Effecting On Biological Behavior Of Human Osteosarcoma Stem Cells By Recombinant IL-24 Protein

In children and adolescents osteosarcoma is the most common primary malignant bone tumor.It has a high malignancy and is prone to metastasizing lung.The traditional treatment ways for osteosarcoma include operation,preoperative and postoperative chemotherapy and radiotherapy,however the overall treatment effect is limited.The study proved that Osteosarcoma Stem Cell?OSC?is the main cause of recurrence and metastasis of tumor.With OSC as a therapeutic target cell a new way for the treatment of osteosarcoma is provided.In recent years,interleukin-24?IL-24?is proved it is a tumor suppressor gene,also known as a melanoma differentiation-related gene,which not only inhibits tumor growth but also has a broad-spectrum anti-tumor effect by apoptosis.Foreign literature reports that hematological malignancies can differentiate into mature monocyte-macrophage cells through IL-24 induction.At present,the impact of IL-24 on the biological behavior of tumor stem cells has not been reported.SO,this paper aims to study the effect of recombinant human IL-24?rhIL-24?protein on the biological behavior of osteosarcoma stem cell..Objective : First,construction of prokaryotic expression plasmid p ET32a-IL-24 utilizing human IL-24 cDNA,by transformating the BL21?DE3?strain,the recombinant protein IL-24 is expressed,identified,purified and recultured to obtain human recombinant IL-24 protein.Second,osteosarcoma stem cells in human osteosarcoma 143-B cell line were cultured and sorted by the way of serum-free suspension culture and immunomagnetic bead sorting.Third,To study the effect of rhIL-24 on the biological behavior of CD133+ osteosarcoma stem cells.Methods : First,using molecular biology techniques,a recombinant plasmid p ET32a-IL-24 was constructed by inserting into the expression plasmid p ET32a?+?human with IL-24 cDNA,and was digested by enzyme,identified by sequencing.The correctrecombined plasmid p ET32a-IL-24 was amplified,purified and transfered into expression strain BL21?DE3?.Rh IL-24 protein was performed and the expression of the target protein was identified by SDS-PAGE and Western Blotting.Rh IL-24 protein inclusion bodies were obtained by induction of recombinant engineering bacteria.After washing,the rhIL-24 protein was purified by the 6×His purification tag and by nickel ion affinity chromatography.The rhIL-24 protein was correctly folded by dialysis.Second,a large number of cell balls containing osteosarcoma stem cells were obtained by suspension culture and Inoculating 143-B cells in serum-free medium containing growth factors,the expression of CD133 in stem cells of osteosarcoma was detected by cellular immunofluorescence.The osteosarcoma stem cells in the stem cell balls were isolated and sorted and purified by means of using CD133-labeled immunomagnetic beads.The proliferative activity of CD133+ osteosarcoma stem cells was tested by the MTT and monoclonal colony formation assay.The proliferative activity of CD133+ osteosarcoma stem cells after treatment with rhIL-24 protein was detected by the MTT assay;the effect of rhIL-24 on the differentiation of CD133+ osteosarcoma stem cells was observed by cell morphology;The effect of rhIL-24 on the expression of CD133 in CD133+ osteosarcoma stem cells was stud Iied by flow cytometry,and the effect of rhIL-24 on the invasive ability of CD133+ osteosarcoma stem cells was tested by transwell chamber invasion assay.By tumor-bearing tumor xenografts in nude mice the colony formation ability of CD133+osteosarcoma stem cells with rhIL-24 treatment was observed.Results:First,the IL-24 expression plasmid p ET32a-IL-24 was constructed.The result of DNA sequencing showed that the sequence of IL-24 was completely consistent with Genebank?NCBI Reference Sequence: NG₀29565.1?sequence.Recombinant expression plasmid p ET32a-IL-24 was transformed into the expression bacterial strain and the bacteria were induced to culture.The total protein was obtained after harvesting and lysing the cells.By analysis SDS-PAGE and Coomassie blue staining,the expressed rhIL-24 protein accounted for approximately 39.68% of total bacterial protein.and rhIL-24 protein is expressed by inclusion bodies.After washing and dissolving,a rhIL-24 protein was obtained by nickel ion affinity chromatography a purity rate is a more than 95%.The renatured rhIL-24 protein was stored at-80°C refrigerator after freeze-dry.Second,143-B cells were founded adherent growth in complete medium containing serum and the proliferation was active.The passaged 143-B cells were inoculated into serum-free medium containing multiplegrowth factors.By microscopic observation most cells adhered to the wall within 24 hours,and a small number of cells present a single cell suspension state;and a part of cells were non-specifically aggregated.After 48 hours culture,small cell spheres began to form,suspended in the culture medium,and then the cultured spheres gradually increased with the prolonged culture period to form typical osteosarcoma stem cell spheres.By cellular immunofluorescence CD133 on stem cells of osteosarcoma showed high expression.The CD133+ cells were purified and sorted by immunomagnetic bead sorting.After sorting by flow cytometry on osteosarcoma stem cells,the proportion of CD133+ cells is 96.91%.The proliferative capacity of CD133+ cells was significantly stronger than that of CD133-cells by MTT assay;the monoclonal clon rate of CD133+ cells was high,and CD133-cells could hardly form typical osteosarcoma stem cell balls.Third,By MTT experiment results wefounded that rhIL-24 protein can inhibit the proliferation of CD133+ osteosarcoma stem cells.Rh IL-24-induced CD133+ osteosarcoma stem cells were cultured in serum-free medium containing growth factors for 7 days,the morphology of the cells was varied and Cell protrusion increased;the expression of stem cell markers CD133 significantly reduced but not completely disappeared,indicating that osteosarcoma stem cells differentiated Incompletly,cells do not reach terminal differentiation.The results of invasion assays showed that invasion ability of CD133+ osteosarcoma stem cells with rhIL-24 inducement was significantly reduced.In the tumor xenografts of nude mice,the tumorigenicity ability of CD133+ osteosarcoma stem cells induced by rhIL-24 in nude mice was significantly reduced.Conclusion: First,the active rhIL-24 protein was successfully obtained by molecular biology and protein recombination techniques,which paid way for the next experiment.Second,143-B cells were cultured in serum-free medium containing growth factors to form typical osteosarcoma stem cells.High-purity CD133+ osteosarcoma stem cells were obtained by technology of immunomagnetic separation.Third,rhIL-24 protein can inhibit the proliferation of osteosarcoma stem cells,reduce the expression of stem cell markers in osteosarcoma stem cells,induce differentiation of osteosarcoma stem cells,and decrease the invasion and tumorigenicity ability of osteosarcoma stem cells.In short,IL-24 plays an important role in the proliferation,differentiation and invasion of osteosarcoma stem cells,and a new ideas and methods is provided for us about treatment of osteosarcoma with IL-24.