| LKB1/AMPK signaling pathway is widely involved in cell proliferation, differentiation, survival. apoptosis and the regulation of energy metabolism.Studies have proved that the close relationship between the mutation of LKBl gene induced human colon cancer and other intestinal diseases by the changes of AMPK signaling pathway.Hence, the aim of the research is to know the mechanism of LKB1/AMPK signaling pathway in rabbit enteritis. In early stage of the research, by using the low fiber (CF<8.96%) to induce the non-specific enteritis model in rabbit, we taken the method of RT-qPCR to detect the genes (LKB1.AMPK. mTOR and NF-??) mRNA expression in seven tissues (Duodenum, Sacculus rotundus,Jejunum, Colon, Ileum. Liver and Spleen) between the normal(30 rabbits)and control group (60 rabbits). Besides,it is first to clone and analyze the LKB1 in rabbit. In the later stage, under the culture of rabbit colonic epithelial cells (CEC). the rabbit CEC model was constructed by the use of LPS medicine. By taking the jamming technology of RNAi to interact LKBl gene, the mRNA expression of genes (LKB1,PRKAA2.NUAK1,mTOR,NF-??,IL-1A,TNF-?, PGC and ATG13) was detected by the method of RT-qPCR between the inflammatory cell group and tranfected inflammatory cell group in order to analyze the changes of LKB1/AMPK signaling pathway in the course of enteritis. The main results were as follows:1. The whole CDS sequence of LKB1 gene (GenBank accession No. KT439400) in rabbit was cloned. The total length of CDS was 1317 bp and the number of encoded amino acids was 438. Compared with the homologous gene in mammal (human. Pika and Rattus etc), the sequence of LKB1 gene in rabbit is consist about 87.85%,89.98% and 85.42%. respectively. Thus the results show that LKB1 gene is conversed in mammal.2. After using RT-PCR method to detect the mRNA expression of LKB1.PRKAA2. mTOR and NF-?? in seven types tissues between the model group, the results showed that both the increasing mRNA expression of LKB1 and mTOR gene was negatively correlated with the mRNA expression of NF-?? gene, in the liver tissue of the enteritis group. LKB1, PRKAA2, and NF-?? were negatively correlated with mTOR in the three tissues of the small intestine of enteritis group. In the three tissues of the small intestine of enteritis group, LKB1,PRKAA2. and NF-?? were negatively correlated with mTOR gene, but LKB1 gene expression was positively correlated with NF-?? while mTOR was increased significantly and negatively correlated with mTOR gene.In the immunohistochemical tissues (rotundus and spleen) of enteritis group, all the genes(LKB1?PRKAA2?mTOR) was negatively correlated with NF-?? .3. After resuscitating CEC cells and using the LPS(1000ng/ml), the model of inflammatory cell succeeded to be established.4. By using RNAi technology to interfer LKB1 gene, we succeeded to choose the siRNA (si-02-LKB1)as a optimum siRNA which the silent rate was 90.7%. Construction of siRNA transfected group5. By constructing siRNA (si-02-LKB1) transfected inflammatory cells group and untransfected siRNA inflammatory cells group, we detected the LKB1/AMPK signaling pathway related genes. The results show that LKB1 gene was interferenced by RNAi technology and it increased the downstream genes (PRKAA2, mTOR, PGC-1? and ATG13) in LKB1/AMPK signaling pathway while the genes (NUAKl IL-1A and TNF-?) was decreased. It concluded that LKB1 affects the downstream genes of AMPK signaling pathway in the inflammatory process and slow down the degree of inflammatory reaction in the rabbit enteritis.In this research, we has cloned the LKB1 gene and explored the LKB1/AMPK signaling pathway in rabbits enteritis. The results showed that the LKB1 gene ralated intestinal inflammation by the AMPK signaling pathway and the LKB1 can slow down the inflammatory response of the non-specific entertis in rabbits, which can be used as an important candidate gene for providing a theoretical basis to the disease resistance breeding of rabbits. |
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