| Background and objective Stem cell transplantation has made remarkable progress in the research field of multi-system diseases such as central nervous system and cardiovascular system.Stem cells are closely related to tumor cells,such as self-renewal and multi-directional differentiation ability.Stem cells and tumor cells not only have similar metabolic activities and there are many same identical silent genes.Reports of stem cell tumor formation have made the safety of stem cell transplantation questionable,and the transplant site often occurs in sensitive sites such as the spinal cord,brain,and eyes.Even if a small tumor is formed,it will cause severe dysfunction,It is especially important to detect the occurrence of tumors as early as possible.by tracing stem cell.Optical,nuclear medicine and magnetic resonance imaging(MRI)are mainly used in the field of stem cell imaging tracing in vivo.Magnetic resonance imaging is the most potential due to the advantages of three-dimensional multi-sequence imaging and high resolution without radiation.MRI tracer stem cells are commonly used to directly and indirectly method to trace stem cells.Directly method commonly labeling the stem cells with magnetic nanoparticles in advance and apply MRI to detect the magnetically labelled cells.For this method,cell division and reuptake limits their long-term longitudinal display.Indirect method label cells with MRI reporter gene and detect cells longitudinally by the persistent expression of reporter gene in stem cells.Although MRI reporter gene imaging can achieve long-term longitudinal tracing after stem cell transplantation,but it can not distinguish early malignant cells from stem cells.The key to solve this problem is to find a differentially expressed gene before and after stem cell tumorigenesis,which is highly expressed in tumorigenic cells,but not in stem cells and normal differentiated tissue cells.The early detection of stem cell tumorigenesis by reporter gene imaging can be achieved theoretically using the differentially expressed gene promoter to drive the specific expression of the reporter gene in the tumorigenic cells.PEG3 promoter is a tumor-specific gene derived from rodents.In cancer cells,PEG3 promoter activity is regulated by two transcription factors,PEA3 and AP-1.It is highly expressed in human cancer cell lines,but rarely expressed in normal tissue cells.At present,PEG3 promoter has been used in the field of targeted therapy of cancer and the mechanism of tumorigenesis.In vivo imaging of stem cell tumorigenesis using PEG-3 promoter-driven reporter gene has not been reported.In this experiment,we combined PEG3 and MRI reporter gene FTH1 to construct PEG3-FTH1-LV lentiviral vector,transferred PEG3-FTH1 system into mesenchymal stem cells(MSCs),and induced MSCs tumorigenesis.Observing the expression of FTH1 gene,Polyferric and changes of MRI signal before and after tumorigenesis.The aim of this study is explore the feasibility of using a tumor-specific promoter PEG3 to drive the reporter gene FTH1 expression for MRI detection in the malignant transformation of bone marrow mesenchymal stem cells.Methods 1 virus vector PEG3-FTH1-LV construction and virus packaging PEG3 promoter,FTH1 Gene(commissioned Han Heng Biology C o.,Ltd.synthesis)and carrier p HBLV-CMV-MCS-3flag-EF1-ZSgreen-pu ro through enzyme cutting,amplification and connection to obtain p HB LV-PEG3-FTH1-3flag-EF1-ZSgreen-puro,sequencing and identification.After successful construction,293 T cells were simultaneously infected with the p SPAX2,p MD2 G and the virus was centrifuged.collected and obtain a virus and labeled as PEG3-FTH1-LV.At the same time,CMV-FTH1-LV was constructed as a positive control,and MSCs cells not in fected with the virus were used as a negative control group.2 MSCs infects with lentivirus and induces tumorigenesis When the cell fusion degree of MSCs reached 30%-40%,the culture containing PEG3-FTH1-LV,CMV-FTH1-LV virus was added respectively,the expression of green fluorescent protein was observed under fluorescence microscope after 48 h infected.The MSCs-PEG3-FTH1,MSCs-CMV-FTH1 stable expression cell line were constructed