| Hepatocellular carcinoma(HCC) as one of the most frequent tumors is life threatening to human being.There are 564,000 new cases every year. The incidence rate of HCC ranks the fifth among all tumors in the world,and the mortality ranks the third. High rates are seen in China and some Asia- Africa countries. The cases in China account for 40-45% of those in the world.As a country with high incidence rate of hepatitis ,the incidence and mortality rate of HCC increase steadily recently in China, IARC reports in 2001 shows age-standardised rate (ASR) of Chinese male is 35.17 per 100,000, ranks the third after lung cancer and gastric cancer;that of female is 13.34 per 100,000, ranks the fouth after gastric cancer breast cancer and lung cancer ; the death cases accout for 22.1% of those of all tumors ,exceeding lung cancer ,ranks the first.Recent researches shows HCC carcinogenesis is a multi-gene and multi-step complicated process. The mechanism of molecular regulation is not completely clear. Most of the patients of HCC in China have hepatitis B history, but the incidence rate of HCC in patients of hepatitis B without cirrhosis is far lower than that with cirrhosis. Nearly 84.6% of HCC come with cirrhosis, whereas 49.9% of cirrhosis will develop into HCC, which hints HBV, cirrhosis, HCC is a three-stepprocess.Therefore, to detect the expression of oncogene and suppressor gene in cirrhotic liver is not only essential to find the gene changes in HCC early stage but also helpful to expound the carcinogenesis of HCC. There are little reports on the effect of liver cirrhosis on HCC carcinogenesis, most of which are mainly focus on known individual oncogene or suppressor gene. There is no reports on screening of differentially expressed genes in large scale and construction of cDNA library. In our study , the differentially expressed genes between normal liver and liver cirrhosis tissue accompanied with hepatocellular carcinoma were screened by suppression subtractive hybridization (SSH), and cDNA library was constructed, 70 colonies (parts of library)were analyzed by restriction enzyme analysis and PCR, 16 of positive colonies were sequenced further and aligned by BLAST(Basic Local Alignment Search Tool) in NCBI ,which set up the foundation of the study on the effect of liver cirrhosis in HCC carcinogenesis.Part I : Screening of differentially expressed genesbetween normal liver and liver cirrhosis tissueaccompanied with hepatocellular carcinomaAim: To screen the differentially expressed genes between normal liver and liver cirrhosis tissue accompanied with hepatocellular carcinoma Methods: 3 normal liver and 3 liver cirrhosis tissue accompanied with hepatocellular carcinoma as comparative materials ,SSH was used. The populations of mRNA was isolated first and reverse transcripted to cDNA, the samples were divided into tester and driver, specific adaptors were ligated to tester .Two-round unbalanced hybridization and nested polymerase chain reaction were performed .The differentially expressedgenes between two tissues were acquired . The subtraction efficiency was also tested.Results: Tests of quantity and quality of mRNA populations , Rsal digestion of cDNA , ligation efficiency of adaptors and Subtraction Efficiency all reached the standards of experimental design. Conclusion: The differentially expressed genes between normal liver and liver cirrhosis tissue accompanied with hepatocellular carcinoma were separated successfully first .Part II: Construction of a subtracted cDNA library of differentially expressed genes and primary identificationAim: To construct a subtracted cDNA library of differentially expressed genes and identify primarily.Methods: TOPO T/A Cloning technique was used, the differentially expressed genes acquired in part I were inserted into TOPO T vector, and transform TOP10 One Shot Competent Cells ( E.Coli), a subtracted cDNA library containing all differentially expressed genes between normal liver and liver cirrhosis tissue with HCC background...
|
PDF Full Text Request