| Neuroblastoma,arising from primordial neural crest cells,is the most common extracranial solid tumors in infants and children and account for approximately 10% of all childhood tumors.It still is one of the most difficult childhood tumors to cure with high malignant degree and the fast progression as most cases have been found for stage III and IV.Currently,more advanced treatment measures,such as the radiation,chemotherapy before surgery,and hematopoietic stem cell transplantation,etc,have carried out,but the cure rate of neuroblastoma does not obvious enhance.Two-year survival rate is only 19% for Stage IV children.It is urgent to search for more effective treatments in neuroblastoma.We have study IL-24 therapeutic effect on neuroblastoma from 2010.The results show that IL-24 can effectively inhibit growth,induce apoptosis and inhibit invasion of neuroblastoma cells.Moreover,our results show that IL-24 recombinant protein can induce the differentiation of neuroblastoma cells.To improve the therapeutic effect of virus for neuroblastoma,we further build a new oncolytic adenovirus ZD55.F35-IL.24.The therapeutic effect of ZD55.F35-IL.24 on NB-CSC will be detected in vivo and vitro.Part ? The construction and amplification of oncolytic adenovirus ZD55.F35-IL.24Objective The plasmid p ZD55.IL-24 and p BHGE3-knob5/35 were transfected into 293 cells for constructing a new oncolytic virus,ZD55.F35-IL.24.Method 293 cells were seeded on a 10 cm dish according to the instructions.The plasmid p ZD55.IL-24 and p BHGE3-knob5/35 were transfected to 293 cells.The viral plaques appeared after 9-14 days.Viruses were purified and amplified to be extracted DNA.The viral DNA extraction was carried out in accordance with the QIAamp DNA Mini Kit instruction manual.The virus was identified by PCR for the insertion of IL-24 and hexon of recombinant virus by design primers.The correct virus was identified to infect HEK293 cell for amplifying.The virus was purified virus by Cs Cl gradient centrifugation.Result We purified and amplified virus plaque and obtained the recombinant adenovirus DNA.The identified virus was amplified in the HEK293 cell,and purified,and storaged at-80 ?.Conclusion The oncolytic adenovirus ZD55.F35-IL.24 was successfully constructed,and further amplified and purified.Part ? Stem cell sorting from neuroblastoma cell line BE(2)-CObjective We induced and purified the CD133+ cells in BE(2)-c cell line by free-serum medium suspension culture and immunomagnetic beads.Method We seeded BE(2)-c cells in containing a lot of growth factors free-serum medium,and obtained the cell spheres containing a large number of neuroblastoma stem cells by suspension culture method.The expression of CD133 and Nestin in the neuroblastoma stem cells were detected by cell immunofluorescence.CD133+ neuroblastoma stem cells was purified by immunomagnetic beads.The positive rate of CD133 expression was detected by flow cytometry.The proliferation activity of CD133+ neuroblastoma stem cells was determined by MTT and cell monoclonal formation rate.Result BE(2)-c cells were seeded in the serum-free medium,and most cells were adhered to the wall within 48 hours.Some cells were in a single cell suspension state,and some of the cells were nonspecific.The cell sphere was formed after 48 hours of culture,suspended in the medium,and then the cell sphere gradually increased and formed a typical stem cell sphere of neuroblastoma.Immunofluorescence assay showed high expression of CD133 and Nestin.CD133+ cells were purified by immunomagnetic beads.The percentage of CD133+ cells was 93.8%.MTT assay showed that CD133+ cells were significantly more proliferative than CD133-cells.CD133+ cell monoclonal formation rate is high,while CD133-cells can hardly form typical neuroblastoma stem cell sphere.Conclusion The cell spheres cultured by the suspension method strongly expressed CD133 and Nestin,possessing stem cell characteristics and strong proliferation.Part ? The effect of oncolytic adenovirus ZD55.F35-IL.24 on the biological characteristics in neuroblastoma stem cellsObjective We studied the effect of oncolytic adenovirus ZD55.F35-IL.24 on the proliferation and differentiation of CD133+ neuroblastoma stem cells.Method To detect the ability of infection and replication of oncolytic adenovirus ZD55.F35-IL.24 in the CD133+ neuroblastoma stem cell,the expression of IL-24 protein was detected by Western blotting.The proliferation activity of CD133+ neuroblastoma stem cells was detected by MTT assay.The expression of Neu N and GFAP were detected by cell morphological observation and western blot.The effects of ZD55.F35-IL 24 on the differentiation of CD133+ neuroblastoma stem cells were studied.Flow cytometry was used to study the effect of ZD55.F35-IL.24 on CD133 expression in CD133+ neuroblastoma stem cells.To observe the tumor growth ability of CD133+ neuroblastoma stem cells in nude mice after infecting of oncolytic adenovirus ZD55.F35-IL.24 by nude mouse transplantation tumor experiment.Result ZD55.F35-IL.24 infected CD133+ neuroblastoma stem cells with high expression of IL-24,which indicated that ZD55.F35-IL.24 can target the CD133+ neuroblastoma stem cells and effectively reproduce IL-24 in cells.MTT assay showed that ZD55.F35-IL.24 could significantly inhibit the proliferation of CD133+ neuroblastoma stem cells.The CD133+ neuroblastoma stem cells infected with ZD55.F35-IL.24 were cultured for 7 days in serum-free medium containing growth factor,and then the cell morphology was varied and the swelling increased.The expression of Neu N and astrocyte marker GFAP was significantly increased.However,the expression of CD133 in the stem cell marker of neuroblastoma was significantly reduced,but it did not disappear completely,indicating that the differentiation of neuroblastoma stem cells was not complete,and the cells could not achieve terminal differentiation.The tumor growth ability of CD133+ neuroblastoma stem cells infected by ZD55.F35-IL.24 was significantly reduced in nude mouse transplantation tumor experiment.Conclusion Oncolytic virus ZD55.F35-IL.24 can target the CD133+ neuroblastoma stem cells,effectively replicate,inhibit the proliferation of CD133+ neuroblastoma stem cells,reduce the expression of CD133 in neuroblastoma stem cells and significantly decrease the tumor formation ability. |
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