Study of pathological processes of meibomian gland dysfunction by in vitro culture airlifting conditions.

Meibomian gland dysfunction (MGD) is a group of disorders linked by functional abnormalities of the meibomian glands. Current studies on MGD pathogenesis focus on meibomian gland cells, providing information on a single cell's response to experimental manipulation, and do not maintain the architecture of an intact meibomian gland acinus and the acinar epithelial cells' secretion state in vivo. In this study, rat meibomian gland explants were cultured by a Transwell chamber-assisted method under an air-liquid interface (airlift) in vitro for 96 h. Analyses for tissue viability, histology, biomarker expression, and lipid accumulation were performed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and TUNEL assays, hematoxylin and eosin (H&E) staining, immunofluorescence, Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), transmission electron microscopy (TEM), and western blotting (WB). MTT, TUNEL, and H&E staining indicated better tissue viability and morphology than the submerged conditions used in previous studies. Levels of MGD biomarkers, including keratin 1 (KRT1) and 14 (KRT14) and peroxisome proliferator-activated receptor-gamma (PPAR-γ), along with oxidative stress markers, including reactive oxygen species, malondialdehyde, and 4-hydroxy-2-nonenal, gradually increased over culture time. The MGD pathophysiological changes and biomarker expression of meibomian gland explants cultured under airlift conditions were similar to those reported by previous studies, indicating that abnormal acinar cell differentiation and glandular epithelial cell hyperkeratosis may contribute to obstructive MGD occurrence.

In vitro study of the nephrotoxicity of total Dahuang (Radix Et Rhizoma Rhei Palmati) anthraquinones and emodin in monolayer human proximal tubular epithelial cells cultured in a transwell chamber.

OBJECTIVE: To examine changes in the morphology and physiological functions of human proximal tubular epithelial cells (HK-2 cells) caused by total Dahuang (Radix Et Rhizoma Rhei Palmati) anthraquinones (TDA) and emodin. METHODS: HK-2 cells were cultured on polycarbonate (PCF) membranes to form a complete monolayer of cells. A fluorescein isothiocyanate-dextran (FITC) permeability assay was conducted and secretion of γ-glutamyltranspeptidase (GGT), lactate dehydrogenase (LDH), N-acetyl-ß-D-glucosaminidase (NAG) and kidney injury molecule 1 (KIM-1) was examined. The reabsorption of glucose and the excretion of para-aminohippuric acid (PAH) by HK-2 cells were also examined. The morphology of HK-2 cells was observed using optical microscopy and scanning electron microscopy. The cytoskeleton of HK-2 cells was observed under a fluorescence microscope. RESULTS: Compared with the results for the dimethyl sulfoxide group, treatment of cells with TDA and emodin showed statistically significant differences in the FITC leakage rate, the apical / basolateral ratio of LDH and GGT, and the secretion of GGT, LDH, NAG and KIM-1. At 64 µg/mL, TDA markedly inhibited blood glucose reabsorption and remarkably suppressed PAH excretion by HK-2 cells. Both TDA and emodin caused various degrees of damage to the morphology and cytoskeleton of HK-2 cells with the degree of damage correlating positively with the dosage of the tested substances. CONCLUSION: Both TDA and emodin caused damage to human renal proximal tubular epithelial cells at certain dosages. At the same dosage, TDA caused more severe damage than emodin to the HK-2 cells.

Downregulation of LSD1 suppresses the proliferation, tumorigenicity and invasion of papillary thyroid carcinoma K1 cells.

The present study aimed to evaluate the effects of lysine-specific demethylase 1 (LSD1) downregulation, induced by small interfering RNA (siRNA) transfection, on the proliferation, colony formation, migration and invasion of the papillary thyroid carcinoma K1 cell line. The siRNA targeting LSD1 and scrambled non-targeting siRNA were each transfected into papillary thyroid carcinoma K1 cells. Downregulation of LSD1 mRNA and protein level was evaluated by reverse transcription-quantitative polymerase chain reaction, and immunocytochemical (ICC) analysis and western blotting, respectively. A Cell Counting kit-8 assay was applied to estimate the effect of LSD1-siRNA on cell growth. Migration and invasion abilities were estimated by Transwell chamber assay. A soft agar colony formation assay was performed to estimate the effect of LSD1-siRNA on tumorigenicity in vitro. ICC data showed that LSD1 protein was strongly expressed in the blank and control K1 cells compared with the LSD1-siRNA cells (F=15.192, P<0.01). Compared with the control cells, cells transfected with siRNA targeting LSD1 exhibited significant downregulation of LSD1 mRNA (t=6.845, P<0.01) and protein (F=53.764, P<0.01) levels. siRNA targeting LSD1 also downregulated cell proliferation following transfection for 24, 48 and 72 h (t=4.777, P<0.001; t=3.302, P=0.003; and t=3.017, P=0.006, respectively). Compared with the control group, the amount of cell invasion was gradually reduced in the LSD1-siRNA group (t=12.301, P<0.01). The number of migrating cells was significantly higher in the negative control group compared with the LSD1-siRNA group (t=7.911, P<0.01), and the ability of colony formation in the LSD1-siRNA cells was notably reduced in the soft agar formation assay (t=3.612, P=0.005). siRNA targeting LSD1 efficiently inhibits the proliferation, colony formation, migration and invasion of papillary thyroid carcinoma K1 cells.

