RESUMO
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.
Assuntos
Corantes Fluorescentes , Lisossomos , Vesículas Transportadoras , AnimaisRESUMO
A large body of evidence indicates that particulate matter (PM)2.5 is associated with various negative effects on human health. However, the impact and molecular mechanism of PM2.5 on the skin have not been elucidated. Therefore, the present study aimed to investigate the effects of two types of PM2.5 [water-soluble extracts (W-PM2.5) and non-water-soluble extracts (NW-PM2.5)] on cell proliferation, cell cycle progression, lipid synthesis, and inflammatory cytokine production of human SZ95 sebocytes. The results demonstrated that NW-PM2.5 and W-PM2.5 exposure dose-dependently inhibited SZ95 sebocyte proliferation by inducing G1 cell arrest. Furthermore, NW-PM2.5 and W-PM2.5 significantly reduced sebaceous lipid synthesis and markedly promoted the production of inflammatory cytokines, including interleukin-1α (IL-1α), IL-6 and IL-8 in SZ95 sebocytes. Additionally, the expression of aryl hydrocarbon (Ah) receptor (AhR), AhR nuclear translocator protein (ARNT), as well as cytochrome P450 1A1 were significantly increased following PM2.5 exposure. Thus, these findings indicate that PM2.5 exerts inhibitory effects on cell proliferation and lipid synthesis, and stimulatory effects on inflammatory cytokine production and AhR signaling activation in human SZ95 sebocytes.
Assuntos
Células Epiteliais/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Interleucina-1alfa/genética , Interleucina-6/genética , Interleucina-8/genética , Material Particulado/farmacologia , Translocador Nuclear Receptor Aril Hidrocarboneto/agonistas , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Misturas Complexas/farmacologia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Regulação da Expressão Gênica , Humanos , Interleucina-1alfa/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Glândulas Sebáceas/citologia , Glândulas Sebáceas/efeitos dos fármacos , Glândulas Sebáceas/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Anemarrhena/química , Antineoplásicos Fitogênicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Saponinas/farmacologia , Animais , Anexina A5/metabolismo , Células Cultivadas , Citometria de Fluxo , Fluoresceína/química , Microscopia Confocal , Músculo Liso Vascular/crescimento & desenvolvimento , Óxido Nítrico/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
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.