| Background and objective Gliomas are the most common malignant tumors in the central nervous system(CNS),with an increasing incidence rate and mortality rate,which seriously threaten human life and health.Glioma is easy to relapse after surgery because of the invasive growth characteristics of tumor cells.Although much progress has been made in the past decades in the treatment of gliomas,including radiotherapy,chemotherapy,immunotherapy,and so on,the prognosis for patients with gliomas is still very poor.In recent years,mesenchymal stem cells(MSCs)have become one of the hot spots in the field of biology and medicine.MSCs,a kind of non hematopoietic stem cells,have the ability of self-renewal,immune regulation and multi-directional differentiation.MSCs have attracted extensive attention of many scientists,because MSCs proliferate rapidly and can selectively migrate to the lesion site,and the cell transplantation is not easy to cause immune rejection.However,their application is limited by the lack of source and the inconvenience in obtaining process.The umbilical cord derived mesenchymal stem cells(UC-MSCs)are the mesenchymal stem cells isolated from the umbilical cord of the newborn.Because of the strong proliferative ability,convenient collection and no ethical problems in their application,UC-MSCs are considered to be the most potential gene therapy vehicles in recent years.Interleukin-24(IL-24),also called melanoma differentiationassociated gene-7(MDA-7),is a kind of immunoregulatory cytokine and belongs to IL-10 family.IL-24 has an apoptotic effect on various kinds of tumor cells,including pancreatic cancer,lung cancer,ovarian cancer and prostate cancer,but has no effect on normal cells.Therefore,IL-24-based strategies have been considered as promising therapeutic options for tumor gene therapy.However,its clinical application was limited by some factors,including the short half-life and serious side effects etc.In this study,the combination of the tropism of UC-MSCs that transduced with lentiviral vectors carrying IL-24 complementary DNA with the antitumor effect of IL-24 was used to explore the therapeutic effect and the potential mechanism of UC-MSCs overexpressing IL-24 on gliomas in vitro and in vivo.Methods(1)The lentivirus vector system including human GFP c DNA or IL-24 c DNA was transfected into UC-MSCs to construct the control UC-MSCs(GFP-UC-MSCs)or IL-24 expressing UC-MSCs(IL-24-UC-MSCs).To determine whether the gene modified UC-MSCs still maintain the characteristics of UC-MSCs,morphology of UC-MSCs,GFP-UC-MSCs and IL-24-UC-MSCs was observed under inverted microscope;the expression of CD29,CD105,CD34,CD45 on the cell surface of UC-MSCs,GFP-UC-MSCs and IL-24-UC-MSCs were identified by flow cytometry;multi-differentiation potentials of UC-MSCs,GFP-UC-MSCs and IL-24-UC-MSCs were tested by osteogenic and adipogenic differentiation.(2)Western blot was used to explore the expression of IL-24 in the whole lysate of UC-MSCs,GFP-UC-MSCs and IL-24-UC-MSCs;ELISA was used to explore the expression of IL-24 in the culture medium of IL-24-UC-MSCs that at different time points.(3)The Transwell migration assay was conducted to detect the tropism of GFP-UC-MSCs or IL-24-UC-MSCs for glioma cells in vitro.(4)CCK-8 assay was used to assess the viability of U251 cells treated with the culture medium of GFP-UC-MSCs and IL-24-UC-MSCs for 48 h.(5)The TUNEL assay was used to observe the apoptosis of U251 cells cocultrued with GFP-UC-MSCs and IL-24-UC-MSCs for 72 h.(6)U251 cells were injected subcutaneously into the left flank near the axillary fossa of Balb/c nude mice to establish the xenografted tumor model.GFP-UC-MSCs or IL-24-UC-MSCs were transplanted into the mice through tail vein to observe the tropism of GFP-UC-MSCs or IL-24-UC-MSCs for glioma cells and the therapeutic effect of these cells on gliomas in vivo.(7)The TUNEL assay was used to observe the apoptosis of glioma cells in vivo;immunohistochemistry(IHC)was used to assess the expression of p-Akt and Bcl-2 to explore the mechanism.Results(1)UC-MSCs,GFP-UC-MSCs and IL-24-UC-MSCs showed a flat spindle-shaped morphology and arranged in cluster or vortex shape,with clear cell membrane,abundant cytoplasm,large nucleus and clear nucleolus;the UC-MSCs,GFP-UC-MSCs and IL-24-UC-MSCs showed the similar expression of CD29,CD105,CD34,CD45;the UC-MSCs,GFP-UC-MSCs and IL-24-UC-MSCs maintained the similar potential to differentiate into adipogenic and osteogenic cells.(2)Western blot showed that IL-24 can express stably in the IL-24-UC-MSCs;ELISA showed that the expression level of IL-24 increased with the extension of transfection time,and reached the peak72 h after transfection(3345.4±211.837 pg/ml).(3)The Transwell migration assay showed that GFP-UC-MSCs and IL-24-UC-MSCs were significantly attracted to the U251 cells and their culture medium.(4)CCK-8 assay showed that the viability of U251 cells in control,GFP-UC-MSCs and IL-24-UC-MSCs groups are 0.82±0.06?0.68±0.04?0.37±0.05 respectively;the viability of U251 cells in IL-24-UC-MSCs group is significantly lower than that in control and GFP-UC-MSCs groups(P<0.05).(5)The TUNEL assay showed that the proportion of apoptotic cells in control,GFP-UC-MSCs and IL-24-UC-MSCs groups are 4.57±0.42?6.00±0.60?13.40±0.91 respectively;the proportion of apoptotic cells in IL-24-UC-MSCs group is significantly higher than that in control and GFP-UC-MSCs groups(P<0.05).(6)Antitumor experiments in vivo showed that GFP-UC-MSCs and IL-24-UC-MSCs could migrate to the gliomas and IL-24-UC-MSCs could inhibit the growth of the tumors;the volume of tumor in IL-24-UC-MSCs group was significantly smaller than that in control and GFP-UC-MSCs groups(P<0.05).(7)The TUNEL assay in glioma tissues showed that the apoptotic index(AI)in control,GFP-UC-MSCs and IL-24-UC-MSCs groups are1.47±0.41?2.37±0.33?5.57±0.54 respectively;the AI in IL-24-UC-MSCs group is significantly higher than that in control and GFP-UC-MSCs groups(P<0.05).(8)The IHC showed that the expression of p-Akt and Bcl-2 were significantly lower than that in control and GFP-UC-MSCs groups(P<0.05).Conclusion In this study,the combination of UC-MSCs with IL-24 showed favorable results in the treatment of gliomas,which provides a theoretical basis for targeted gene therapy of glioma by UC-MSCs.(1)IL-24-UC-MSCs that transduced with lentiviral vectors carrying IL-24 complementary DNA could express IL-24 stably and their characteristics were similar to UC-MSCs.(2)IL-24-UC-MSCs could migrate to glioma cells and inhibit their growth in vitro and in vivo.(3)IL-24-UC-MSCs exert an apoptotic effect via the Akt/Bcl-2pathway on glioma cells in vivo. |
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