Automated evaluation of liver fibrosis in thioacetamide, carbon tetrachloride, and bile duct ligation rodent models using second-harmonic generation/two-photon excited fluorescence microscopy.

Animal models provide a useful platform for developing and testing new drugs to treat liver fibrosis. Accordingly, we developed a novel automated system to evaluate liver fibrosis in rodent models. This system uses second-harmonic generation (SHG)/two-photon excited fluorescence (TPEF) microscopy to assess a total of four mouse and rat models, using chemical treatment with either thioacetamide (TAA) or carbon tetrachloride (CCl4), and a surgical method, bile duct ligation (BDL). The results obtained by the new technique were compared with that using Ishak fibrosis scores and two currently used quantitative methods for determining liver fibrosis: the collagen proportionate area (CPA) and measurement of hydroxyproline (HYP) content. We show that 11 shared morphological parameters faithfully recapitulate Ishak fibrosis scores in the models, with high area under the receiver operating characteristic (ROC) curve (AUC) performance. The AUC values of 11 shared parameters were greater than that of the CPA (TAA: 0.758-0.922 vs 0.752-0.908; BDL: 0.874-0.989 vs 0.678-0.966) in the TAA mice and BDL rat models and similar to that of the CPA in the TAA rat and CCl4 mouse models. Similarly, based on the trends in these parameters at different time points, 9, 10, 7, and 2 model-specific parameters were selected for the TAA rats, TAA mice, CCl4 mice, and BDL rats, respectively. These parameters identified differences among the time points in the four models, with high AUC accuracy, and the corresponding AUC values of these parameters were greater compared with those of the CPA in the TAA rat and mouse models (rats: 0.769-0.894 vs 0.64-0.799; mice: 0.87-0.93 vs 0.739-0.836) and similar to those of the CPA in the CCl4 mouse and BDL rat models. Similarly, the AUC values of 11 shared parameters and model-specific parameters were greater than those of HYP in the TAA rats, TAA mice, and CCl4 mouse models and were similar to those of HYP in the BDL rat models. The automated evaluation system, combined with 11 shared parameters and model-specific parameters, could specifically, accurately, and quantitatively stage liver fibrosis in animal models.

[Methylenetetrahydrofolate reductase and methionine synthase reductase gene polymorphisms in ethnic Han women from Linyi].

OBJECTIVE: To explore the distribution of genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) 677C/T, 1298A/C and methionine synthase reductase (MTRR) 66A/G among ethnic Han females from Linyi, and to correlate it with serum level of homocysteine (Hcy). METHODS: A cross-sectional study was carried out. Oral epithelial cell samples were collected from 825 subjects. MTHFR and MTRR gene polymorphisms were determined with a Taqman-Minor Groove Binder (MGB) method. Distribution of gene polymorphisms was analyzed and compared with others regions of China including Weifang, Zhengzhou, Deyang and Hainan. A biochemical assay was also carried out to determine the total Hcy in plasma of 281 subjects. The reductase activity of MTHFR was classified into decreased and stable groups according to genetic polymorphism of MTHFR. Correlation between MTHFR groups and total Hcy level were also explored. RESULTS: (1) The frequencies of MTHFR677CC, CT and TT genotypes of the selected subjects were 16.7%, 48.3% and 35.0%, respectively. The frequencies of MTHFR 1298AA, AC and CC genotypes were 76.0%, 21.6% and 2.4%, respectively. And those of MTRR 66AA, AG and GG genotypes were 54.7%, 39.4% and 5.9%, respectively. For the selected subjects, their frequency of MTHFR 677TT genotype was higher than that of Deyang and Hainan (P< 0.01), whilst the frequency of MTHFR 1298CC genotype was lower than that of Deyang and Hainan (P < 0.01), and the frequency of MTRR 66 GG genotype was lower than that of Hainan (P< 0.01). (2) The Hcy level for those with decreased MTHFR activity was significantly higher than those with stable MTHFR activity (P< 0.05). CONCLUSION: MTHFR gene 677C/T, 1298A/C and MTRR 66A/G polymorphisms in ethnic Han women from Linyi have differed significantly from other regions of China. Decreased MTHFR activity caused by genetic polymorphisms is a risk factor for raised Hcy level.

[Study of genetic susceptibility in 198 children with asthma].

OBJECTIVE: To analyze the frequency distribution of single nucleotide polymorphisms (SNPs) of four asthma-related gene loci (ACE I/D; ADRB2 Arg16Gly; TNF-α G-308A; MS4A2 Glu237Gly) in 198 asthmatic children, and to investigate its association with genetic susceptibility to childhood asthma and some clinical phenotypes of asthma. METHODS: Polymerase chain reaction product electrophoresis identification and real-time quantitative PCR detecting system were used to determine the frequency distributions of the SNPs of the four asthma-related gene loci in 198 asthmatic children and 110 healthy controls. The serum total IgE (TIgE) levels and blood eosinophil proportion (%EOS) of the asthmatic children were measured. Different genotypes at the four asthma-related gene loci were compared in terms of TIgE and %EOS. RESULTS: The genotype DD of angiotensin-converting enzyme (ACE) had a significantly higher frequency in the asthmatic children than in the healthy controls (χ2= 30.667, P<0.01), and the frequency of D allele was also significantly higher in the asthmatic children than in the healthy controls (χ2=7.151, P<0.01). No correlation was found between the polymorphism of each gene locus and serum TIgE level and %EOS (P>0.05). CONCLUSIONS: Genotype DD of ACE is related to genetic susceptibility to childhood asthma and may be the risk factor for childhood asthma.