Purification Of Heat Shock Protein 70 Peptide Complex And The Effect Of HSP70-peptide Complex On The Proliferation Of HepG-2 Cells

ObjectiveThe heat shock proteins are evolutionary ancient families of molecules that are present in, and considered essential to the survival of, all prokaryotic and eukaryotic cell types. A lot of studies show that HSP70 was overexpressed in cells of various cancers, include hepatocellular carcinoma. They act as cytoprotective agents by binding to misfolded proteins and thus protecting them from denaturation, and are of special relevance in human cancer inhibiting apoptosis. Howerer, studies also suggest that hsp70 might have immunotherapeutic potential, as hsp70 purified from malignant and virally infected cells can transfer and deliver anti-genic peptides to antigen—presenting cells to elicit peptide—specific immunity. Our aims were to separate and purify heat shock protein 70 peptide complex from hepatocellular carcinoma tissues by fast protein liquid chromatography (FPLC) system, and study the effect of HSP70 —peptide complex on the proliferation of HepG—2 cells and the expression of JNK and c—Jun in HepG—2 cells.Methods1. The preparation of mixed protein: the protein drived from hepatocellular carcinoma tissues by means of spliting, centrifuge.2. Purification of HSP70 — peptide complex: the mixed protein was separated by affinity chromatography on concanavalin A — Sepharose and DEAE—Sephacel exchange chromatography.3. The qualitative and quantitative analysis of HSP70 — peptide complex: the molecular weight and identity of the HSP70 was confirmed by SDS—PAGE and Western blot, the concentration was measured by the means of Bradford.4. Cells were observed by MTT examine the growth of HepG — 2 cells.5. Expression levels of JNK, p —JNK, c — Jun, p —c —Jun protein were determined by Western—blot technique.Results1. A sharp stained protein band with a molecular weight of about 70kD was obtained and shown to be HSP70 as confirmed by Western blot, the quantity of HSP70 obtained from lOg hepatocellular carcinoma tissues was 8mg by the means of Bradford.2. The value of OD in HSP70 group was significantly higher than control group (p<0. 01). The cell livability of HSP70 group was higher than control group.3. The expression levels of JNK, p—JNK, c — Jun, p —c —Jun protein were downregulated in the HSP70 group than control group.Conclusion1. The results suggest that the pure HSP70 — peptide complex have been be obtained through this procedure.2. HSP70 — peptide complex can promote HepG—2 cells growth.3. HSP70 — peptide complex can downregulate the expression of JNK, p—JNK, c—Jun, p—c—Jun protein and inhibit apoptosis of HepG — 2 cells.