| Part I. Construction and Charaterization of cDNA Libraries from Liver Tissues of Chronic Liver DiseasesIn China, the number of patients with chronic hepatitis B accounts for nearly 10% of the population, due to the high ratio of chronic HBV infection. The natural course of chronic HBV infection is characterized by a series of hepatitic flares or exacerbations and remissions. The HBV replication cycle is not directly cytotoxic to hepatocytes. It is now thought that host immune responses to viral antigens displayed on infected hepatocytes are the principal determinants of hepatocellular injury. The immune responses to HBV and their role in the pathogenesis of hepatitis B are incompletely understood.HBV infection is a serious clinical problem because of its wide distribution and possible adverse consequences, such as hepatic decompensation, cirrhosis and/or hepatocellular carcinoma. The molecular pathogenesis of hepatitis B and that from the initial stage of chronic hepatitis B to end-stage liver disease with liver failure remains largely unknown.Constructing a cDNA library will lay solid foundation for finding relevant genes and investigating their functions. Unlike genome DNA due to the introns in it.cDNA is not difficult to be expressed. So a cDNA library can be used not only to screen the target genes required, but also to express them, with the application oflarge scale EST sequencing, SEREX, yeast 2-hybrid system and phage display.Thus, We constructed three cDNA libraries from Asian Chinese liver tissue withchronic hepatitis B, liver cirrhosis and hepatocellular carcinoma, and this will help in not only understanding the gene spectrums of chronic hepatitis B, liver cirrhosis and hepatocellular carcinoma, but also screening the relevant genes and interesting proteins associated with them. It will lay solid foundation not only for better understanding of the molecular pathogenesis of chronic hepatitis B, liver cirrhosis and hepatocellular carcinoma and the development from chronic hepatitis B to the end-stage liver disease, but also for searching more effective theraputic strategies.Methods: The total RNA from liver tissue with chronic hepatitis B, liver cirrhosis and hepatocellular carcinoma were extracted and the mRNAs were purified using TRIZOL method. SMART technique and COSIII/3' primer were used for first-strand cDNA synthesis. LD PCRs were then used to synthesize the double-strand cDNAs that were then digested by Sfi I and fractionated by CHROMA SPIN-400 columns. The longer than 0.4 kb cDNAs were collected and ligated to X TriplEx2 vector. Then X phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. Fourteen, eleven and fourteen plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector.Results: The titers of unamplifed libraries of chronic hepatitis B, liver cirrhosis and hepatocellular carcinoma were 1.94×l06pfu/ml 1.03×106pfu/ml and 1.37×106pfu/ml respectively. The percentages of recombinants from three libraries were 98.15%, 97.24% and 97.46% respectively. While the amplified libraries of chronic hepatitis B, liver cirrhosis and hepatocellular carcinoma were 1.49×109pfu/ml, 1.36× 109 pfu/ml and 1.28× 109pfu/ml respectively, the percentages of recombinants from three libraries were 98.76%, 99.02% and 99.06% respectively. The lengths of the library inserts of chronic hepatitis B were 1.23 kb in average, 1kb~2 kb in 64.29%, and 0.5 kb~1.0 kb in 35.71%. The lengths of the library inserts of chronic hepatitis B were 1.23 kb in average, 1 kb~2 kb in 64.29%, and 0.5 kb~1.0 kb in 35.71%. The lengths of the library inserts of liver cirrhosis were 1.02 kb in average, 1 kb~2 kb in 36.36%, and 0.5 kb~1.0 kb in 63.64%. The lengths of the library inserts of hepatocellular carcinoma were 0.96 kb in average, 1 kb~2 kb in 40%, and 0.5 kb~1.0 kb in 60%.Conclusion: Three high quality cDNA libraries from human liver tissue wi...
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