| Endoplasmic reticulum ( ER) is a specified membranous organell in eu-karyotes, which is associated with the synthesis of proteins and lipids and also is an important Ca2* pool inside the cell. So the study of the microstructure of ERs, especially the studies of membrane proteins, secretory proteins and molecular chaperones synthesized by ERs have become the frontiers of the molecular biology all over the world. Ilie glucose - regulated proteins ( GRPs) are important resident proteins of the ERs, including GRF78 and GRP94. GRP94 is a member of the heat shock protein90 ( HSP90) family. It is a highly conservative protein and sharing 50% homology with the HSP90. The genetic location of grp94 is clarified, whose coding gene is on 12q24. 2 - 12q24. 3. GRP94 functions as molecular chaperones, which are ( poly ) peptide binding proteins that assist in protein folding, assembly and transport. Moreover, its role of the transporter for antigen presentation has been discovered. It delivers the false ?folding peptides for presentation to proteasomes in normal cells, while in tumor cells it acts as main tumor - resistant antigen, inducing specific immune response. Recent research found that GRP94 also relates with cell cycles, differentiation and apoptosis. As tumors are regarded as the most severe one in uncontrolled proliferation and impaired differentiation cellular diseases, the study of how the changes of GRP94 expression level affect tumor cell biological characteristics may broaden the understanding of GRP94 function.GRP94 is a stress protein whose transcriptional level increases under several physiologial and pathological conditions. This is the unfolded protein response ( UPR) pathway which associates with the signal transferring from ERs to nucle-us. The UPR pathway in mammals links with several branches, including the increased transcription of a series of genes that encode folding - related proteins, the ubiquitous down - regulation of protein translation, programmed cell death mediated by CHOP and degradation of ER - related proteins, etc. Though somewhat independent, these branches do have multiple connections. So any change of one effective component in one branch may affect the whole UPR pathway, e-ven the fate of the cell. The study of GRP94 expression changes affecting UPR pathway branches will lead to more understanding of, not only GRP94 function, but also changes in UPR pathways.Based on this consideration, we chose Human Colorectal Cancer cell line CCL229 as the experimental object, down - regulating its GRP94 expression by specific ribozyme targeting. We observed its effects on the biological characteristics of CCL229 and UPR pathway, and gained further understand of the GRP94, UPR and tumor relationship.Method1. Constructing Human Colorectal Cancer Cell line with stably down - regulated GRP94(1) The plasmid pRc/RSV - ribol that contains specific GRP94 - targeting ribozyme and the control plasmid pRc/RSV were miniprepared, respectively, cleaved by endoenzyme PvuII.(2) After the gene sequences of pRc/RSV - ribol and pRc/RSV were i-dentified, the plasmids DNA were maxiprepared and purified.(3)The CCL229 cells were transfected by these two plasmids according to the instruction of Fugene?6 transfection reagent.(4)The mRNA and protein expression GRP94 were detected by RT -PCR and Western bloting respectively to find the cell clones that have been transfected successfully.2. Measuring the effects of low expression of GRP94 on human colorectal cancer cell biological characteristics(1) Light microscopic observation of the cell morphology, cell adhesion a-bility, and growth rate.(2)drawing the cell growth curves, and calculating the population doubling time.(3) Detecting cell aggregate ability, and calculating the aggregate degree of cells.(4) cell cycle distribution by flow cytometry.3. Measuring expression of the associated molecular chaperone GRP78 and the enzyme protein disulphide isomerase PDI affected by low expression of GRP94.(1) Detecting mRNA and p...
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