| Dysfunction of endoplasmic reticulum (ER) maintaining proteinhomeostasis can result from various disturbances, including hypoxia oroxidative stress,which lead to an imbalance between protein-folding capacityand protein-folding load. This in turn leads to ER stress and induction of theunfolded protein response (UPR). The UPR initially serves as an adaptiveresponse, but also induces apoptosis in cells under severe or prolonged ERstress. Accumulating evidence indicates that ER stress contributes to tissuesdamages in liver disease. These findings emphasize the importance of ERstress as a new progression factor and the interesting future possibility ofprotective strategies targeting ER stress.These therapeutic approaches may actby breaking the vicious cycle of oxidative stress, hypoxia, and ER stress.Part1Normal liver cells HL-7702is divided into five groups, named A, B, C, D, E group; group A was control group, the remaining four groups of normalculture medium was changed after48hours (respectively containing theconcentration of tunicamycin0.05,0.10,0.20,0.40μg/ml culture medium)and48hours later, replace the normal medium, and immediately transferred to thehypoxic incubator and were cultured2,4,8,16hours,after that,all cells wereturned into a normal incubator and cultured for6hours after the cell morphologycells were collected to measure the apoptosis rate.Conclusion:the cells of D and E groups are all dead;B group cells have nodifferences significantly campared to A group,the apoptosis rate of C group is20%.Concentration of0.1μg/ml of tunicamycin, hypoxia4hours reoxygenationcultured for6hours of hypoxia is considered as a normal liver cell H/R injurymodel standard time limit.Part2According to the model of the experiment1, the cells were divided intofour groups: normal control group (control group); endoplasmic reticulum stressgroup (ERS Group); hypoxic injury group (H/R group); endoplasmic reticulumstress+hypoxic injury group (ERS+H/R group). Collect cells in each group toextract protein,test the specific molecular chaperone GRP78of ER and threepathways of the UPR, including the PERK and RACK1and ATF6expressionsby Western-blot.Conclusion: Under the conditions of ER stress, GRP78expressions areraised,showing that tunicamycin was successfully induced the theendoplasmic reticulum stress in the liver cells. Three pathways specific proteinsexpressions are raised compared to the control groups.Our studies focus on theIRE1pathway.We confirm that GRP78protein to enhance the survival rates ofER stress in hepatocytes induced by TM. UPR three pathways of proteinexpression was also improved within varying degrees Our topic focuses on the IRE1downstream molecular RACK1against hepatocytes hypoxia injuries.Part3.According to the result of experiment1,we divide the cells into5groups: Group A: control; B: endoplasmic reticulum stress+H/R injury group;group C: only the endoplasmic reticulum stress group; Group D: Endoplasmicreticulum stress+no-load transfection group; Group E: endoplasmic reticulumstress+siRNA transfection group. Collecting cells in each group after theexperiment, and detect the apoptosis rate by flow cytometry, and detect theendoplasmic reticulum stress-specific protein RACK1protein and RNAexpression levels with Western-blotting and RT-PCR, and observed bytransmission electron microscopy the ultrastructural changes of cells in eachgroup.Conclusion: E group apoptosis rate was significantly higher; RACK1RNAand protein in the BCD group has a high expression, in contrast to the E-cellRNA and protein expression were decreased.Endoplasmic reticulum stresspreconditioning on liver cells in hypoxia-reoxygenation injury has a significantprotective effect, silencing RACK1expression in cells will greatly enhance thevalue of apoptosis in liver cells in hypoxia-reoxygenation injury, endoplasmicreticulum stress-specific protein RACK1may play as a key protective protein inthe hypoxia-reoxygenation injury of liver cells. |
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