Glucose transporter 4 (GLUT4) is responsible for insulin-stimulated glucose transporting into the insulin-sensitive fat and muscle cells. The dynamics of GLUT4 storage vesicles (GSVs) remains to be explored and it is ...Glucose transporter 4 (GLUT4) is responsible for insulin-stimulated glucose transporting into the insulin-sensitive fat and muscle cells. The dynamics of GLUT4 storage vesicles (GSVs) remains to be explored and it is unclear how GSVs are arranged based on their mobility. We examined this issue in 3T3-L1 cells via investigating the three-dimensional mobility of single GSV labeled with EGFP-fused GLUT4. A thin layer of cytosol right adjacent to the plasma membrane was illuminated and successively imaged at 5 Hz under a total internal reflection fluorescence microscope with a penetration depth of 136 nm. Employing single particle tracking, the three-dimensional subpixel displacement of single GSV was tracked at a spatial precision of 22 nm. Both the mean square displacement and the diffusion coefficient were calculated for each vesicle. Tracking results revealed that vesicles moved as if restricted within a cage that has a mean radius of 160 nm, suggesting the presence of some intracellular tethering matrix. By constructing the histogram of the diffusion coefficients of GSVs, we observed a smooth distribution instead of the existence of distinct groups. The result indicates that GSVs are dynamically retained in a continuous and wide range of mobility rather than into separate classes.展开更多
AIM： To investigate a dual labeling technique, which would enable real-time monitoring of transplanted em- bryonic stem cell （ESC） kinetics, as well as long-term tracking. METHODS： Liver damage was induced in C57/...AIM： To investigate a dual labeling technique, which would enable real-time monitoring of transplanted em- bryonic stem cell （ESC） kinetics, as well as long-term tracking. METHODS： Liver damage was induced in C57/BL6 male mice （n = 40） by acetaminophen （APAP） 300 mg/kg administered intraperitoneally. Green fluores- cence protein （GFP） positive C57/BL6 mouse ESCs were stained with the near-infrared fluorescent lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbo- cyanine iodide （DiR） immediately before transplantationinto the spleen. Each of the animals in the cell therapy group （n = 20） received 5 x 106 ESCs 4 h following treatment with APAP. The control group （n = 20） re- ceived the vehicle only. The distribution and dynamics of the cells were monitored in real-time with the IVIS lumina-2 at 30 rain post transplantation, then at 3, 12, 24, 48 and 72 h, and after one and 2 wk. Immunohisto- chemical examination of liver tissue was used to identify expression of GFP and albumin. Plasma alanine amino- transferase （ALT） was measured as an indication of liver damage.RESULTS： DiR-stained ESCs were easily tracked with the IVIS using the indocyanine green filter due to its high background passband with minimal background autofluorescence. The transplanted cells were confined inside the spleen at 30 min post-transplantation, gradu- ally moved into the splenic vein, and were detectable in parts of the liver at the 3 h time-point. Within 24 h of transplantation, homing of almost 90% of cells was confirmed in the liver. On day three, however, the DiR signal started to fade out, and ex vivo IVIS imaging of different organs allowed signal detection at time-points when the signal could not be detected by in vivo imag- ing, and confirmed that the highest photon emission was in the liver （P 〈 0.0001）. At 2 wk, the DiRsignal was no longer detectable in vivo; however, immuno- histochemistry analysis of constitutively-expressed GFP was used to provide an insight into the distribution of the cells. GFP ＋ve