Activation, isolation, identification and in vitro proliferation of oval cells from adult rat livers.

Oval cells, putative hepatic stem cells, could potentially provide a novel solution to the severe shortage of donor livers, because of their ability to proliferate and differentiate into functional hepatocytes. We have previously demonstrated that oval cells can be induced to differentiate into cells with morphologic, phenotypic, and functional characteristics of mature hepatocytes. In this study, we have established a new model combining ethionine treatment with partial hepatectomy to activate oval cells, then developed a procedure utilizing selective enzymatic digestion and density gradient centrifugation to isolate and purify such cells from heterogeneous liver cell population. We identified oval cells by their morphological characteristics and phenotypic properties, thereby providing definitive evidence of the presence of hepatic stem-like cells in adult rat livers. Viewed by transmission electron microscopy, they were small cells with ovoid nuclei, a high nucleus/cytoplasm ratio and few organelles, including mitochondria and endoplasmic reticulum. Flow cytometric assay showed that these cells highly expressed OV-6, cytokeratin-19 (CK-19) and albumin. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis displayed that the freshly isolated cells co-expressed albumin, cytokeratin-7 (CK-7) and CK-19 mRNA, indicating that they were essentially bipotential hepatic stem-like cells. Furthermore, we set up a culture system containing growth factors and a fibroblast feeder layer, to provide nourishment to these cells. Thus, we were able to culture them in vitro for more than 3 months, with the number of cells doubling 100 times. Gene expressions of albumin, CK-7 and CK-19 in the cells derived from the expanding colonies at day 95 were confirmed by RT-PCR analysis. These data suggested that the hepatic oval cells derived from adult rat livers possess a high potential to proliferate in vitro with a large increase in number, while maintaining the bipotential nature of hepatic stem cells.

Differentiation of putative hepatic stem cells derived from adult rats into mature hepatocytes in the presence of epidermal growth factor and hepatocyte growth factor.

Oval cells, putative hepatic stem cells, can differentiate into a wide range of cell types including hepatocytes, bile epithelial cells, pancreatic cells and intestinal epithelial cells. In this study, we used different growth factor combinations to induce oval cells to differentiate into mature hepatocytes. We isolated and purified oval cells utilizing selective enzymatic digestion and density gradient centrifugation. Oval cells were identified by their morphological characteristics and the strong expressions of OV-6, albumin, cytokeratin (CK)-19 and CK-7. Using a 2-step induction protocol, we demonstrated that oval cells first changed into small hepatocytes, then differentiated into mature hepatocytes. Small hepatocytes were distinguished from oval cells by their morphological features (e.g. round shape and nuclei) and the lack of CK-19 mRNA expression. Mature hepatocytes were identified by their ultrastructural traits and their expressions of albumin, CK-18, tyrosine aminotransferase (TAT), and alpha-1-antitrypsin (alpha-1-AT). Differentiated cells acquired the functional attributes of hepatocytes in that they secreted albumin and synthesized urea at a high level throughout differentiation. Oval cells can thus differentiate into cells with the morphological, phenotypic and functional characteristics of hepatocytes. This 2-step induction procedure could provide an abundant source of hepatocytes for cell transplantation and tissue engineering.

Adherence of human monocytes and NK cells to human TNF-alpha-stimulated porcine endothelial cells.

Discordant xenograft models undergoing delayed rejection response are characterized by xenograft infiltration with host monocytes and NK cells, associated with the release of large quantities of pro-inflammatory cytokines, such as TNF-alpha. In the present study, human monocytes (PBMo)/NK cells (PBNK) isolated from peripheral blood and cultured porcine aortic endothelial cells (PAEC) treated with recombinant human TNF-alpha (rhTNF-alpha) were used to investigate their adhesive interactions and mAbs against porcine E-selectin, human CD11a and CD49d were used to test their relative contributions to such intercellular adhesions. The PBMo exhibited significantly greater adherence to resting (unstimulated) PAEC than PBNK. The rhTNF-alpha upregulated E-selectin and vascular cell adhesion molecule-1 (VCAM-1) expression on PAEC and augmented the adhesiveness of PAEC for PBMo and PBNK in a time- and dose-dependent manner. In mAb blocking assays, anti-E-selectin, anti-CD11a and anti-CD49d mAbs did not inhibit PBMo adherence to rhTNF-alpha-stimulated PAEC when used singly, but resulted in a maximal inhibitory effect when used in combination. Regarding PBNK, anti-E-selectin mAb had no marked influence on PBNK adherence. The combined use of anti-CD11a and anti-CD49d mAbs produced additive reduction in the PBNK binding to rhTNF-alpha-stimulated PAEC, even to far below baseline (unstimulated) levels. Therefore, it is concluded that human TNF-alpha promotes the adhesiveness of PAEC for human monocytes and NK cells and that the mechanism underlying the increased adherence differs for PBMo and PBNK.

Synthesis, characterization, biodegradation, and drug delivery application of biodegradable lactic/glycolic acid polymers: I. Synthesis and characterization.

