Evaluation of the Stress Effect on Expression of BEST-5 Gene in Cultured Rat Hepatocytes and Cloning of This Gene

Liver has important roles in body metabolic regulation and for this reason hepatocytes are used worldwide. Investigations showed that isolation of hepatocytes causes activation of stress related genes. The aim of this study was to study the stress related expression of BEST-5 following hepatocytes isolation and culture. The BEST-5 gene is cloned and analyzed for the first time from isolated and cultured rat hepatocytes. Very little is known about this gene and almost nothing is known about its function. RNA was isolated from hepatocytes after 3h culture and used for generation of PCR products corresponding to the BEST-5. cDNA generated was cloned into pCR®2.1 plasmid vector. Following transformation into TOPO10 oneshot® cells, the cells were grown in LB agar plates containing X-Gal and ampicillin, overnight at 37ºC. To confirm that the plasmids contained inserts of the correct size, the vectors obtained from mini-preparations were digested with the desired restriction enzymes. Sequencing was performed for the gene. RT-PCR and Northern blotting analysis showed that BEST-5 mRNA is expressed, 3h after isolation and culture of primary hepatocytes (3h) BEST-5 mRNA was observed until 5h of culture and then there was no detectable band of BEST-5 at further time points. Comparison of expression of the level of mRNA of BEST-5, when data statistically were analyzed, showed a significant difference between the expression of BEST-5 mRNA expression at 3h with 0h, 24h, 35h and 48h of culture (P<0.001). According to the results the stress induced by hepatocytes isolation and culture leads to the expression of Best-5 time-dependently.