[Show abstract][Hide abstract] ABSTRACT: Mechanical pressure plays an important role in many physiological and pathological processes. Mimicking the mechanical pressure present in vitro is necessary for related research, but usually requires expensive and complicated equipment. In this study we created a simple pressure culture system based on the transwell culture system. By cutting off the top rim of the transwell insert, the cells were compressed between the insert membrane and the well floor. The new pressure culture system was proven effective in that it induced cell morphological change, integrin β1 upregulation, actin polymerization and growth change in rat retinal ganglion cells, human nasopharyngeal carcinoma cells and mice embryonic fibroblasts. Though the pressure value is immeasurable and inhomogeneous, the easily available culture system still provides a choice for the laboratories that do not have access to the better, but much more expensive pressure culture equipment.
Biological research 01/2013; 46(1):47-52. · 1.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate whether dexamethasone has an effect on functional expression of p-glycoprotein in cultured human RPE and, if so, whether this occurs through interaction with glucocorticoid receptor (GR) and pregnane X receptor (PXR).
The human RPE D407 was treated with increasing concentrations of dexamethasone and/or RU486 for various time periods up to 24 hours. Treated cells were collected for cell viability, expressions of p-glycoprotein and PXR, and rhodamine 123 accumulation assays. GR expression plasmid and rifampicin were chosen to investigate the relationship of GR/PXR activation and p-glycoprotein expression.
Significant increases in p-glycoprotein, as indicated by mRNA and protein levels, as well as by functional activity, were induced within 12 hours of dexamethasone treatment, persisted as long as 24 hours, and were dose-dependent and attenuable with coculture of RU486. In parallel, a dose-dependent upregulation of PXR was notable at both mRNA and protein levels by 24 hours of dexamethasone treatment, and was partially reversible with RU486 coculture. Additionally, transfection of GR expression plasmid increased the transitional expressions of PXR and p-glycoprotein in untreated cells, and enhanced PXR transcriptional expression in dexamethasone-treated cells. Further, PXR silencing inhibited the dexamethasone-induced p-glycoprotein alterations; however, rifampicin had no apparent effects on the dexamethasone-induced p-glycoprotein alterations.
Our results suggest for the first time that expression and activation of p-glycoprotein involve GR and PXR in human RPE.
[Show abstract][Hide abstract] ABSTRACT: Inhibition of p-glycoprotein under hyperglycemic conditions has been reported in various barrier tissues including blood-brain barrier, intestine, and kidney, and has been linked to significant clinical complications. However, whether this is also true for the outer blood-retinal barrier constituted by retinal pigment epithelium, or has a role in pathogenesis of diabetic retinopathy is not yet clear. In this study, using cultured human retinal pigment epithelium cell line D407, we found that high glucose exposure induced a significant decrease in p-glycoprotein expression both at mRNA and at protein levels, accompanied by an attenuated p-glycoprotein activity determined by intracellular rhodamine 123 retention. In marked contrast, the expressions of both mRNA and protein levels of inducible nitrate oxide synthase (iNOS) increased, and were accompanied by increased extracellular nitrate/nitrite production by Griess reaction. In addition, mRNA levels of nuclear receptors revealed a decreased expression of pregnane X receptor after the exposure of high glucose. However, the subsequent alterations in production of nitrate/nitrite, functional expression of p-glycoprotein, and mRNA levels of pregnane X receptor were partially blocked when pretreated with S,S'-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea•2HBr (PBITU), a selective iNOS inhibitor. Moreover, the effects of PBITU were antagonized with the addition of L-arginine, a substrate for NO synthesis. Our in vitro results suggest for the first time that iNOS induction plays a novel role in decreased p-glycoprotein expression and transport function at the human outer blood-retinal barrier under hyperglycemic conditions and further support the concept of inhibiting iNOS pathway as a therapeutic strategy for diabetic retinopathy.
PLoS ONE 01/2012; 7(2):e31631. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Retinal progenitor cells (RPCs) are the most valuable seed cells in replacement therapy for neural retinal diseases. The competence of RPCs changes with retinal development. Gene expression plays a fundamental role in determining the competence. To improve the selection of the right-timing RPCs for replacement therapy, we compared the gene expression between embryonic day (E) 13.5 and E17.5 RPCs and further explored their gene expression and differentiation capacity in vitro.
Timed-pregnant E13.5 and E17.5 RPCs were freshly harvested and cultured in proliferation conditions for 4 days and then in differentiation conditions for 8 days. At different time points, the expression of key genes involved in retinal development was investigated by quantitative reverse transcription-PCR or immunofluorescence.
