Effecting On Proliferation And Differentiation Of Human Glioblastoma Stem Cells By Recombinant Human IL-24

Gliomas are the most common human brain tumors. The traditional treatmentmethods are including surgery, radiotherapy and chemotherapy, but the effect is limited.The study found that it is the main reason that the brain tumor stem cells (BTSC) lead torecurrence, metastasis in the glioma. It provides a new way to target the BTSC for thetreatment of glioma, especially inducing the BTSC differentiation therapy. Interleukin-24(IL-24) is a unique member of the IL-10gene family that displays nearly ubiquitouscancer-specific toxicity, with no harmful effects toward normal cells or tissues. The recentstudies have found that IL-24can induce the cancer cells in hematopoietic malignancies tothe mature monocyte-macrophage differentiation. IL-24can induce the cancer-specificapoptosis in a broad-spectrum of human cancers, but it is unknown for the treatment effecton the tumor stem cells by IL-24. Therefore, we investigated the effect of proliferation anddifferentiation in the BTSC by IL-24, as the following:Part Ⅰ：Expression, purification and refolding of the recombinanthuman IL-24proteinObjective：The expression plasmid pET28a-IL24was constructed, and used totransform E.coli strain BL21(DE3). The IL-24recombinant proteins were obtainedthrough purification and renaturation.Methods：IL-24cDNA were inserted into expression plasmid pET28a (+), andidentified by the recombination enzyme digestion and sequencing. The successfullyconstructed plasmid was used to transform E. coli strain BL21(DE3) competent cells. Thepositive clone cells were inoculated to overnight express instant TB medium containing15μg/ml kanamycin. The recombinant proteins were purified by His-tag affinitypurification under denaturing conditions using BugBuster Ni-NTA His.Bind PurificationKit (Novagen, U.S.A), and then renatured using Protein Refolding Kit (Novagen, U.S.A). All kits were used according to the manufacturer’s instruction. Protein purity and identitywas checked by sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)and Western blotting.Results: We constructed the recombined express vectors pET28a-IL-24andconfirmed by DNA sequencing. The transformed BL21(DE3) cells with pET28a-IL-24were inoculated to overnight express instant TB medium at37°C for16hours. Afterinduction, the cells were dissolved in SDS-PAGE sample buffer and subjected toSDS-PAGE. The result showed that the recombinant IL-24proteins were expressedefficiently with the yield accounting for36.47%of total bacterial proteins. SDS-PAGEanalysis also showed that the recombinant proteins were mainly in the precipitation of celllysate, indicating that the recombinant proteins existed predominantly in the inclusionbodies. After washing with2mol/L urea, the recombinant proteins were purified underdenaturing conditions with Nickel metal-affinity resin columns for single-step purificationsof His-tagged in IL-24, and the purity of purified proteins was about90%. The purifiedproteins were renatured by step and step dilution using protein refolding kit, and stored in-20℃. IL-24was recognized with anti-IL-24monoclonal antibody by western blotting.Conclusion: We successfully constructed the expression plasmid pET28a-IL24, andobtained the recombinant proteins IL-24by purification and renaturation.Part Ⅱ：Isolation and identification of brain cancer stem cells fromglioma cell line U251Objective：To isolate and culture of brain cancer stem cells from glioma cell lineU251using suspension culture and magnetic activated cell sorting (MACS)．Methods：First through suspension culture of U251cells were inoculated intoserum-free medium with growth factor to obtain brain tumor sphere (BTS) which containsa lot of BTSC. Then, we detected the expression of CD133and Nestin in BTS byimmunefluorescence. We separated and collected the BTS for further separation andpurification through immunomagnetic beads sorting and verification for the CD133positive rate by flow cytometry, and then, determined the proliferation activity of CD133+BTSC by MTT.Results: The adherent growth and proliferation of U251cells were cultured in themedium containing serum. When the U251cells were cultured in medium with serum-free, part of the cells was single suspension or nonspecific adhesion at24to48hours, and then,cells formed brain tumor sphere, suspended in the medium, increased gradually, and finallyformed a typical brain tumor stem cells sphere after72hours. The high expressions ofCD133and Nestin in BTSC were tested by immunofluorescence. With the methodcombining suspension culture with MACS the CD133+positive rate of BTSC can beraised over95.8％．The proliferation of CD133+cells showed significantly stronger thanthe CD133-cells by MTT assay, and the monoclonal formation rate of CD133+cells ishigher than that of CD133-cells.Conclusion: The brain cancer stem cells have been successfully isolated from gliomacell line U251．They formed typical neurospheres in serum-free medium，and had strongcapacity to self-renew and proliferation．Part Ⅲ：rhIL-24effect on human glioma stem cells proliferation anddifferentiationObjective：To investigate the effect of rhIL-24on the proliferation and differentiationof brain tumor stem cells (BTSCs) in vitro.Methods：The cell proliferation activity of CD133+BTSC treatment with rhIL-24was detected by MTT. The cell differentiation of CD133+BTSC treatment with rhIL-24was detected by immunofluorescence and observation of cell morphology change. TheCD133expression of was detected by flow cytometry analysis in CD133+BTSC treatmentwith rhIL-24. We evaluated the effect of rhIL-24on xenograft formation by CD133+BTSC in nude mice．Results: The results from MTT assays showed that the rhIL-24can significantlyinhibit the proliferation of CD133+BTSC. The observation of cell morphology showed theCD133+BTSC treated with rhIL-24were morphological diversity and bumps growinglonger in serum-free culture medium containing growth factors after7days. In CD133+BTSC treated with rhIL-24, the expression of NeuN (neuronal markers) and GFAP(astrocytes markers) were significantly higher, while the expression of CD133BTSCmarkers significantly reduced, but not completely disappear, which showed BTSCdifferentiation is not complete, cannot achieve terminal differentiation. The ability ofxenograft formation of CD133+BTSC treated with rhIL-24significantly reduced in nude mice.Conclusion: rhIL-24can inhibit proliferation and induce the differentiafion of BTSCs.This may provide a new ideas and methods in the treatment of glioma.