Cytotoxic Effects Of CuInS2/ZnS Quantum Dots On Human Ovarian Granulosa Cells

ObjectiveThe cumulus cells around superovulated oocytes were cultured in vitro,and 5-fluorouracil(5-Fu)was used as a positive drug to detect its cytotoxic effect on human egg particles,and a human egg particle cytotoxicity detection system was established.Then,using this system,CuInS2/ZnS is detected Cytotoxicity and molecular mechanism of ZnS quantum dots.This project provides a theoretical basis for the biosafety of fluorescent nanoparticles.Method:Hyaluronidase was used to remove cumulus cells outside human superovulation,and primary culture was carried out to detect 5-Fu and PEGylated CuInS,respectively The cytotoxic effects of ZnS quantum dots and explore the molecular mechanism of their toxicity.Using CCK8 method to detect different concentrations of layered 5-fluorouracil(5-Fu)and PEGylated CuInS2/ZnS.The effect of ZnS quantum dots on cell viability was calculated by IC50;Annexin V/PI double staining method was used to detect the apoptosis of human egg granule cells by flow cytometry;LC3-GFP plasmid was transfected to observe the changes of autophagy after drug action in real time;mitochondrial structure changes and membrane potential technology were analyzed by electron microscopy to detect mitochondrial function changes;different kits were used to detect sex hormone release and oxidative stress changes;then real-time quantitative PCR and Western blotting were used to analyze the molecular mechanism of autophagy and apoptosis.Results:Superovulated human oocyte granule cells(cumulus cells)are very important reproductive helper cells around the egg.In this experiment,primary cultured cumulus cells in vitro were established and used to detect PEGylated CuInS2/ZnS Cytotoxic effects and molecular mechanisms of CuInS2/ZnS quantum dots.1.The cytotoxic effect of 5-FU induced by primary cultured granulosa cells in vitro was detected:5-FU promoted the apoptosis of granulosa cells in a dose-dependent manner;the IC50 value of 5-FU on granulosa cells was 1.227 μM.2.5-FU significantly induces cumulus cytotoxicity and causes autophagy:it mainly damages the physiological structure of mitochondria and produces a large number of autophagosomes and apoptotic bodies,which mainly activate the mitochondrial apoptosis pathway inside cells and cause cell apoptosis.3.CuInS2/ZnS Cytotoxic effect of ZnS quantum dots on human egg particles:The quantum dots can enter cumulus cells and significantly inhibit cell viability,with an IC50 value of 66.72 μg/mL.4.Quantum dots can induce oxidative damage of granular cells.Toxic effects:4.1 Detection of changes in enzymes involved in oxidative stress:The total antioxidant capacity(T-AOC)and total superoxide dismutase(T-SOD)activities of the high-dose(100 μg/mL)quantum dot group were significantly lower than those of the treatment group;The malondialdehyde(MDA)and glutathione(GSH)activities in cumulus cells of the high-dose(100 μg/mL)quantum dot group were significantly higher than those of the treatment group.4.2 Detection of mitochondrial functional changes:after CuInS2/ZnS quantum dot treatment,quantum dots induce a significant increase in intracellular calcium ion concentration while mitochondrial membrane potential is significantly reduced.Quantum dots therefore significantly reduce intracellular ATP content,increase cAMP content,and significantly increase reactive oxygen species(ROS)levels.Increase the level of autophagy in the cell.5.Quantum dots induce granule cell sex hormone release:quantum dots promote sex hormones progesterone(P)and estradiol(E2)in cumulus cell culture medium)may be related to the release of endogenous hormones due to impaired cell function.6.Quantum dots induce the release of calcium ions from the endoplasmic reticulum of granular cells:quantum dots induce a significant increase in calcium ions in granular cells,and the endoplasmic reticulum-specific calcium channel inhibitor BAPTA can effectively inhibit the increase in calcium ion concentration induced by quantum dots.The results suggest that the endoplasmic reticulum Calcium channel is the target of CuInS2/ZnS quantum dot toxicity.7.Quantum dots induce mitochondrial apoptosis in granulosa cells:After the action of quantum dots,the related proteins Caspase-3,Bcl-2 and P53 in the mitochondrial apoptosis pathway are significantly changed.CuInS2/ZnS quantum dots can induce mitochondrial apoptosis in granulosa cells,and BAPTA can effectively alleviate this apoptosis.It is suggested that the molecular mechanism of quantum dot toxicity may be related to its inducing the release of endoplasmic reticulum calcium ions into mitochondria and causing mitochondrial apoptosis.Conclusion:Successfully established in vitro human egg granule cytotoxicity detection system,CuInS2/ZnS quantum dots affect cell function by enhancing cell oxidative damage and endoplasmic reticulum calcium release,which in turn leads to mitochondrial functional damage and promotes cell apoptosis.Our experimental results will be CuInS2/ZnS quantum dot biosafety assessment provides valuable information.