The expression of 14 key genes involved in retinal development was investigated in freshly harvested E13.5 and E17.5 RPCs. The freshly harvested E13.5 RPCs showed a high expression of retinal ganglion cell (RGC)-related genes, including Math5, Brn3b, Islet1, and Nfl, while the freshly harvested E17.5 RPCs displayed a high expression for Nrl, GFAP, and Thy1, the key genes involved in rod photoreceptor development, glial cell development, and synaptogenesis, respectively. During proliferation culture in vitro, the gene expression changed dramatically in both RPCs. After the 4 days of proliferation culture, the expression levels of most genes (11 of the 14 genes) in E13.5 RPCs came close to those in the freshly harvested E17.5 RPCs. Differentiation of RPCs in vitro was verified by the significant decrease in Nestin expression and BruU incorporation efficiency. After the 8 days of differentiation in vitro, the expression level of RGC-related genes (Math5, Brn3b, and Islet1) was still significantly higher in E13.5 RPCs than in E17.5 RPCs. In contrast, the expression level of Nrl and GFAP was significantly higher in E17.5 RPCs than in E13.5 RPCs. In morphology, the differentiated E13.5 RPCs displayed more robust process outgrowth than did the differentiated E17.5 RPCs. Immunofluorescence showed that, after the 8 days of differentiation, E13.5 RPCs contained more Brn3b- and Map2-positive cells, while E17.5 RPCs contained more GFAP-, GS-, and Rhodopsin-positive cells.
The results implied that E13.5 RPCs might be a better choice for RGC replacement therapy, while E17.5 RPCs might be better for photoreceptor replacement therapy. The duration of in vitro culture should be timed, since the expression of key genes kept changing in the proliferating RPCs.
[Show abstract][Hide abstract] ABSTRACT: Microinjection of adult stem cells (ASCs) into blastocysts provides a classic model for studying ASC plasticity. To explore the molecular mechanisms that govern the reprogramming of ASCs, we evaluated the experimental model through microinjection of human epidermal stem cells (hEpiSCs) into mouse blastocysts. Mouse blastocysts underwent regular embryogenesis after microinjection of allogeneic cells, confirmed by morphological observation and embryo cell counting. hEpiSCs survive and integrate into mouse embryos, by monitoring the migration of injected cells at 2, 4, 12, 16 and 24 h. In this xenogeneic system, hEpiSCs could be reprogrammed within 24 h, as evidenced by the silencing of CK15 and Integrin beta 1 gene expression, without activation of Oct4 and Nanog. Microinjection of hEpiSCs into mouse blastocysts provides an efficient model for studying the molecular mechanisms of their plasticity. Moreover, the possibility of inducing pluripotent stem cells without transgenes or viruses can be entertained.
Cell Biology International 10/2008; 32(12):1567-73. · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Primary open-angle glaucoma (POAG) is a leading cause of irreversible blindness worldwide, and its pathogenesis is still unknown. The purpose of this study was to determine molecular changes in membrane proteins in trabecular meshwork (TM) cells from POAG patients compared to those of age-matched normal controls.
Two-dimensional (2-D) gel electrophoresis profiles of membrane extracts from normal and glaucomatous TM cells were compared. The desired spots were identified after trypsin digestion and mass spectrometric analysis. Based on the results, a calcium-dependant membrane-binding protein, copine1, was further approached for a possible role in glaucomatous TM cells. The intracellular calcium concentration ([Ca(2+)]i) of TM cells was increased by incubating with calcium ionophore, A23187. Relative quantification real-time polymerase chain reaction (PCR) and western blot analysis measured copine1 expression and localization both in untreated and A23187-treated TM cells.
Real-time PCR and western blot analysis confirmed that copine1 mRNA and protein expression were upregulated in glaucomatous TM cells when compared to normal ones. The cell distribution studies further showed that copine1 existed both in the membrane and cytoplasm fractions of glaucomatous TM cells but existed exclusively in cytoplasm fractions of their normal counterparts. More importantly, an influx of Ca(2+) markedly promoted the translocation of copine1 from the cytoplasm to membranes in glaucomatous TM cells.
Copine1 is upregulated in plasma membranes of TM cells in individuals with POAG, which may be partly explained by its Ca(2+)-dependent translocation from the cytoplasm to the membranes. Investigation of the role and functions of copine1 in TM cells should offer new insight into the abnormal intracellular Ca(2+)-signaling pathway in glaucomatous TM and help to clarify the molecular mechanism of POAG.