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[Objective] In this study, by studying diethylnitrosamine (DEN) induced Wistar rat liver cancer model liver tumor tissue specimens in vitro high-resolution magic angle spinning 1H spectral analysis, and control the histological examination results from the molecular level metabolic changes in liver tumors and normal tissue. [Method] 1. Establishment of animal models: Wistar 1-month-old rats were 65, 50 were randomly divided into two groups: the experimental group and the control group 15. Experimental group (n = 50), according to the weight intraperitoneal injection of 70mg/kg DEN (99.9% purity), once a week for a total injection of 10 weeks from the weekend of the 17th week randomly 5-6 only tumor tissue samples extracted until 25 on weekends. Which from the late start, one after another, nine died. Control group: (n = 15) to give the normal basic diet. The weekend of the 17th, every other week to organize random 1-2 sample extraction up to 25 weekend. The end of the experiment, and no death. Throughout the experiment for 27 weeks. Sample collection and in vitro tissue block sample preparation: collection of tumor samples will be divided into one of quickly into the second half of placement frozen in liquid nitrogen at -80 ℃ sent to the analysis of high-resolution magic angle spinning proton spectroscopy vitro and the other half sent to pathology; same control group. Line of high-resolution magic angle spinning proton spectroscopy, cut about weight is about 15mg ZrO2 rotor load spectral analysis of liver tissue samples. Vitro tissue blocks of high-resolution spectral analysis: dedicated spectral analysis software, baseline correction, noise filtration, concentration correction, the correction of the chemical shift, filter out may cause errors in the peak after peak detection and peak matching use of the OPLS, wilcoxon rank and test and Hottelings'T2 test methods of data for statistical analysis, combined with the chemical shifts of the peaks identified compounds. [Results] 1. Pathological findings: the total process of the experiment was 27 weeks, which post-experimental rats in the experiment began to die 9, the survival rate was 82%. The control group had no deaths. Liver pathology confirmed the control group were normal liver; the experimental group pathological prompted three cases of cholangiocarcinoma, six cases of atypical hyperplasia, adenomatous hyperplasia, 31 cases of hepatocellular carcinoma. 14 cases of hepatocellular carcinoma grade Ⅰ, Ⅱ grade, Ⅲ grade seven cases, Ⅳ grade six cases. Effective spectroscopy 2.HR-MAS MRS results: the resulting number of 11 cases, 26 cases (normal control group; 15 cases of liver tumors, including hepatocellular carcinoma 10 cases of cholangiocarcinoma cases of adenomatous hyperplasia, not atypical hyperplasia 3 cases). Spectroscopic analysis and crest identified the following results: ① normal control group, choline, glycerophosphocholine, β-glucose, α-glucose, triglycerides relative concentrations were 11.2 ± 3.4,7.04 ± 3.10,11.7 ± 4.81,29.1 ± 4.71,13.3 ± 12.1, DEN treated its relative concentration is 2.41 ± 2.00,1.18 ± 1.26,2.40 ± 3.57,11.9 ± 7.32,1.24 ± 2.16, the above metabolites in normal liver group relatively high content of liver tumors by DEN treatment group (P <0.05). DEN treatment group of glutamic acid, isoleucine, lysine relative concentrations were higher than the normal control group was 4.57 ± 2.28,6.34 ± 3.40,5.83 ± 3.88, 0.245 ± 0.400,0.406 ± 0.59,0.425 ± 0.618 (P <0.05). ② hepatocellular carcinoma and other pathological type lesions (bile duct carcinoma, atypical hyperplasia, adenomatous hyperplasia): the relative concentrations of choline HCC was 2.39 ± 1.42, higher than other pathological types of 0.242 ± 0.387 (P <0.05). Other differences larger lysine, it is a relatively high concentration in hepatocellular, but P> 0.05, this may be because of fewer samples than cause. ③ Because hepatocellular carcinoma contained in the different levels of the small sample size, can not line statistical tests. Metabolite analysis in the classification of hepatocellular carcinoma, showing the relative concentrations of glycogen, glucose was significantly higher than in hepatocellular carcinoma grade Ⅰ Ⅱ - Ⅳ grade; alanine essentially not detected in Ⅰ - Ⅲ grade Ⅳ level relative content significantly increased Board. [Conclusion] The present study results show that successfully prepared by DEN-induced rat liver cancer model, high-resolution magic angle spinning proton spectroscopy provides a reliable animal model for liver tumors in vitro. 2 In this study, the error factors introduced in vitro high-resolution magic angle spinning nuclear magnetic resonance techniques to avoid tissue extraction complex process, at the same time to obtain high-resolution spectra, both to improve the accuracy of the spectra peaks identified, but also reduce the statistical analysis error. OPLS (orthogonal signal correction partial least squares) of liver cancer and other pathological types of liver tissue metabolites and different grading liver metabolite differences well distinguish. Compared with normal liver tissue in rats, DEN treatment of liver tumor tissue glutamate (Glu), lysine (Lys), iso-leucine (Ile) relative concentration (P <0.05); normal liver choline (Cho), glycerophosphocholine (GPC), glucose the glycogen (GLC Gly), α-glucose (α-Glc), β-glucose (β-Glc) and triglyceride (TG) content is higher than the tumor tissue ( P <0.05); hepatocellular carcinoma has a relatively high content of choline (P <0.05) compared with other pathological types. Poorly differentiated HCC with well-differentiated hepatocellular carcinoma the metabolite differences reflect trends.
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