Font Size: a A A

Human Recombinant Protein RhPGRN Promotes Cell Proliferation By Regulating Autophagy And Er Stress Through MAPK/PI3K Pathway

Posted on:2021-04-21Degree:MasterType:Thesis
Country:ChinaCandidate:R LuoFull Text:PDF
GTID:2370330620474975Subject:Genetics
Abstract/Summary:PDF Full Text Request
Objective:To investigate the effect and mechanism of human recombinant protein Progranulin(rhPGRN)on proliferation,autophagy and endoplasmic reticulum stress in C28I2 chondrocyte and RAW264.7 macrophage.Methods:The histidine-tagged protein was specifically affinity-purified using Ni-NTA agarose resin,the purity and concentration of the target protein were verified by Coomassie blue staining,BCA method and Western blot.The effects of rhPGRN on cell proliferation,autophagy and ER stress in C28I2 and RAW264.7 cells were detected by cell counting,Western blot and RT-PCR in the control group and different treatment groups,including rhPGRN,U0126,3-methyladenine,rhPGRN + U0126,rhPGRN + 3-MA,rhPGRN + bafemycin A1 and rhPGRN + sodium 4-phenylbutyrate group,.Results:(1)The human-derived recombinant protein rhPGRN with high purity and biological activity was successfully extracted from the stable cell lines containing His-tagged PGRN.(2)rhPGRN promotes cell proliferation,increases the mRNA level of cell cycle related genes PCNA,Cyclin B1(CCNB1),Cyclin D1(CCND1),and the protein level of cell cycle regulatory protein Ki67,and activates the phosphorylated level of proliferation-related signaling molecules ERK and AKT in C28I2 and RAW264.7 cells.(3)The rhPGRN-induced cell proliferation,autophagy and ER stress is inhibited after ERK pathway inhibitor U0126 treatment;the rhPGRN-induced cell autophagy is significantly suppressed after PI3 K / Akt pathway inhibitor 3-MA treatment;the rhPGRN-induced Ki67 protein enhancement is significantly down-regulated after autophagy inhibitor Baf-A1 And ER stress inhibitor 4-PBA treatment.Conclusion:rhPGRN may regulate autophagy and ER stress through MAPK,PI3K/Akt pathway to promote the proliferation of C28I2 and RAW264.7 cells.
Keywords/Search Tags:rhPGRN, eukaryotic expression, proliferation, autophagy, ER stress
PDF Full Text Request
Related items