Transcriptional profiling of the human liver during the reperfusion phase of transplantation.

摘要

Liver transplantation continues to be the only remedy for end-stage liver disease. Moreover, the number of recipients far exceeds the number of donors and patients die on waiting lists. Unfortunately, not all grafts survive the process of transplantation and marginal livers are discarded, as they would not tolerate the stresses of ischemia-reperfusion and therefore would not survive the process of transplantation. If we are to resolve these problems, and decrease the chasm between the donor and recipient numbers, we need to characterize how a normal liver survives the process of transplantation.;The underlying hypothesis of my thesis is that the surviving liver invokes protective mechanisms to moderate the damage that could occur as a result of transplantation in part by regulating the level and type of expressed genes. Using microarray technology, we determined the identity of the mRNAs that revealed a degree of regulation, either up- or down-regulation during the reperfusion phase of transplantation. Furthermore, because ischemia precedes reperfusion, the process of reperfusion per se includes all of the stresses associated with ischemia, e.g. all reperfused livers were ischemia preconditioned. Thus, to conduct our analysis biopsy specimens were taken at three time-points during the peri-reperfusion phase of the operation. Our methodology not only permitted us to identify regulated genes, it also allowed us to control for recipient blood-borne contribution of messenger RNA. Because the last biopsy specimen was taken 1h post-reperfusion, our list was comprised of immediate early genes.;Of the other immediate-early genes that were on our list, we found an upregulated gene that coded for map kinase phosphatase-1 (MKP-1). Immunohistochemistry preformed on frozen human liver sections revealed expression of MKP-1 in hepatocytes. MKP-1 is a phosphatase is best known for JNK-1, p38MAPK and ERK1/2/5 dephosphorylation. Using transplantation relevant stresses in vitro, and HepG2 as a cellular model for hepatocytes, we characterized mkp-1 mRNA regulation. Furthermore, using the same protocol and MKP-1 shRNA expressed in HepG2, we found that a lack of MKP-1 protein expression increased apoptosis. The second gene, als2, we investigated was slightly down-regulated and coded for a protein called alsin. The latter is a RhoGEF, a guanine nucleotide exchange factor that activates Rac1, Rab5 and Ran. We proceeded to characterize alsin mRNA regulation in vitro using ischemia-reperfusion relevant stresses, as we did for MKP-1. Finally, we are seeking to determine if or how the Rho proteins (Rac1, Rab5 or Ran) are implicated during the "reperfusion" phase of the operation. All in all, our results indicate a hepatic coping mechanism invoked for the purpose of reducing the damage caused by the trauma of ischemia and reperfusion.;We have developed a protocol that allows us to characterize the normal liver's response to transplantation. Indeed, a liver that endures the process of transplantation must be able to limit the amount of damage caused by the various stresses related to cold ischemia and warm oxygenated reperfusion. Unfortunately, the process of transplantation is not easily amenable to the reduction much less the elimination of these stresses. Therefore, a means of investigating what happens to the liver during the process of transplantation was needed that would take into account the global effect of these variables on the graft's survival.