PLGA-Based Cell Sensors For Monitoring The Cytotoxicity Of Persistent Organic Pollutants

Persistent organic pollutants(POPs)are a class of organic compounds,including pesticides,dioxins,furans,polychlorinated biphenyls and perfluorinated compounds.POPs persist in the environment and have long half-life,long residual,bioaccumulative,semi-volatile and highly toxic characteristics.In recent years,numerous studies have shown that high-dose short-term exposure to POPs can lead to major diseases such as reproductive system damage,immune system damage,endocrine disorders,neurotoxicity and cancer.Since POPs are difficult to degrade in the natural environment,humans are exposed to low doses of POPs for a long time with the gradual ban of POPs.The concentration of POPs used in current toxicology studies is higher than the concentration of POPs in human blood samples.Thus the low-dose long-term cytotoxicity of cells exposed to POPs has been an important scientific issue and causing significant global concerns.The current cytotoxicity analysis method has two drawbacks.First,the traditional end-point analysis methods fail to monitor the cell responses in real-time,and they are impossible to observe subtle changes of cellular responses in a lower time resolution.Second,the cancer cell line can not be representative of the original cells that is more relavent to human health.In response to the above problems,this paper designs sensors that can be integrated into cells for monitoring the cellular responses stimulated by toxic pollutants in real time.At the same time,human bone marrow mesenchymal stem cells(BMSCs)were used as the research object to study the response of specific biochemical indicators(cell survival,reactive oxygen species production and fat differentiation related genes)and function under long-term co-culture of typical POPs.The main works are as follows:1.A biodegradable polylactic acid glycolic acid copolymer(PLGA)-based sensor was synthesized by the double emulsion.The preparation conditions were optimized and the surface positive charge was achieved by polylysine(PLL)modification.The sensor size control(1 ?m)and real-time function monitoring of human mesenchymal stem cells up to 30 days were achieved.2.The PLGA@calceinAM and PLGA@CellROX sensors were successfully synthesized by the aformentioned method,and the sensitive detection of intracellular lysate lipase(indicating cell viability)and reactive oxygen species was successfully achieved.The change of hMSCs cell activity and intracellular reactive oxygen species under long-term exposure to PFOS were monitered in short-term(0-48 hours)and long-term(0-14 days)periods,and two doses of 0.1 and 10 ?M(blood concentration in the general population and occupational population)PFOS were chosed.3.Kinesin(?-actin),peroxisome proliferator-activated receptor(PPAR?)and adipocyte-type fatty acid binding protein(ap2)molecular beacons were designed successfuly,and three molecular beacon pairs for their respective target strands were verified,and specific response of the concentration.A PLGA-based molecular beacon sensor was prepared to simultaneously monitor mRNA expression levels of ?-actin,PPAR? and ap2 in cells.The adipose differentiation of hMSCs was monitored by long-term exposure of two doses of PFOS at 0.1 and 10 ?M.The stable expression of ?-actin was used as an internal reference for monitoring two genes related to cell adipose differentiation(i.e.PPAR? and ap2).In this thesis,the human mesenchymal stem cells integrated by PLGA sensor were used as the research platform for txoxicity screening.It brings a new stratergy for investigation of the cytotoxicity of POPs under low-dose and long-term exposure.