Construction Of A Subtracted CDNA Library And Cloning Of Differentially Expressed Genes In Silicosis Of Rats

Introduction: Pulmonary fibrosis is a sort of destructive lung diseases induced by multiple agents, which has poor prognosis. The pathological processes of pulmonary fibrosis include inflammatory injury, tissue damage and repair. Many kinds of cells such as alveolar macrophages, epithelial cells and fibroblasts et al play important roles in the progress of pulmonary fibrosis. These cells educe direct or indirect effects by secreting many bioactive compounds such as cytokines and mediators of inflammation and constitute a complicate cellular network in which the cells interacting each other and promoting the development of pulmonary fibrosis. However, the critical molecular mechanism in the development of the pulmonary fibrosis is still unknown, it is vital to search for better treatment plan of pulmonary fibrosis, based on the understanding of the pathogenesis of these diseases. As the developing of strategies and methods of identification of novel genes, various methods to compare patterns of gene expression have been described, it is becoming possible to identify some novel genes related with pulmonary fibrosis. The aim of our studies is to screen and identify differentially expressed gene of pulmonary fibrosis.Methods: The SD (Sprage-Dawley) rats were poured into suspension of crystalline silica (200mg/kg body weight) through trachea cannula to construct the animal model of silicosis. The suppression subtractive hybridization was performed; two different forward and reverse subtractions were performed to compare gene expression between lung tissue with silicosis and normal lung tissue. The SSH PCR products from the library were cloned into pMD20-T vector; the positive clones were randomly selected, sequenced and compared to the database in GenBank of the differentially expressed gene fragments from the libraries. Seven novel cDNA sequences were examined by reverse transcription- polymerase chain reaction (RT-PCR) in order to confirm the differentially expression of cDNA fragments in rat's lung tissue with silicosis and normal lung tissue.Results:1. After 360 days, radiograph of chest showed that rat's pulmonary markings was thickened, some scattered high-density shadows appeared in lung field. From the surface and cross section of lung, some scattered nodules could be seen whose sizes were inequable. Under microscope, typical fibrosing siliconic nodules were formed in lung, alveolar epithelial cells and bronchial epithelial cells proliferated around partial fibrosing siliconic nodules, and some cells of which showed atypical hyperplasia. Diffused pulmonary interstitial fibrosis also could be seen.2. Two different forward and reverse subtractions were performed to compare gene expression between lung tissue with silicosis and normal lung tissue. For the lung tissue with silicosis vs. normal lung tissue comparison forward subtraction (named as SNA), lung tissue with silicosis was the tester and normal lung tissue the driver. For the reverse subtraction (named as SNB), normal lung tissue was the tester and lung tissue with silicosis the driver. With the methods of T/A cloning and blue-white screening, the cDNA fragments generated by SSH were cloned into a T/A cloning plasmid (pMD20-T), the subtracted cDNA libraries being constructed. The polymerase chain reaction (PCR) using liquid which containing competent bacteria JM109 was initially used to validate the insert cDNA fragments, many of which contained 200-600bp inserts. seven novel cDNA sequences(named as SNA-11, SNA-28, SNA-43, SNA-48, SNB-13, SNB-30, SNB-42) were examined by reverse transcription- polymerase chain reaction(RT-PCR), the results showed that the expressions of these seven cDNA sequences in rat's lung tissue with silicosis were all different from which in normal lung tissue(Fig. 7). According to the quantitative analysis of image scanning, the gene expression quantity was 2-5 times between two lung tissues.3. Bioinformatics analysis showed that 47 positive clones represented 35 genes containing 2 putative proteins and 4 predicted similar proteins. Most genes whose function known had important roles in the structure and movement of cell or extracellular matrix, the defense of cell and body, material transportation, the regulation of expression and metabolism.Conclusions:1. The animal model of silicosis is successfully constructed.2. Two different forward and reverse subtraction libraries of silicosis are successfully constructed.3. Some genes may be related to pulmonary fibrosis are cloned, if we can study these genes further, it is useful to understand the mechanism of pulmonary fibrosis.4. It is confirmed that silicosis is related to lung cancer and may be a dangerous cause of lung cancer.