Metabolic Characteristics Of Hepatocellular Carcinoma In Rat Induced Via DEN By In Vitro High-Resolution Magic-angle-spinning ~1H NMR Spectroscopy

[Purposes]In this research, by researching diethylnitrosamine (DEN)-induced liver cancer model(Wister rat liver tissue) in vitro high-resolution magic angle spinning 1H MRS analysis, and control the results of histological examination, get metabolism information of liver cancer and normal tissue from the molecular level study. Aim to explore the mechanism of liver cancer.[Method]1. Animal model: 65 Wistar 1-month-old rats are randomly divided into two groups:experimentalgroup 50, control group 15. The experimental group (n=50), according to the weight, injecting 70mg/kg DEN (purity 99.9%) in the intraperitoneal, about once a week, inject totally of 10 weeks, from the 17 weeks we start to collect tumor tissue samples, weekly random kill 5-6 until the 25th week. This group totally die 9.Control:(n=15), fed the normal basis. from the first 17 week, every week only 1-2 are organized random sample extraction, until the 25th week. To the end of the experiment, totally died 0. The experiment cost 27 weeks.2. Sample collection and in vitro tissue sample preparation:Tumor samples will be divided into two, one half is placed in liquid nitrogen and is rapidly placed in-80℃refrigerator, sent to carry out in vitro high-resolution magic angle spinning proton spectroscopy. One half is sent to pathology. The control group is the same.High-resolution magic angle spinning proton spectroscopy analysis, when the liver tissue samples are stored in liquid nitrogen thawed slightly, washing with D2O, cut into approximately the weight of approximately 15mg, put it into the ZrO2 rotor, and then adding 10ul D2O for spectral analysis.3. High-resolution magic angle spinning MRS analysis method:The effective Spectra are 26 cases (normal control group 11 cases,15 cases of liver cancer.In liver tumor group,10 with hepatocellular carcinoma,and other 5 cases) through a dedicated spectral analysis software, after baseline correction, noise filtra-tion, concentration correction, chemical shift correction after the spectral peak detec-tion and spectral peak matched filter may cause the error after the post-peak, using of OPLS, wilcoxon rank sum test and Hottelings' T2 testing methods for multivariate statistical analysis of data. Combining with chemical shifts of the database for peak identification.[Results]1. Pathologic results:Experiment total process is 27 weeks, of which the latter part of the experiment experimental 9 rats began to die, the survival rate is 82%. The control group died 0. The control group were normal liver, confirmed by pathology; experimental group, there are four different pathology, namely,3 cases of bile duct cell carcinoma,6 cases of atypical hyperplasia, adenomatous hyperplasial case, and 31 cases of hepatocellular carcinoma.,of which 14 cases of gradeⅠ,Ⅱgrade 4 cases,Ⅲgrade 7 cases,Ⅳgrade in 6 cases.2. HR-MAS MRS results:①Normal liver groups, the relative concentration of the choline, Glycerophosphocholine,β-glucose, a-glucose, triglyceride are11.2±3.4,7.04±3.10, 11.7±4.81,29.1±4.71,13.3±12.1, while the liver tumor groups, the relative concentration of 2.41±2.00,1.18±1.26,2.40±3.57,11.9±7.32,1.24±2.16, normal liver group were higher than the liver tumor group (P<0.05). On the contrary, the liver tumor group the relative concentration of glutamate, isoleucine, lysine are 4.57±2.28,6.34±3.40,5.83±3.88 higher than the normal group 0.245±0.400, 0.406±0.59,0.425±0.618 (P<0.05).②Hepatocellular carcinoma and other pathological types of lesions (bile duct cell carcinoma, atypical hyperplasia, adenomatous hyperplasia):The relative concentration of choline in hepatocellular carcinoma is 2.39±1.42, which is higher than that in other pathological types of 0.242±0.387 (P<0.05). There are other great differences in isoleucine and lysine, which is relatively high concentrations in human hepatocellular carcinoma, although the P value is greater than 0.05, but this may be because the sample is relatively small.③As the samples of different levels of hepatocellular carcinoma are less, that can not do statistical testing. The different levels of hepatocellular carcinoma metabo-lite analysis can only play a reference role in the relative concentration of glyco-gen+glucose was significantly higher than that in theⅠ-levelⅡ-Ⅳclass. And alanine in theⅠ-Ⅲlevel, basically can not be detected, while in theⅣlevel is significantly higher. [Conclusion]1. The results show that rat liver cancer model induced by DEN is successful,and it provides a reliable animal modeliver tumors in vitro high-resolution magic angle spinning pro-ton spectroscopy study.2. This study uses high-resolution magic angle spinning nuclear magnetic resonance techniques, in order to avoid error factors in the extraction of complex organizational processes. High-resolution spectra can not only improve the accuracy of peaks, but also reduce the statistical analysis of error..3. OPLS method can distinguish metabolites of liver cancer and other pathological types of liver,also different pathological levels in a very good, We conclude that compared with rats with normal liver tissues, he relative concentration in liver tumor tissues of glutamate (Glu), lysine (Lys), isoleucine (Ile) (P<0.05)are higher. Normal liver choline(Cho), Glycerophosphocholine(GPC), glucose+glycogen(Glc+Gly), a-glucose(α-Glc),β-glucose(β-Glc), triglyceride(TG) were higher than in tumor tissue (P<0.05). Between the two groups lactate (Lac) has no significant difference. Hepa-tocellular carcinoma has a relatively high choline (P<0.05)than other histological types. Comparing well-differentiated with hepatocellular carcinoma, the metabolic characteristics are also different.