In situ fluorogenic reaction for ratiometric fluorescent detection of alkaline phosphatase activity.

Assays utilizing in situ fluorogenic reactions provide a simple and convenient alternative approach for the detection of biological molecules and activities. In this work, a novel ratiometric fluorescent probe based on in situ fluorogenic reaction is explored and developed for alkaline phosphatase (ALP) activity sensing. An intriguing fluorogenic reaction between 2,3-diaminonaphthalene (2,3-DAN) and ascorbic acid (AA) in alkaline aqueous solutions could generate the fluorescent quinoxaline derivative. The resultant quinoxaline emits intense yellow fluorescence, differing from the blue fluorescence of 2,3-DAN. Thus, a ratiometric fluorescent probe based on this fluorogenic reaction is constructed for ALP activity sensing, combining with ALP-triggered hydrolysis of AA2P into AA. Meanwhile, the addition of copper(II) acetate into the reaction system largely improves reaction rate and efficiency. This sensing strategy shows high sensitivity for ALP activity with detection limit of 0.08 U/L, and excellent selectivity towards ALP out of various interferences. This method is extended to human salivary ALP detection. The present method provides a simple and reliable alternative for the detection of ALP activity and has the potential for clinical applications. It also shows a feasible way to construct ratiometric fluorescent methods.

CD146 mediates the anti-apoptotic role of Netrin-1 in endothelial progenitor cells under hypoxic conditions.

Modulating the biological status of endothelial progenitor cells (EPCs), such as function and survival, is essential for therapeutic angiogenesis in ischemic vascular disease environments. This study aimed to explore the role and molecular mechanisms underlying Netrin­1 in the viability and angiogenic function of EPCs. EPCs were isolated from the bone barrow of adult C57/BL6 mice. The apoptosis and various functions of EPCs were analyzed in vitro by manipulating the expression of Netrin­1. The TUNEL assay was performed to detect apoptotic EPCs. Cell migration and tube formation assays were performed to detect EPC function. Trypan blue staining was performed to detect cell viability. Western blot analysis was performed to detect the protein expression levels of Netrin­1, CD146 and apoptotic factors. Quantitative PCR analysis was performed to detect the expression levels of Netrin­1 receptors. The results demonstrated that treatment with exogenous Netrin­1 promoted EPC migration and tube formation, whereas transfection with small interfering (si)RNA targeting Netrin­1 exhibited the opposite effects. Exogenous Netrin­1 protected EPCs from hypoxia­induced apoptosis, whereas the interruption of endogenous Netrin­1 enhancement under hypoxia by Netrin­1­siRNA exacerbated the apoptosis of EPCs. Furthermore, CD146, one of the immunoglobulin receptors activated by Netrin­1, was screened for in the present study. Results demonstrated that CD146 did not participate in Netrin­1­promoted EPC function, but mediated the anti­apoptotic effects of Netrin­1 in EPCs. In conclusion, Netrin­1 enhanced the angiogenic function of EPCs and alleviated hypoxia­induced apoptosis, which was mediated by CD146. This biological function of Netrin­1 may provide a potential therapeutic option to promote EPCs for the treatment of ischemic vascular diseases.