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二次谐波/双光子激发荧光显微成像技术定量评估非酒精性脂肪性肝病小鼠模型肝纤维化的价值
Value of second harmonic generation/two-photon excitation fluorescence in quantitative evaluation of liver fibrosis in mice with nonalcoholic fatty liver disease
文章发布日期:2019年07月05日  来源:  作者:王晓晓,赵洁,李晓鹤,等  点击次数:134次  下载次数:7次

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【摘要】:目的通过二次谐波(SHG)和双光子激发荧光(TPEF)显微成像技术分析非酒精性脂肪性肝病(NAFLD)小鼠模型胶原参数动态变化,找出并建立适合蛋氨酸胆碱缺乏饲料(MCD)诱导的NAFLD小鼠的自动化定量评估参数,为SHG/TPEF显微成像技术应用于临床提供实验依据。 方法获取MCD饮食小鼠不同时间点(0、4、8、12、16、20和24周)的肝组织标本,行HE、Masson和天狼猩红(SR)染色,并计算胶原蛋白比例面积(CPA)和羟脯氨酸(HYP)。使用SHG/TPEF纤维成像技术分析100个胶原参数。以造模后不同时间点和不同纤维化分期(S0~S4)为标准,采用支持向量机算法(SVM)模型分析胶原参数,并对参数行受试者工作特征曲线(ROC 曲线)分析,同时与CPA、HYP进行比较。结果在MCD小鼠模型中,HE和SR染色后可以观察到随着造模时间的延长,肝小叶内脂肪变形成,纤维化逐渐加重。分别基于造模不同时间点和不同肝纤维化分期,选出26和27个参数;进一步采用SVM模型分析筛选出7个共同参数(#StrCV、#ShortStrCV、#ThickStrCV、#StrPTAgg、#StrPSAgg、#LongStrPSAgg和StrLengthPSAgg)并进行论证,7个参数与不同肝纤维化分期相关的ROC曲线下面积(AUC)为:0.857~0.923,P<0.05,与不同时间点相关的AUC为:0823~0.976,P<0.05。进一步比较7个参数和CPA及HYP对肝纤维化的预测价值,发现7个参数的AUC在分期0 vs 1~4时,与CPA和HYP的AUC值接近,其他分期比较,7个参数的AUC值均高于CPA和HPA;同样在造模后不同的时间点,发现7个参数的AUC在造模早期0 vs 4周时,与CPA和HYP的AUC值接近,造模4周后比较,7个参数的AUC值均高于CPA和HPA。结论7个与纤维化阶段和不同时间点相关的参数组合,可以准确反映MCD诱导的NAFLD模型不同分期和不同时间点的肝纤维化变化,可用于该模型中以定量的方式具体、准确地监测肝纤维化进展。
【Abstract】:ObjectiveTo investigate the dynamic changes in collagen parameters in mice with nonalcoholic fatty liver disease (NAFLD) by second harmonic generation (SHG)/two-photon excitation fluorescence (TPEF), to establish the parameters for automatic quantitative evaluation of mice with NAFLD induced by methionine- and choline-deficient diet (MCD), and to provide an experimental basis for the application of SHG/TPEF in clinical practice. MethodsLiver tissue specimens of MCD mice were collected at weeks 0, 4, 8, 12, 16, 20, and 24, and HE staining, Masson staining, and sirius red (SR) staining were performed. Collagen proportionate area (CPA) and hydroxyproline (HYP) were calculated. SHG/TPEF was used to analyze 100 collagen parameters. With time points and fibrosis stage (S0-S4) as criteria, the SVM model was used to analyze the collagen parameters. A receiver operating characteristic (ROC) curve analysis was performed for the collagen parameters, and these parameters were compared with CPA and HYP in terms of the area under the ROC curve (AUC). ResultsIn the MCD mice, HE and SR staining showed that over the time of modeling, hepatic steatosis was observed in hepatic lobules, with gradual aggravation of fibrosis. A total of 26 parameters were screened out based on their correlation with time point, and 27 parameters were screened out based on their correlation with liver fibrosis stage. The analysis based on the SVM model identified 7 shared parameters (#StrCV, #ShortStrCV, #ThickStrCV, #StrPTAgg, #StrPSAgg, #LongStrPSAgg, and StrLengthPSAgg). These 7 parameters had an AUC of 0.857-0.923 (P<0.05) in predicting different stages of liver fibrosis and an AUC of 0.823 -0.976 (P<0.05) in predicting liver fibrosis at different time points. These 7 parameters were compared with CPA and HYP in terms of their value in predicting liver fibrosis, and it was found that the 7 parameters had a similar AUC as CPA and HYP in predicting S0 fibrosis, while the 7 parameters had a significantly higher AUC than CPA and HYP in predicting S1-S4 fibrosis. The 7 parameters had a similar AUC as CPA and HYP at week 0 of modeling, while at week 4 of modeling, the 7 parameters had a significantly higher AUC than CPA and HYP. ConclusionSeven parameters associated with fibrosis stage and time point can accurately reflect the changes in liver fibrosis in different stages and at different time points in a model of MCD-induced NAFLD, and therefore, they can be used for specific and accurate monitoring of liver fibrosis in a quantitative manner in this model.
【关键字】:非酒精性脂肪性肝病; 肝硬化; 显微成像技术; 小鼠, 近交C57BL
【Key words】:non-alcoholic fatty liver disease; liver cirrhosis; excitation fluorescence imaging; mice, inbred C57BL
【引证本文】:WANG XX, ZHAO J, LI XH, et al. Value of second harmonic generation/two-photon excitation fluorescence in quantitative evaluation of liver fibrosis in mice with nonalcoholic fatty liver disease[J]. J Clin Hepatol, 2019, 35(8): 1786-1790. (in Chinese)
王晓晓, 赵洁, 李晓鹤, 等. 二次谐波/双光子激发荧光显微成像技术定量评估非酒精性脂肪性肝病小鼠模型肝纤维化的价值[J]. 临床肝胆病杂志, 2019, 35(8): 1786-1790.

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