Construction Of Fluorescence Detection System For Stress Response Capacity Of Organisms To Chemical And Its Application In Visual Evaluation For Aging

Cell senescence is beneficial to prevent abnormal cell proliferation,while excessive accumulation of senescent cells in tissues aggravates senescence.The evaluation of cell senescence usually relies on the detection of senescence markers in resting state.The latest research shows that the gradual decline of stress response capability is the essential feature of aging,which can more accurately reflect the aging degree.However,to the best of our knowledge,there are few reports on the fluorescence detection system capable to accurately evaluate the response capability of cells to stress.Therefore,we here present a fluorescence detection system which can logically identify the stressor thiophenol(Ph SH)and hypochlorous acid(HOCl)as a stress response product induced by Ph SH to detect aging degree more accurately by indicating the individual's stress response capability under the dynamic stress.The specific research contents are as follows:In this thesis,the N,N-diethylamino group in the scaffold of rhodafluor(rhodol)was modified to be a cyclic amino group structure and 2-(2'-hydroxyphenyl)benzothiazole(HBT)was further introduced into this scaffold to construct a fluorescent dye ROB.Then,the ROB-based compound ROCL was constructed by using spirocyclic hydrazide structure as the HOCl recognition group,and the ROCZ was constructed by introducing spirocyclic hydrazone structure to turn off HOCl recognition function.Our pioneering probe ROKS and ROZ were prepared by introducing 2,4-dinitrobenzoxy into the molecular scaffolds of ROCL and ROCZ to turn on the recognition function of Ph SH,and ROE was constructed by introducing ethyl into the scaffold of ROCL to turn off the recognition function of Ph SH.The structures of all the above compounds were characterized by NMR,HRMS and IR,and their absolute configurations were all confirmed by X-ray crystallography.The experimental results showed that compared with ROZ and ROE,the probe ROKS achieves excellent logical recognition by a green fluorescence “turn-on” signal and then a ratio fluorescence signal from green to red.ROCL and ROCZ were successfully used for cell imaging experiments stimulated by LPS.The results showed that the fluorescence signal of ROCL in the red channel increased gradually with the increase of stimulation time and the fluorescence signal of ROCZ in the red channel remained unchanged,indicating that the structure of spirocyclic hydrazide was sensitive to HOCl.At the same time,ROZ was applied to cell imaging experiments stimulated by Ph SH.The results showed that only the fluorescence signal in green channel gradually increased with the increase of stimulation time,which confirmed that the structure of dinitrobenzoxy was sensitive to Ph SH.ROKS was applied to the imaging experiments of cells and C.elegans at different degrees of senescence.The results showed that the accumulation level of reactive oxygen species(ROS)in young system was higher under the stimulation of Ph SH,indicating that the stress response capability of young system was stronger than that of aging system.The ROKS system developed in this paper is a single probe system with dual detection functions for chemical stressors and stress products.Compared with the system only detecting stress products,it is more suitable for complex biological systems because quantifications of both stressors and the corresponding stress products can provide more accurate evaluation of the stress response capability to different biological samples.