| Exosomes are disk-shaped vesicles secreted by various kinds of cells and usually are 30 to 200 nm in diameter.They can carry many important donor cells-derived biological functional molecules,such as lipids,proteins,messenger RNAs?mRNAs?,microRNAs?miRNAs?and non-coding RNAs?ncRNAs?,which would changes under stress or pathological conditions.As natural delivery vehicles,thesemicrovesicleshave unique advantages,such as low immunogenicity,high stability in the blood,high efficiency of drug delivery to cells and stronger enhanced penetration and retention effect?EPR?,etc[2-4].Therefore,exosomes can be used as natural endogenous carriers of RNA,proteins and chemical drugs to treathuman diseases.However,there is a lack on exosomes loading protein drugs.In this study,using CHO cells stably expressed exogenous recombinant proteins,we analyzed the load of recombination proteins in exosomes secreted by CHO cells to study the feasibility of using CHO cells as exosomes producer cells in large scale.Firstly,the recombinant CHO cells stably expressing Her2 antibody were cultured and the highest cell density reached 2.08×107 cells/m L,andthe expression of Her2antibody protein reached 2.9 g/L at day18.Cell culture medium was collected and purified by Protein A column,then the penetrating solution was collected to obtain exosomesby ultracentrifugation,followed by analysis with transmission electron microscope and nanometer particle size analyzer.It was foundthat the extracted vesicles showed a"cup-like"structure with a bilayer membrane.And about 100 nm in diameter,which belonged to the size range of exosomes.Furthermore,it was identified by Western blot using antibodies of marker proteins in exosomes including CD63 and TSG101.At the same time,the recombianant Her2 antibody was found in exosomes.Protein profiles of recombianant CHO cells and exosomes were analyzed with mass spectrometer,and it was found that the Her2 antibody was enriched in CHO-derived exosomes.Further this antibody was identified to have activity to inhibit growth of Her2 over-expressing cells BT474.In order to further verify that these results are not specific for Her2 antibody,we tested another CHO cells which stably expressed recombinant TNFR-Fc fusion protien.Exosomes were extracted from the cell culture medium after purified by Protein A chromatography.TNFR-Fc was also found in these exosomes and has activity to antagonize TNF-?with L929 cells.These results mean that CHO cells-derived exosomes could load active recombianant proteins expressed in these cells.Secondly,in order to investigate the factors affecting recombinant proteins loading in exosomes,three EGFP recombinant plasmids with three different signal peptides with different secretion efficiency was constructe,followed by transfecting CHO cells for transient expression,and cell supernatants were harvested to extract exosomes.It was found that the higher of secretion efficiency of the recombinant protein,the greater of probability of being loaded into exosomes.At the same time,the higher ofprotein expression levels in cells,the greater loading in exosomes which was consistent with the results of protein profile analysis.At present,CHO cells are the most important expression system for the production of recombinant proteins,and both the protein expression and culture technology are very mature.The protein expression level can reach 3-5 g/L,while the cells can be cultured with serum-free medium in large scale,which could supply a large number of exosomes without the interference of serum in exosome extraction if using CHO cells as exosome producers.Our study,provides the possibility to use CHO cells to obtain exosomes for protein drug delivery. |
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