A series of lactic/glycolic acid polymers with various molar ratios of lactic to glycolic acid and various molecular weights were synthesized using the ring-opening polymerization method. The polymerization conditions for the lactic/glycolic acid polymer synthesis were as follows: 150 degrees C, 700 microm Hg, 3 h, 0.03 wt% of catalyst (stannous 2-ethyl-hexanoate) concentration. The molecular weight of these polymers was controlled by using a molecular weight controller, lauryl alcohol. The synthesized polymers have been characterized with respect to polymer composition, molecular weight, inherent viscosity, and glass transition temperature. The characterization experiments show a good correlation between the polymer compositions and the feed ratios of lactic to glycolic acid. The molecular weight of the lactic/glycolic polymers, ranging from 10,876 to 166,630 D and the intrinsic viscosity of the polymers, ranging from 0.16 to 0.86 dl g(-1), are controlled by the amount of molecular weight controller used. The effect of the amount of the molecular weight controller on the polymer molecular weight and the polymer inherent viscosity was studied. Results indicate that the molecular weight and inherent viscosity of the polymers have a log-log linear relationship with the amount of molecular weight controller used. The lactic/glycolic acid polymers are amorphous, glassy, and transparent. The glass transition temperature of the polymers range from 21.95 to 51.29 degrees C, depending on the polymer molecular weight and the composition.

[Fluorescent mRNA differential display technique].

AIM: To apply fluorescent mRNA differential display technique. METHODS: Total RNA samples were extracted from human monocyte line U937 treated/untreated with IFN and LPS, and were used as templates in differential display PCR. The anchored primers used were labeled with the fluorescent tag. After running on 5.6% denaturing PAGE gel, differentially expressed bands were excised and recovered, and finally reamplified. RESULTS: Three tested samples all showed amplified bands differed from 300 bp to 2.0 kb, the bands were bright and clear, the background was low. Both yes/no changes and upregulated/downregulated happenings were shown simultaneously. The reamplification bands were sharp and pure. CONCLUSION: We have successfully practiced fluorescent differential display technique in our lab. It is a fast, safe and cost-effective method used to sereen unknown expressed genes.

[Properties of the GM-CSF-induced outward K+ current in murine peritoneal exudate macrophages].

Whole-cell patch-clamp technique was used to study the changes of ionic currents in murine peritoneal exudate macrophages (PEMs) prestimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF, 16 ng/ml) for periods from 0.5 up to 6 d. The GM-CSF-treated PEMs developed a GM-CSF-induced transient inactivating outward K+ current (IA). IA showed steady-state inactivation over the physiological voltage range and possessed frequency dependence of inactivation when depolarizing pulses were applied at a frequency of 0.5 Hz. IA was selectively inhibited by extracellular 4-AP (3 mmol/L). When [Ca2+]i was increased (from pCa 8 to 6), the amplitudes of IA were depressed significantly. When the PEMs were exposed to cycloheximide (0.3 microgram/ml), a protein synthesis inhibitor, for 12 h, IA expression was completely suppressed. It was notable that the changes of the current expression, activation behavior and kinetic properties occurred during GM-CSF treatment. When PEMs were pretreated for a 2-d period, the frequency of IA expression reached a peak value (55% in a total of 27 cells), PEMs exhibited the highest density of the corresponding channel proteins, half-maximal activation of IA was most easily achieved with a value of -27.55 mV, and the time course of activation and inactivation during depolarization proceeded rapidly. However, along with continuous incubation with GM-CSF, the number of PEMs expressing IA decreased, the channel proteins were down regulated constantly, the activation curve for IA shifted to positive potentials, and the activation time and inactivation time of IA slowed down. These results indicated that GM-CSF could induce a transient inactivating outward K+ current in PEMs, which may have a close relation to the state of functional activation of macrophages primed with GM-CSF.

Role of T-lymphocyte subsets in recovery from herpes simplex virus infection.

Our investigations probed the nature of different T-lymphocyte subsets effecting clearance of herpes simplex virus after infection of the pinna. Cell populations from animals recently infected subcutaneously or intraperitoneally (acute population) or from animals infected 6 weeks previously (primed population) or the latter cells reimmunized in vitro with virus (memory population) were studied. Viral clearance was a function of the Lyt 1+2- subset in the acute population, but with the memory population both Lyt 1+ and Lyt 2+ cells affected clearance. In primed populations, viral clearance was effected only by the Lyt 2+ subset. The ability of the various cell populations to adoptively transfer delayed-type hypersensitivity was also studied. Only acute population cells from animals infected subcutaneously and memory population cells transferred delayed-type hypersensitivity. In both cases, the cell subtype was Lyt 1+2-. Our results demonstrated that the delayed-type hypersensitivity response does not always correlate with immunity to herpes simplex virus. Multiple subsets of T cells participate in viral clearance, and their respective importances vary according to the stage of the virus-host interaction.