cells were detected in tissue sections resembling hepatocytes and were dispersed throughout the hepatic parenchyma, with the presence of a larger number of GFP ＋ve cells incorporated within the sinu- soidal endothelial lining. Very faint albumin expression was detected in the transplanted GFP ＋re cells at 72 h; however at 2 wk, few cells that were positive for GFP were also strongly positive for albumin. There was a significant improvement in serum levels of ALT, albumin and bilirubin in both groups at 2 wk when compared with the 72 h time-point. In the cell therapy group, serum ALT was significantly （P = 0.016） lower and al- bumin （P = 0.009） was significantly higher when com- pared with the control group at the 2 wk time-point;however there was no difference in mortality between the two groups. CONCLUSION： Dual labeling is an easy to use and cheap method for longitudinal monitoring of distribu- tion, survival and engraftment of transplanted cells, and could be used for cell therapy models.展开更多
Identification of fluorescent biomarkers with peptide ligand-directed receptors for diagnosis or theranostic of pancreatic ductal adenocarcinoma (PDAC) is still challenging. As potential prognostic/predictive bioimagi...Identification of fluorescent biomarkers with peptide ligand-directed receptors for diagnosis or theranostic of pancreatic ductal adenocarcinoma (PDAC) is still challenging. As potential prognostic/predictive bioimaging targets, both aminopeptidase N(APN, known as CD13) and Caveolin-1 are found as upregulation on the cell membrane surface of PDAC, in which APN is the principal receptor of the cyclic peptide cNGR (Asn-Gly-Arg, NGR) and Caveolin-1 can synergistically mediate endocytosis in this receptor-targeted process. Herein, we conjugate cNGR to dicyanomethylene-4H-pyran (DCM) chromophore to develop a synergistic-targeted near-infrared (NIR) fluorescent probe DCM-cNGR with strongly intrinsic NIR fluorescence, stable optical performance, low cytotoxicity, and rapid accumulation in PANC-1 cells with the synergistic overexpressed APN receptor-targeted and Caveolin-1-mediated endocytosis. As demonstrated, DCM-cNGR can realize noninvasive NIR imaging for targeting PANC-1 tumor in vivo after intravenous injection into PANC-1 xenograft tumor of nude mice, making a great promise to improve the precision diagnosis and therapy of pancreatic cancer with real time tracing and bioimaging of PDAC in vitro and in vivo.展开更多
文摘Glucose transporter 4 (GLUT4) is responsible for insulin-stimulated glucose transporting into the insulin-sensitive fat and muscle cells. The dynamics of GLUT4 storage vesicles (GSVs) remains to be explored and it is unclear how GSVs are arranged based on their mobility. We examined this issue in 3T3-L1 cells via investigating the three-dimensional mobility of single GSV labeled with EGFP-fused GLUT4. A thin layer of cytosol right adjacent to the plasma membrane was illuminated and successively imaged at 5 Hz under a total internal reflection fluorescence microscope with a penetration depth of 136 nm. Employing single particle tracking, the three-dimensional subpixel displacement of single GSV was tracked at a spatial precision of 22 nm. Both the mean square displacement and the diffusion coefficient were calculated for each vesicle. Tracking results revealed that vesicles moved as if restricted within a cage that has a mean radius of 160 nm, suggesting the presence of some intracellular tethering matrix. By constructing the histogram of the diffusion coefficients of GSVs, we observed a smooth distribution instead of the existence of distinct groups. The result indicates that GSVs are dynamically retained in a continuous and wide range of mobility rather than into separate classes.
