| Objective:To illuminate the function of SOCS-3 in the chronic liver injury, study the expression of SOCS-3 in the liver tissue of rats with chronic liver injury and construct the eukaryotic expression plasmid carrying rat suppressors of cytokine signaling-3(SOCS-3) gene. Methods:1. Male Wistar rats (48 cases) were randomly divided into the Control group (12 cases) and the Model group (36 cases). The chronic liver injury was induced by 40% CCL4 hypodermic injection in the Model groups; the Control groups were given arachis oil hypodermical injection at the coordinational condition. Liver tissues and serum were collected at the same time at the end of the fourth, the sixth, the eighth and the tenth week.2. The expression of SOCS-3 mRNA was measured by real-time fluorescent quantitive RT-PCR.3. SOCS-3 protein in liver tissues was assayed by immunohistochemistry.4. The function of liver was measured in Department of Laboratory Medicine of Affiliated Hospital of Lu Zhou Medical College.5. The levels of tumor necrosis factor-a (TNF-a) in serum was tested in Department of Nuclear Medicine of Affiliated Hospital of Lu Zhou Medical College.6. Liver tissue pathological examination:Liver tissues were Stained by HE, the liver inflammation and fibrosis was measured with histological activity index (Knodell HAI).7. Specific primers was designed, the total RNA was extracted from rat liver, The SOCS-3 fragment was cloned into pcDNA3.1 to construct the recombinant pcDNA3.1-SOCS-3 after RT-PCR. The pcDNA3.1-SOCS-3 was vertified by restriction digestion and gene sequencing. 8. Statistical methods:The results were showed by means plus or subtracting stand deviation. Means between the Model groups and Control groups were compared by Independent-Samples T test. One way (AONVA) was utilized in multiple comparisons further. All the statistical methods were accomplished by software SPSS16.0. Results:1. In the Model group, the function of liver and the inflammation of hepatic tissue weren't obviously abnormal at the fourth week. The function of liver, inflammation of hepatic tissue and hepatic fibrosis at the sixth week, the eighth week, and the tenth week were worse than those of the fourth week, and hepatic cirrhosis was found at the tenth week. However, there were no inflammation and fibrosis in Control group.2. The relative expression level of SOCS-3 mRNA was gradually increased with the increasing degree of liver injury in the Model groups, the levels of SOCS-3 mRNA were 2.13±0.79,3.42±0.49,4.24±0.48,6.09±1.08 in the Model groups at the 4th,6th, 8th,10th week, in the Normal control groups, the levels of SOCS-3 mRNA were 0.62±0.34,0.71±0.30,0.71±0.31,0.83±0.24 at the 4th,6th,8th,10th week. The levels of SOCS-3 mRNA in the Model groups were higher than those in the Control groups, and the differences between them were significant.3. The SOCS-3 proteins tested by immunohistochemistry were positively stained to brown, and existed mainly in the cytoplasm of cells. The SOCS-3 proteins were rare in the Control group. Compared with the Control group, the SOCS-3 proteins were significantly increased in the Model groups. The levels of SOCS-3 proteins were analyzed by average optical density, The significant differences were found between the Model groups and the Control groups.4. A positive correlation was found between SOCS-3 mRNA and ALT,AST,TNF-αand histological activity index of liver inflammation and fibrosis, A positive correlation was also found between SOCS-3 protein and ALT,AST,TNF-αand histological activity index of liver inflammation and fibrosis.5. By restriction digestion and gene sequencing, the DNA fragments of SOCS-3 were inserted correctly into pcDNA3.1 vector as expected. Conclusions:1. SOCS-3 expression was increased gradually with the increasing of liver injury and it related with the degree of inflammation, chronic liver injury was protected by SOCS-3.2. The construction of eukaryotic expression plasmid carrying rat suppressors of cytokine signaling-3 gene was successful. |
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