Effect of AT-1 alpha receptor gene silencing on nuclear factor-kappaB activity in hepatic Kupffer cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of angiotensin II type-1 (AT-1) alpha receptor gene silencing on nuclear factor-kappaB (NF-kappaB) activity in hepatic Kupffer cells.</p><p><b>METHODS</b>The expression of AT-1 alpha receptors in primary isolated cultured hepatic Kupffer cells was detected by immunohistochemistry. pSilencer/AT-1 alpha receptor siRNA plasmids were transfected into Kupffer cells, which were subsequently exposed to 10(-6) mol/L angiotensin II (Ang II) for 60 min. The changes in the DNA binding activity of NF-kappaB in the cells was assessed using electrophoretic gel mobility shift assay (EMSA).</p><p><b>RESULTS</b>AT-1 alpha receptor expression was detected in Kupffer cells. NF-kappaB DNA binding activity was markedly increased in Kupffer cells after Ang II stimulation, and obviously inhibited by transfectiom with pSilencer/AT-1 alpha receptor siRNA plasmid.</p><p><b>CONCLUSION</b>Ang II stimulation of Kupffer cell results in increased activation of NF-kappaB via AT-1 alpha receptor.</p>

Effect of angiotensin II type 1 receptor and angiotensin-converting enzyme gene silencing on nuclear factor-kappaB activity in hepatic stellate cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of angiotensin II (AngII) type 1 (AT-1) receptor and angiotensin-converting enzyme (ACE) gene silencing on nuclear factor-kappaB (NF-kappaB) activity in hepatic stellate cells (HSCs).</p><p><b>METHODS</b>pSilencer/AT-1 alpha receptor siRNA and pSilencer/ACE siRNA plasmids were transfected into cultured HSC-T6 cells, which were subsequently stimulated by 10(-6) mol/L AngII or ACE inhibitor (ACEI). The DNA binding activity of NF-kappaB in the transfected cells was analyzed using electrophoretic gel mobility shift assay (EMSA).</p><p><b>RESULTS</b>s Gel shift studies showed that stimulation of the HSCs by AngII markedly increased the DNA-binding activity of NF-kappaB, which was inhibited by the transfection with pSilencer/ AT-1 alpha receptor siRNA plasmid or pSilencer/ACE siRNA plasmid.</p><p><b>CONCLUSION</b>AT-1 alpha receptor and ACE gene silencing result in inhibition of NF-kappaB activity in HSCs in vitro.</p>

Intrasplenic heterotransplantation of in vitro cultured human fetal hepatic stem cells for treatment of acute liver injury in mice with severe combined immunodeficiency / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the in vivo colonization, migration, and differentiation of in vitro cultured human fetal hepatic stem cells (HSCs) following intrasplenic transplantation for treatment of acute liver injury in mice with severe combined immunodeficiency (SCID).</p><p><b>METHODS</b>Human fetal HSCs were isolated from the normal fetal liver (16-24 weeks) and purified, and the morphology of HSCs was observed under optical and transmission electron microscopes. The expressions of stem cell markers were examined in these HSCs by means of immunocytochemistry and flow cytometry. The passaged human fetal HSC suspension (0.2 ml) were injected into the spleen of SCID mice with acute liver injury induced by two-third partial hepatectomy, and 15, 30, 60, and 90 days after cell transplantation, immunohistochemistry was performed to examine the location and expressions of human hepatocytes, alpha1-AT and AFP antigen in the spleen and liver of the recipient SCID mice. PAS staining was used to examine the expression of glycogen and RT-PCR employed for detection of the expressions of AFP and albumin mRNA in the spleen of the mice on the scheduled time points.</p><p><b>RESULTS</b>Under optical microscope and transmission electron microscope, most of the HSCs were small, about 1/6 to 1/3 of the size of the hepatocyte, with relatively large nucleus-cytoplasm ratio and only small quantities of endocytoplasmic reticulum, chondriosome, and ribosome. Immunohistochemistry and flow cytometry identified positive expressions of AFP, Thy-1, C-kit, CD34 and CK19 in the HSCs, and after cell transplantation, positive expressions of human hepatocyte, alpha1-AT, and AFP antigen occurred in the liver and spleen of the recipient SCID mice. PAS staining confirmed the presence of glycogenosome in the spleen of the mice following cell transplantation. RT-PCR on days 30, 60, and 90 showed positive expressions of human AFP and albumin mRNA in the spleen of the mice.</p><p><b>CONCLUSION</b>Human fetal HSCs can survive and settle in the spleen and liver, and migrate to the damaged liver of the recipient mice after intrasplenic transplantation, with the capacity of proliferation and differentiation into hepatocytes in the recipient target organs.</p>