Building A Cellular Aging Model And Tentatively Screening Anti-aging Drugs

Aging is characterized by a progressive loss of physiological integrity,which is the inexorable law of vital process.This deterioration is the primary risk factor for major human pathologies,including cancer,diabetes,cardiovascular disorders,and neurodegenerative diseases.Last five years,more and more attention had been paid for the accumulation of senescent cells and their secretory phenotype(SASP),then the identification of small molecules with potential to selectively induce death of senescent cells(called senolytics)had become one of promising therapeutic approaches in anti-aging research.This theory is based on the common anti-apoptotic,pro-survival mechanism of senescent cells and tumour cells.Our study started from four aspects.1)Building a cellular aging model.Considering the easily-aging characteristic of primary cell,we monitored the aging process by continuously culture.We got the corresponding portion of senescent cell by SA-?gal staining,then observed the growth arrest by PI staining and flow cytometry.And we got the variation curve between culture time and senescent ratio.After 7 or 8 days' culture half of the cells got old,and more than 90% after the ninth day,with the increased G0/G1 cell cycle arrest.The innovative senescent cell model can avoid the widespread problem of current premature senescent(SIPS)model,which can't easily differentiate cell damage and cell senescence caused from acute or chronic cytotoxicity.2)After the building of the senescent model,we tested 19 drugs to see if they can affect the aging progress between the 7th day and the 9th day.Currently we found a combination of superoxide dismutase(SOD),glutathion peroxidase and catalase were able to delay the aging process apparently,with 29% reduction of senescent portion(from 92.23% to 63.55%).0.5-2.5 ?M ABT263(existing senolytics)could selectively kill senescent cells.And 5 ?M amifostine disulfide,250 ?g/m L cardiomypeptidin(CMP),250 ?M cinepazide performed the same results.3)We screened 5 drug candidates and analyzed the cytotoxicity difference between the primary and senescent tubular endothelial cell.The existing senolytics ABT263 was regarded as positive control with NC/SC value(the ratio between cell viability of normal cell and that of senescent cell)3.5(>1).10 ?M amifostine disulfide performed 89,and 1000 ?M cinepazide 4.67,showing excellent selectivity.4)Besides that,we also found cardiomypeptidin and related compounds of amifostine could promote proliferation of non-tumor cell line,then rebuilt cell number and prevented aging cooperate with senolytics.50 ?g/m L CMP,50 ?M WR-1064,and 1 ?M amifostine disulfide could respectively improve cell viability by 34%,12% and 9%(p<0.001).