Luan-Feng Pan

Fibrillar accumulation of A53T mutant α-synuclein (A53T-AS) in Lewy bodies is a symptom of Parkinsonism. Inhibitions of the overexpression and fibrillar aggregation of α-synuclein (AS) in vivo could be a promising strategy for treating Parkinson’s disease (PD). In this study, at concentrations lower than 1 mM, trehalose decreased the A53T-AS expression level in transduced PC12 cells. Although H₂O₂ and aluminum ions increased the expression level and neurotoxicity of A53T-AS in cells, proper trehalose concentrations inhibited the event. These studies adequately prove that trehalose at an appropriate dose would be potentially useful for PD treatment.

Cell affinity is one of the important issues required for developing tissue engineering materials. Although the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) has been attractive for its controllable mechanical properties recent years, its cell affinity is still necessary to be improved for the requirements. For this purpose, the regenerated silk fibroin (SF) was coated on the PHBHHx films and its porous scaffolds. The mechanical test showed that SF-modified PHBHHx (SF/PHBHHx) film has a maximum tensile strength of 11.5 ± 0.5 MPa and elongation at break of 175 ± 5%. ATR-FTIR spectroscopy demonstrated that SF firmly attached on the scaffold by the hydrogen bonding interaction between SF and PHBHHx even flushed for 21 days in the phosphate-buffer saline (PBS) solution (pH = 7.4). In order to characterize the cell affinity of the SF-modified material, endothelial-like cell line ECV304 cells were seeded on the SF/PHBHHx films and its porous scaffolds. The histochemical analyses of cells stained by the hematoxylin and eosin (HE) as well as cell nuclei stained by the 4′,6-diamindine-2′-phenylindole (DAPI) demonstrated that cell attached and reached nearly 100% confluence on the SF/PHBHHx films when cultured for 4 days, which was much faster than that on the pure PHBHHx film. Moreover, the assay of cell activity by the 3-(4, 5-dimethyl thiazol -2-yl)-2, 5-diphenyl terazolium bromide (MTT) showed quantitatively that the number of cells on the SF/PHBHHx porous scaffolds was significant more than that on the unmodified ones after 4, 8, and 14 days culture, respectively. Scanning electron microscopy (SEM) revealed the similar results. Therefore, the SF-modified PHBHHx material is maybe a potential material applicable in the cardiovascular tissue engineering.

A major shortcoming in tissue engineered blood vessels (TEBVs) is the lack of healthy and easily attainable smooth muscle cells (SMCs). Smooth muscle progenitor cells (SPCs), especially from peripheral blood, may offer an alternative cell source for tissue engineering involving a less invasive harvesting technique. SPCs were isolated from 5-ml fresh rat peripheral blood by density-gradient centrifugation and cultured for 3 weeks in endothelial growth medium-2-MV (EGM-2-MV) medium containing platelet-derived growth factor-BB (PDGF BB). Before seeded on the synthesized scaffold, SPC-derived smooth muscle outgrowth cell (SOC) phenotypes were assessed by immuno-fluorescent staining, Western blot analysis, and reverse transcription polymerase chain reaction (RT-PCR). The cells were seeded onto the silk fibroin-modified poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (SF-PHBHH _x ) scaffolds by 6×10⁴ cells/cm² and cultured under the static condition for 3 weeks. The growth and proliferation of the seeded cells on the scaffold were analyzed by 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay, scanning electron microscope (SEM), and 4,6-diamidino-2-phenylindole (DAPI) staining. SOCs displayed specific “hill and valley” morphology, expressed the specific markers of the SMC lineage: smooth muscle (SM) α-actin, calponin and smooth muscle myosin heavy chain (SM MHC) at protein and messenger ribonucleic acid (mRNA) levels. RT-PCR results demonstrate that SOCs also expressed smooth muscle protein 22α (SM22α), a contractile protein, and extracellular matrix components elastin and matrix Gla protein (MGP), as well as vascular endothelial growth factor (VEGF). After seeded on the SF-PHBHH _x scaffold, the cells showed excellent metabolic activity and proliferation. SPCs isolated from peripheral blood can be differentiated into the SMCs in vitro and have an impressive growth potential in the biodegradable synthesized scaffold. Thus, SPCs may be a promising cell source for constructing TEBVs.

To detect the expression of IgG receptors (FcgammaR) on cytokine-stimulated human umbilical vein endothelial cells (HUVECs). By using ELISA, immunocytochemical staining, immunofluorescent staining and RT-PCR, the expression and subtypes of FcgammaR were detected. Non-stimulated HUVECs expressed very low level of FcgammaRIIa. FcgammaRIIa mRNA was dramatically up-regulated upon 24 hour stimulation with tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma(IFN-gamma). ELISA results indicated that the expression of FcgammaRIIa increased 16 folds after stimulation with TNF-alpha and IFN-gamma for 3 days (P<0.01). Immunofluorescent staining showed that FcgammaRIIa was expressed on the surface of the stimulated HUVECs. TNF-alpha and IFN-gamma could increase FcgammaRIIa expression on HUVECs. The enhanced expression of FcgammaRIIa may mediate the deposition of immune complexes to blood vessels under vasculitic conditions.