Therapeutic potentials of oligodendrocyte precursors in the animal model of multiple sclerosis

published_or_final_versionAnatomyDoctoralDoctor of Philosoph

Identification of two immortalized cell lines, ECV304 and bEnd3, for in vitro permeability studies of blood-brain barrier

To identify suitable cell lines for a mimetic system of in vivo blood-brain barrier (BBB) for drug permeability assessment, we characterized two immortalized cell lines, ECV304 and bEnd3 in the respect of the tightness, tight junction proteins, P-glycoprotein (P-gp) function and discriminative brain penetration. The ECV304 monoculture achieved higher transendothelial electrical resistance (TEER) and lower permeability to Lucifer yellow than bEnd3. However, co-culture with rat glioma C6 cells impaired the integrity of ECV304 and bEnd3 cell layers perhaps due to the heterogeneity among C6 cells in inducing BBB characteristics. The immunostaining of ZO-1 delivered distinct bands along cell borders on both cell lines while those of occludin and claudin-5 were diffused and weak. P-gp functionality was only proved in bEnd3 by Rhodamine 123 (R123) uptake assay. A permeability test of reference compounds displayed a similar rank order (digoxin \u3c R123 \u3c quinidine, verapamil \u3c propranolol) in ECV304 and bEnd3 cells. In comparison with bEnd3, ECV304 developed tighter barrier for the passage of reference compounds and higher discrimination between transcellular and paracellular transport. However, the monoculture models of ECV304 and bEnd3 fail to achieve the sufficient tightness of in vitro BBB permeability models with high TEER and evident immunostaining of tight junction proteins. Further strategies to enhance the paracellular tightness of both cell lines to mimic in vivo BBB tight barrier deserve to be conducted

Resveratrol Inhibits the Invasion of Glioblastoma-Initiating Cells via Down-Regulation of the PI3K/Akt/NF-κB Signaling Pathway

Invasion and metastasis of glioblastoma-initiating cells (GICs) are thought to be responsible for the progression and recurrence of glioblastoma multiforme (GBM). A safe drug that can be applied during the rest period of temozolomide (TMZ) maintenance cycles would greatly improve the prognosis of GBM patients by inhibiting GIC invasion. Resveratrol (RES) is a natural compound that exhibits anti-invasion properties in multiple tumor cell lines. The current study aimed to evaluate whether RES can inhibit GIC invasion in vitro and in vivo. GICs were identified using CD133 and Nestin immunofluorescence staining and tumorigenesis in non-obese diabetic severe combined immunodeficient (NOD/SCID) mice. Invasive behaviors, including the adhesion, invasion and migration of GICs, were determined by tumor invasive assays in vitro and in vivo. The activity of matrix metalloproteinases (MMPs) was measured by the gelatin zymography assay. Western blotting analysis and immunofluorescence staining were used to determine the expression of signaling effectors in GICs. We demonstrated that RES suppressed the adhesion, invasion and migration of GICs in vitro and in vivo. Moreover, we proved that RES inhibited the invasion of GICs via the inhibition of PI3K/Akt/NF-κB signal transduction and the subsequent suppression of MMP-2 expression

Immunofluorescent staining of tight junction proteins occludin, claudin-5 and ZO-1 in ECV304 and bEnd3 cells.

<p>The immunofluorescence of ZO-1 gave distinct strands on cell membrane while the staining of occludin and claudin-5 were diffused and weak in both cell lines. The confocal images were acquired at 20 × magnification.</p

Permeability coefficient values of reference compounds measured in ECV304 and bEnd3 monoculture models.

<p>Permeability coefficient values of reference compounds measured in ECV304 and bEnd3 monoculture models.</p

R123 uptake in ECV304 and bEnd3 cells in the absence or presence of P-gp inhibitor verapamil.

<p>The P-gp inhibitor verapamil delivered no significant effects on R123 uptake in ECV304 cells but significantly increased R123 uptake in bEnd3 cells in comparison with that in the absence of the inhibitor. Data represent means ± SD (n = 3). ** <i>p</i> < 0.01.</p