基金Supported by Citadel Capital Scholarship Foundation,EgyptDr. Leslie Borthwick/Ms. Anita Holme,Charitable Research Fund East and North Herts NHS TrusHertfordshire,United Kingdom
文摘AIM： To investigate a dual labeling technique, which would enable real-time monitoring of transplanted em- bryonic stem cell （ESC） kinetics, as well as long-term tracking. METHODS： Liver damage was induced in C57/BL6 male mice （n = 40） by acetaminophen （APAP） 300 mg/kg administered intraperitoneally. Green fluores- cence protein （GFP） positive C57/BL6 mouse ESCs were stained with the near-infrared fluorescent lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbo- cyanine iodide （DiR） immediately before transplantationinto the spleen. Each of the animals in the cell therapy group （n = 20） received 5 x 106 ESCs 4 h following treatment with APAP. The control group （n = 20） re- ceived the vehicle only. The distribution and dynamics of the cells were monitored in real-time with the IVIS lumina-2 at 30 rain post transplantation, then at 3, 12, 24, 48 and 72 h, and after one and 2 wk. Immunohisto- chemical examination of liver tissue was used to identify expression of GFP and albumin. Plasma alanine amino- transferase （ALT） was measured as an indication of liver damage.RESULTS： DiR-stained ESCs were easily tracked with the IVIS using the indocyanine green filter due to its high background passband with minimal background autofluorescence. The transplanted cells were confined inside the spleen at 30 min post-transplantation, gradu- ally moved into the splenic vein, and were detectable in parts of the liver at the 3 h time-point. Within 24 h of transplantation, homing of almost 90% of cells was confirmed in the liver. On day three, however, the DiR signal started to fade out, and ex vivo IVIS imaging of different organs allowed signal detection at time-points when the signal could not be detected by in vivo imag- ing, and confirmed that the highest photon emission was in the liver （P 〈 0.0001）. At 2 wk, the DiRsignal was no longer detectable in vivo; however, immuno- histochemistry analysis of constitutively-expressed GFP was used to provide an insight into the distribution of the cells. GFP ＋ve cells were detected in tissue sections resembling hepatocytes and were dispersed throughout the hepatic parenchyma, with the presence of a larger number of GFP ＋ve cells incorporated within the sinu- soidal endothelial lining. Very faint albumin expression was detected in the transplanted GFP ＋re cells at 72 h; however at 2 wk, few cells that were positive for GFP were also strongly positive for albumin. There was a significant improvement in serum levels of ALT, albumin and bilirubin in both groups at 2 wk when compared with the 72 h time-point. In the cell therapy group, serum ALT was significantly （P = 0.016） lower and al- bumin （P = 0.009） was significantly higher when com- pared with the control group at the 2 wk time-point;however there was no difference in mortality between the two groups. CONCLUSION： Dual labeling is an easy to use and cheap method for longitudinal monitoring of distribu- tion, survival and engraftment of transplanted cells, and could be used for cell therapy models.
基金supported by the National Basic Research Program of China (2013CB733700)the National Natural Science Foundation of China for Creative Research Groups (21421004)+6 种基金Key Project (21636002)Distinguished Young Scholars (21325625)NSFC/Chinathe Oriental ScholarshipScience and Technology Commission of Shanghai Municipality (15XD1501400)the Fundamental Research Funds for the Central Universities (222201717003)Program of Introducing Talents of Discipline to Universities (B16017)
文摘Identification of fluorescent biomarkers with peptide ligand-directed receptors for diagnosis or theranostic of pancreatic ductal adenocarcinoma (PDAC) is still challenging. As potential prognostic/predictive bioimaging targets, both aminopeptidase N(APN, known as CD13) and Caveolin-1 are found as upregulation on the cell membrane surface of PDAC, in which APN is the principal receptor of the cyclic peptide cNGR (Asn-Gly-Arg, NGR) and Caveolin-1 can synergistically mediate endocytosis in this receptor-targeted process. Herein, we conjugate cNGR to dicyanomethylene-4H-pyran (DCM) chromophore to develop a synergistic-targeted near-infrared (NIR) fluorescent probe DCM-cNGR with strongly intrinsic NIR fluorescence, stable optical performance, low cytotoxicity, and rapid accumulation in PANC-1 cells with the synergistic overexpressed APN receptor-targeted and Caveolin-1-mediated endocytosis. As demonstrated, DCM-cNGR can realize noninvasive NIR imaging for targeting PANC-1 tumor in vivo after intravenous injection into PANC-1 xenograft tumor of nude mice, making a great promise to improve the precision diagnosis and therapy of pancreatic cancer with real time tracing and bioimaging of PDAC in vitro and in vivo.
