[Improvement and application of a fast labeling system for bulk internalized vesicles].

To study trafficking of bulk internalized vesicles such as macropinosome and lysosome in live cells, an efficient and convenient assay was established according to the axon turning assay. By injecting indicator or fluorescent dyes through a micropipette with air pressure into cell cultures to create a stable gradient around the micropipette tip, vesicles were indicated and labeled. With live cell imaging, the whole process was recorded. Without wash-out of fluorescent dyes and transferring, this assay is an effective, fast labeling system for bulk internalized vesicles, and can also be combined with imaging system.

[In vivo imaging of blood flow using two-photon laser scanning fluorescent microscopy].

OBJECTIVE: To observe the three-dimensional distribution of vessels, and to establish a new method for measurement of blood flow velocity in mice cerebral cortex using two-photon laser scanning microscopy and fluorescence probe labeling technique. METHODS: The mouse was made cranial window surgery and injected Texas-Red through tail vein after anesthetized. The three-dimensional imaging of vessel was obtained through z-stack scanning, and blood flow velocity was quantified through line scanning. RESULTS: We could detect vascular distribution for more than 500 µm depth using two-photon microscopy. The velocity of blood flow was (0.59 ± 0.12) mm/s in capillary. CONCLUSION: The method for observing the brain blood flow by two-photon microscopy was established, which could achieve quantification of single vascular blood flow velocity and provide experimental evidence for basic research and medical applications.

Inhibitory effects of saponins from Anemarrhena asphodeloides Bunge on the growth of vascular smooth muscle cells.

OBJECTIVE: To investigate the effects of saponins from Anemarrhena asphodeloides Bunge (SAaB) (Botanical Name: Anemarrhena Asphodeloidis Rhizoma) on the growth of vascular smooth muscle cells (VSMCs). METHODS: Cell proliferation was measured by a newly developed cell proliferation reagent, WST-1. Cell apoptosis was assayed by flow cytometry through detecting annexin V. Nitric oxide production was evaluated using confocal laser scanning microscopy with diaminofluorescein diacetate (DAF-2, DA). Cell aldose reductase (AR) activity, as well as the effect of Epalrestat and interleukin-1beta were also explored. RESULTS: WST assay showed that cell proliferation induced by serum was significantly inhibited by SAaB (P<0.01). Flow cytometry analysis revealed that SAaB could enhance apoptotic rate of VSMCs (P<0.01). Nitric oxide production was significantly enhanced after administration of SAaB and interleukin-1beta. Moreover, AR activity of VSMCs was also remarkably inhibited by both SAaB and Epalrestat (P<0.01). CONCLUSION: SAaB can inhibit proliferation and enhance apoptosis of VSMCs. It may protect vascular cells by inhibiting VSMC proliferation and augmenting apoptotic rate of VSMCs via NO-dependent pathway.