| Hepatocellular carcinoma(HCC) is one of common malignant tumors in the world and high incidence malignant tumors in Guangxi.For several yeas many animal experiments and human epidemiological studies illuminate that: long-term chronic exposure to aflatoxin B1 (AFB1)with liver cancer have a clear relationship. The reasons could be that the end products are generated by AFB1 in vivo metabolic activation, resulting in changes of cells in genetic characteristics, and to bring its mutagenic effects of carcinogens. However, there are still unclear of molecules mechanism in the process of AFB1 exposure, metabolism, detoxification and DNA repair, still a lack of stability, convenient and accurate biomarkers that can be used for early diagnosis the liver cancer of AFB1, so it is very important to investigate and find the biomarkers about occurrence and development of diseases .With the post-genome era, people come to realize: the complexity interactions of the gene and protein, intracellular activity and the environment will influence the gene expression and protein translation process. AFB1 damage the target gene mainly through its combination of the end of carcinogens and DNA adduct formation. Gene's product is a protein, cell phenotype changes in the final through the protein expression, which is a major life function of enforcement. Proteomic researches in oncology currently has become a powerful tool, Surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is the emergence of new proteomics research methods in recent years, and has been used for a variety of biomarkers of early disease detection and diagnosis research with its high-throughput and high sensitivity.Peri-cancer tissue of hepatocellular carcinoma is divided into high and low AFB1 exposure groups by immunohistochemical detection of aflatoxin B1-DNA adduct. To identify proteins in this two groups that could be further screened different expression protein related to AFB1-induced HCC by SELDI-TOF-MS, at the same time, normal liver tissue as the control.To clarify the specific environmental factors in the course of the pathogenic mechanism, to screen early diagnosis, prevention and early treatment measures for AFB1-induced HCC in AFB1 exposure areas, this research will provide an important theoretical and application of basis.Part one: Immunohistochemical detection of aflatoxin B1-DNA adduct in peri-cancer tissue of hepatocellular carcinomaObjective: To group for the second part according to AFB1-DNA adducts level in peri-cancer tissue of hepatocellular carcinoma.Methods: 36 cases peri-cancer tissue of hepatocellular carcinoma in paraffin sections were detected by immunohistochemical techniques by reference to Regina M. Santella.The positive granular of AFB1-DNA adduct were Purple in hepatic cell nucleus, membrane or cytoplasm. The cumulative scores in accordance with the extent and the percentage of positive cells will determine AFB1-DNA adduct levels. Each batch of Immunoperoxidase staining,at the same time the use of positive and negative control.Results: AFB1 were detected in varying degrees, which mainly concentrated in the hepatic cell nucleus, showing staining Purple granular of AFB1-DNA adducts. Liver cell membrane andcytoplasm also was detected, but not in bile duct epithelial cells. The distribution of positive staining of granulosa cells spread with high power microscope, for 5～90%. By the statistics, 23 cases peri-cancer tissue of hepatocellular carcinoma were in a high level of AFB1- DNA adducts(score of 3～6) and 13 cases were in a low level(score of 0～2).Conclusion: AFB1-DNA adduct levels help to assess the situation of AFB1 exposure in HCC.Part two: Using SELDI protein chip technology to screen for tissue different proteomic AFB1-related hepatocellular carcinoma Objective: Screening for tissue different proteomic AFB1-related hepato- cellular carcinoma from Guangxi by SELDI protein chip technology, and discoverying proteins related to AFB1-induced hepatocellular carcinoma.Methods: With the part one of the immunohistochemical detection AFB1- DNA adduct levels in peri-cancer tissue of hepatocellular carcinoma, patients were divided into high AFB1 exposure (AFB1-DNA adducts in a high level, 23 cases) and low exposure (AFB1-DNA adduct in a low leve, 13 cases), selecting normal liver tissue as control(14 cases). Three groups samples of liver tissue were profiled by SELDI-TOF-MS technology and CM10 protein chip. Biomarker Wizard software was used for statistical analysis.Results:1. Comparison of the groups of hepatocellular carcinoma and the normal controls: detect 130 protein peak,among them 9 protein peaks show the difference obviously in two groups(P<0.05).Compared with the normal controls, 5 proteins were up-regulated expression and the other 4 proteins were down- regulated expression in hepatocellular carcinoma group.2. Comparison of the groups of AFB1 high-exposure hepatocellular carcinoma and the normal controls: detect 132 protein peak,among them 10 protein peaks show the difference obviously in two groups(P<0.05).Compared with the normal controls, 5 proteins were up-regulated expression and the other 5 proteins were down-regulated expression in AFB1 high-exposure hepato- cellular carcinoma group.3. Comparison of the groups of AFB1 low-exposure hepatocellular carcinoma and the normal controls: detect 132 protein peak,among them 13 protein peaks show the difference obviously in two groups(P<0.05).Compared with the normal controls, 6 proteins were up-regulated expression and the other 7 proteins were down-regulated expression in AFB1 low-exposure hepatocellular carcinoma group.4. Comparison of the groups of AFB1 low-exposure and high-exposure hepatocellular carcinoma: detect 132 protein peak,among them 7 protein peaks show the difference obviously in two groups(P<0.05).Compared with the AFB1 low-exposure group, 3 proteins were up-regulated expression and the other 4 proteins were down-regulated expression in AFB1 high-exposure group.5. By comparing of the three groups profile, it found that the protein m/z value is 1126.90, which expression level was AFB1 high-exposure group>AFB1 low-exposure group> the normal control group (P<0.05); The protein m/z value is 10134.59, 10881.76, which expression level were higher in the AFB1 high- exposure and normal controls group than AFB1 low-exposure group (P<0.05); The protein m/z value is 3734.96, 8096.21, which expression level were lower in AFB1 high-exposure group than the AFB1 low-exposure and normal controls group (P<0.05). It suggested these proteins may be involved in abnormal metabolic of AFB1-related hepatocellular carcinoma. Conclusion: Screening the protein difference obviously by SELDI technology combined with bioinformatics, it apply to precancerous prevention and screen early diagnosis markers for AFB1-related hepatocellular carcinoma from Guangxi.
|
PDF Full Text Request