preparation,and the experimental data will not be affected within 7 days.Dry lung tissue of mice can improve the detection level of HYP.The improved experimental protocol was applied to the bleomycin-induced mouse pulmonary fibrosis model,and the HYP measurement results were significantly higher than that of the original protocol.Conclusion An accurate and reliable method for the determination of hydroxyproline in lung tissue of mice was established.