| Liver injury and regeneration are often associated with many liver diseases, including chronic and acute hepatesis, acute liver failure, alcoholic liver disease, etc. The inhibition of hepatocyte death or stimulation of hepatocyte proliferation would benefit to relieve symptoms of these diseases, and the study of liver regeneration would also be helpful to develop new drugs or treatments for various liver diseases. Here, we applied a cDNA microarray technique to analyze gene expression prolife during liver regeneration induced by CCl4 in mice, under the hypothesis that the process of liver regeneration is a homeostatic event of gene regulation. This homeostatic model of liver regeneration will be tested at the levels of mRNA expression and physiological evaluation of the process. This thesis study includes two parts: the first part is the study of gene expression prolife during liver regeneration; the second part is the study on verification of spp1 function during liver regeneration of mice.In the first part, 9-week-old male C57BL/6 mice were injected intraperitoneally with 1ml of carbon tetrachloride (CCl4) per 1kg of body weight as a 25% solution in peanut oil. Three mice were sacrificed at each point (0, 0.5, 1.5, 4.5, and 7 days after CCl4 treatment), and their alanine aminotransferase (ALT) levels were measured. The mouse liver was removed for total mRNA extraction. For the histological studies, a piece of liver tissue was fixed in 10% formalin, and liver sections were cut 5μm and stained with hematoxylin-eosin. The PCNA staining was also performed to count the numbers of proliferating hepatocytes. Genechip? Mouse genome 430.2 micro-array was used to analyze the gene expression prolife at different time-points. Changes of gene expression were log2-transformed using signal value at the 0 day time-point as base line. Genes with expression levels that varied at least 2-fold compared to that of 0 day time-point were subjected to hierarchical clustering, physiological evaluation, and gene analysis. In addition, amino acid metabolism pathway was identified. Results showed that 2110 genes whose expression were changed during liver regeneration, and they were divided into 12 clusters. 35.45% genes expression at mRNA levels decreased initially and remained below the base line. The mRNA levels of 11.51% genes decreased gradually then increased toward the base line. These genes with differential expression patterns were grouped into 117 transcription factors encoding genes and 217 signal transduction genes, including spp1. Only those genes with their expression patterns in comply with our homeostasis hypothesis were collected, and profiles of these gene expression were constructed and further analyzed.In the second part, the mouse spp1 gene was cloned into the pcDNA3.1 expression vector (spp1-pCDNA3.1) and the pET28a vector (spp1-pET28a), respectively. The spp1-pCDNA3.1 plasmid was transfected to the muscle of mouse leg under an electrical pulse, and the effect of Spp1 over-expression in liver injury and regeneration model was studied in vivo. The spp1-pET28a vector was transformed to Ecoli BL21, and recombinant mouse Spp1 (rmSpp1) protein was expressed and purified from bacteria. The purity of rmSpp1 reached to 98%, and it had biological activity. For understanding the mechanism of Spp1 regulating liver regeneration, mice were immediately administrated rmSpp1 (dose was 1mg/kg body weight, in control group, mice was injected an equal volume of PBS) or anti-Spp1 (dose was 20μl for a mouse, in control group, mice was injected an equal volume of preserum) subcutaneously after injection of CCl4 intraperitoneally. Five mice were sacrificed at each point (1, 3, 5 and 7 days) of every group. The sample collection, fixation, and staining were carried out. Results showed that ALT activity and the ratio of necrotic to normal areas of liver were significantly lower in mice treated with rmSpp1 than that in control (78.6±59.0 vs 186.6±109.0 IU/L, p=0.018; 11.7±3.4% vs 21.0±4.4%,p=0.0006 ) at 3 days time-point. But positive nuclear staining of PCNA was more in rmSpp1 treatment group than in control at the same time-point. Contrarily, ALT activity was higher in anti-Spp1 treatment group than in control group (426.9±166.5 vs 90.8±61.9, p=0.008). The ratio of liver necrotic to normal areas was higher in anti-Spp1 treatment group than in control at 3 and 5 days time-points (24.1±4.4% vs 8.6±1.4%,p=0.014; 3.4±0.29% vs 1.7±0.45%,p=0.007, respectively). The number of proliferating hepatocytes in anti-Spp1 treatment mice was higher than that in control mice (6.0±1.7 vs 3.3±0.7/ 0.01mm2,p=0.03).Conclusion: We identified genes according to our hypothesis of homeostasis model, analyzed those genes with differential expression profiles during liver regeneration, and refined the search for liver regeneration regulatory candidate genes. In addition, the function of spp1 was studied, as an example, in controlling liver regeneration induced by CCl4. Our data indicated that Spp1 could inhibit hepatic necrosis and stimulate liver regeneration in mice. |
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