siRNA targeting RBP2 inhibits expression, proliferation, tumorigenicity and invasion in thyroid carcinoma cells.

In order to estimate the effects of small interfering RNA (siRNA) targeting retinoblastoma binding protein 2 (RBP2) on the proliferation, expression, invasion, migration and tumorigenicity abilities of papillary thyroid carcinoma K1 cells, siRNA targeting RBP2 (RBP2-siRNA) and negative control siRNA were transfected into K1 cells. The mRNA levels of RBP2 in the transfected cells were estimated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and the protein levels of RBP2 in these cells were evaluated by western blot analysis and immunocytochemical (ICC) analyses. The growth, tumorigenicity, migration and invasion abilities of the transfected cells were measured by Cell Counting Kit-8 (CCK-8), soft agar colony formation and transwell chamber assay, respectively. The ICC results demonstrated that the protein expression levels of RBP2 were lower in the RBP2-siRNA-transfected cells than in the blank and control cells (analysis of variance, F=26.754, P<0.01). RBP2-siRNA downregulated RBP2 at the mRNA (t=8.869) and protein level (F=60.835) (P=0.000 vs. control cells). In addition, the transfection of RBP2-siRNA into K1 cells also suppressed cell proliferation at 24, 48 and 72 h post-transfection (t=7.650, P<0.01; t=2.606, P=0.016; and t=2.377, P=0.027, respectively). Compared with the control group, the number of invasive and migrated cells were significantly reduced in the RBP2-siRNA-transfected group (t=4.774 and t=6.366, respectively; P<0.01). Furthermore, the tumorigenic potential of the cells transfected with RBP2-siRNA was markedly reduced, as indicated by the soft agar formation assay (t=2.749, P=0.014 vs. control cells). In conclusion, the transfection of RBP2-siRNA into papillary thyroid carcinoma K1 cells suppressed the expression of RBP2 in these cells, and reduced their proliferation, invasion, migration and tumorigenic potential. Therefore, targeting RBP2 may be an efficient approach to control thyroid carcinoma.

In vitro model of the blood-brain barrier established by co-culture of primary cerebral microvascular endothelial and astrocyte cells.

Drugs for the treatment and prevention of nervous system diseases must permeate the blood-brain barrier to take effect. In vitro models of the blood-brain barrier are therefore important in the investigation of drug permeation mechanisms. However, to date, no unified method has been described for establishing a blood-brain barrier model. Here, we modified an in vitro model of the blood-brain barrier by seeding brain microvascular endothelial cells and astrocytes from newborn rats on a polyester Transwell cell culture membrane with 0.4-µm pores, and conducted transepithelial electrical resistance measurements, leakage tests and assays for specific blood-brain barrier enzymes. We show that the permeability of our model is as low as that of the blood-brain barrier in vivo. Our model will be a valuable tool in the study of the mechanisms of action of neuroprotective drugs.

Chemokine receptor 4 gene silencing blocks neuroblastoma metastasis in vitro.

This study investigated the effects of small interfering RNA (siRNA)-mediated silencing of chemokine receptor 4 (CXCR4) on the invasion capacity of human neuroblastoma cell line SH-SY5Y in vitro. Three siRNAs targeting CXCR4 were chemically synthesized and individually transfected into SH-SY5Y cells. Expression of CXCR4 mRNA and protein was significantly suppressed in transfected cells by all three sequence-specific siRNAs compared with control groups. Furthermore, the invasion capacity of SH-SY5Y cells was significantly decreased following transfection with CXCR4-specific siRNA compared with the control groups. These data demonstrate that down-regulation of CXCR4 can inhibit in vitro invasion of neuroblastoma.