by Purine Mycin.MSCs,MSCs-PEG3-FTH1 and MSCs-CMV-FTH1 cells were transformed into TMSCs,TMSCs-PEG3-FTH1,TMSCs-CMV-FTH1 by indirect co-culture with rat glioma cells C6 for 2 weeks respectively.3 Identification of cells before and after induction The morphology of the cells before and after co-culture was observed.The expression of cell surface antibodies CD29,CD34,CD45 and CD90 were detected by flow cytometry.The cell migration ability was detected by crystal violet staining.The cell proliferation rate before and after co-culture was detected by CCK-8.Nude mouse subcutaneous tumor test were performed to verify the tumorigenesis of MSCs,and the tumors formed after transplantation were stained with HE to observe the pathological condition.4 FTH1 expression and polyferric effect before and after cell induction Western blot was used to detect the expression of FTH1 in the cells before and after tumor transformation.The cells were cultured 48 h in FAC medium,and the T2 WI signal was observed by 3.0T MRI,Prussian blue staining and cell transmission electron microscopy showed the aggregation of iron before and after cell tumor transformation.5 Iron concentration detection in graft of cells after induction 4-6W,20 g healthy male nude mice were selected,and the cells after induction were subcutaneously implanted in the neck of nude mice,and fed with 500 mmol/L FAC solution every day.The 3.0T MR was used to observe the tumor mass signal on the 1st,3d,7d,14 d,21d,28 d,35d after cell implantation.Prussian blue staining and transmission electron microscopy were used to observe the iron particles in the tumor and the pathological structure of tumor was observed by HE staining.Results 1 Virus vector PEG3-FTH1-LV construction and virus packaging After PEG3-FTH1-LV lentiviral vector was successfully constructed by DNA sequencing,PCR amplification,agarose gel electrophoresis.the virus was packaged,collected and detected to have a titer of 1×108 TU/ml.2 Establishment of stable strains After the cells were infected with lentivirus for 48 hours,the expression of green fluorescent cells was observed.After screening for purinemycin,the MSCs-PEG3-FTH1 and MSCs-CMV-FTH1 stable strains were successfully constructed.3 Identification of cells before and after induction The cells of MSCs were arranged neatly and fish-like growth and the TMSCs cells arranged disorderly and smaller than MSCs cells,the nucleoplasmic ratio larger than MSCs cells.The proliferation of TMSCs cells accelerated by CCK-8 was faster than that of MSCs.The results of crystal violet staining showed that the migration ability of TMSCs cells was stronger than that of MSCs cells.The results of flow cytometry showed that the expression of CD34 and CD45 was decreased and the expression of CD90 was decreased in TMSCs cells compared with MSCs.The MSCs cells was not formed tumor in nude mice but TMSCs cells were positive for subcutaneous tumor formation in nude mice.4 FTH1 expression and polyferric effect before and after cell induction Western blot analysis showed that MSCs,MSCs-PEG3-FTH1,TMSCs cells did not express or expressed FTH1 protein in a small amount,but MSCs-CMV-FTH1,TMSCs-PEG3-FTH1 and TMSCs-CMV-FTH1 cells were abundantly expressed,and MRI showed the T2 WI signal of TMSCs-PEG3-FTH1 cells was significantly lower than that of MSCs-PEG3-FTH1 cells.Iron particles in MSCs-CMV-FTH1,TMSCs-PEG3-FTH1 and TMSCs-CMV-FTH1 cells were observed by Prussian blue staining and cell transmission electron microscopy,but no iron particles were found in MSCs,MSCs-PEG3-FTH1 and TMSCs cells.5 Iron detect of grafts MRI showed that the signal of TMSCs-PEG3-FTH1 cell grafts was similar to the signal TMSCs-CMV-FTH1 cell grafts and was significantly lower than the signal of grafts TMSCs cell.Obvious iron particles were observed by prussian blue staining and cell transmission electron microscopy in TMSCs-PEG3-FTH1 and TMSCs-CMV-FTH1 cell grafts but not observed in TMSCs cell grafts.Conclusion The tumor-specific promoter PEG3 can trigger FTH1 expression,allowing for MRI detection in the malignant transformation of mesenchymal stem cells. |
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