[Cloning, sequencing and evaluation of expression of SDF-1 alpha in rat hepatocytes]

High constitutive levels of SDF-1 alpha have been observed in the non-inflamed biliary epithelium of the liver. SDF-1 alpha is also produced by ductal plate cells [biliary epithelial cells progenitors] and it is involved in maturation and homing of B cells in fetal liver. Giving the above brief introductory remarks, this chemokine was chosen to analysis in isolated and cultured hepatocytes. We employed gene cloning methods to clone and northern analysis to show the expression of the gene in hepatocytes. In the first stage of this work, we have sequenced cloned fragment of SDF-1 in cultured rat hepatocytes and compared with database to prove that the studied and cloned gene is SDF-1. we showed that the fragment, which we have used as probe for our northern blotting analysis is SDF-1 by sequencing methods. Following using specific produced probe for SDF-1, in this study we showed that SDF-1 is expressed by hepatocytes after isolation and early culture of hepatocytes. Isolation of hepatocytes by enzymatic methods causes liver injury and therefore hepatocytes may stimulate to response to the injury. The expression of this chemokine may involve in stress response of hepatocytes to isolation

[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[R]2.1 plasmid vector. Following transformation into TOPO10 oneshot [R]cells, the cells were grown in LB agar plates containing X-Gal and ampicillin, overnight at 37[oC]. 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