| The liver is one of vital organs of the human body, and its powerful regenerative capacity has become a hotpot for scholars. Generally, the mechanism of liver regeneration (LR) is studied by using regenerating liver tissues after 2/3 hepatectomy operation, which was established by Higgins and Anderson. However, the liver is composed of a variety of hepatic cells, and the direct or indirect interactions between hepatic parenchymal cells and nonparenchymal cells constitute the dynamic recovery process of LR. There is significant variation among types of liver cells regard to the physiological and biochemical events occurred during LR, so it will make results more clear and simplified if a variety of cell types are isolated for study. Pit cells, the liver-specific natural killer cells, not only directly kill tumor cells and virus-infected liver cells, but also participate in regulating the extent of LR. Although the role of pit cells in hepatic reconstitution has been being investigated, there are very few studies comprehensively addressing its detailed relevance with LR at transcriptional level at present.In this study, rat partial hepatectomy (PH) model was made as described by Higgins et al. Then conventional two-step method was applied for scattering liver cell suspension, and hepatic pit cells were isolated and purified by density gradient centrifugation with 60% percol1 and immunomagnetic beads. Immunocytochemistry method was used to quantify and localize CD8 and CD56 in isolated hepatic cells and purified pit cells. The expressions of CD8 and CD56 were quantified using real-time RT-PCR and western blotting. The results showed that markers-positive cell rates were statistically over 95% for isolated pit cells, and mRNA levels of CD8 and CD56 and the corresponding proteins were stable in purified pit cells during LR.This study then started with highly purified and active pit cells, measured the temporal expression profiles of pit cells at 10 time points post PH using Rat Genome 230 2.0 Array composed of 25020 distinct rat liver cDNA clones, evaluated the reliability of chip results by real-time PCR, and analyzed their relevance with rat LR by bioinformatics and systems biology methods. It was found that 612 known and 358 unknown genes, a total of 970 genes, were identified to be associated with LR. The 612 known genes were roughly clustered into up-regulation and down-regulation patterns, and classified into 8 clusters by k-means according to their minor expression differences. GO analysis indicated that up- and down-regulated genes primarily participated in at least 23 biological activities. Together with gene function enrichment analysis, it was found that secretion of active substances, including cytokines and growth factors, and their signal pathways in pit cells were activated at the initiation phase of LR; pit cell proliferation occurred between 24-72h after PH; immune/inflammatory response was enhanced at the termination phase of LR; pit cells apparently did not undertake the functions of wound healing and basic material metabolism.Of thousands of LR-related genes in pit cells, osteopontin (OPN) is a kind of active cytokines secreted by activated lymphocytes and macrophages. Its mRNA level increased at 2-72h, and peaked 31-fold higher than the control. By using Pathway Studio 7.0, a relational gene network focusing on opn was constructed from the LR-related genes in pit cells, and a disease network was identified to be highly related to OPN. It was found that more than 30 genes were identified as OPN’s regulators or targets, and many inflammatory diseases are among the pathological processes associated with this gene network. Together, OPN was found to be closely related to the functions of pit cells in LR.To study the roles of OPN in LR, we cloned the ORF (open read fragment) of opn, and established its expression vector pEGFP-N1-opn and two interference vectors pGenesil-1.0-opn(313) and pGenesil-1.0-opn(887). Based on above these vectors, three test vectors pGenesil-1.0-HK-opn、pGenesil-1.0-opn(313)-opn and pGenesil-1.0-opn(887)-opn were subsequently constructed. The constructed test vectors were administrated using a hydrodynamics-based procedure, and it was found that opn(313) could significantly decrease more mRNA level of opn than opn(887) by observing decreased rate of green fluorescent protein (GFP)-positive cells compared with the control. The peak expression time points of exogenous opn and interference fragment were obtained by administration of expression and interference vectors into rat liver tissues via hydrodynamics. Combined with expression peak of endogenous opn at 6-12h after PH, the time points of 30h before PH and 0h after PH were chose to be the best transgenic time for administrating of expression and interference vectors respectively according to the principle of making the expression of endogenous opn and exogenous vectors overlying. By observing and analyzing rat death rate, expression kinetics, morphology structure of liver tissue, liver coefficient, and liver regenerating rate, inflammation response and liver injury were found to be dramatically aggravated after transfection of pEGFP-N1-opn, while opn(313) reduced the liver coefficient and liver regenerating rate, and decreased expression levels of pcna, myc, bcl2, icam1, and mmp2. In conclusion, opn, as an important LR-related gene, might promote liver regeneration through facilitating cell proliferation, inhibiting cell apoptosis, and regulating extracellular matrix synthesis. |
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