| Objective : To observe the regulatory rule of proliferation, differentiation and angiogenesis of vascular endothelial progenitor cells(EPCs) by the inhibition and overexpression of interferon inducible protein 204(Ifi204).Method: Using density gradient centrifugation to get bone marrow mononuclear cells from C57BL/6 mice, EPCs were finally selected from EBM-2 culture medium. EPCs were identified through DAPI staining and DiI-Ac-LDL endocytosis and flow cytometry. The third day’s to 14 th day’s EPCs total RNAs were extracted to reverse transcription-quantitative polymerase chain reaction(RT-qPCR). It turned out that on the 7th day EPCs expressed the highest of gene ifi204 for transfection and transduction. To obtain low expression of ifi204 and overexpression of ifi204 of EPCs, on the 7th day we performed siRNA transfection and Lenti-Virus transduction, respectively, and verified the effect by RT-qPCR. Three groups of cells, including the normal EPCs group, overexpression of ifi204 EPCs group and low expression of ifi204 EPCs group, were used to extract total RNA expression. Then we examined the expression level of EPCs’ specific molecular markers(including CD31, VE-Cadherin, vWF, etc.) by RT-qPCR, flow cytometry and immunofluorescence staining. Three groups of cells were carried out angiogenesis experiment and colony formation assay to verify EPCs’ ability of angiogenesis and proliferation. Finally, in vivo experiments, the C57BL/6 mouse hindlimb ischemic models were established, three groups of EPCs, normal group and siRNA group cells treated with TPE-11(a newly synthesized nanofluorogen, a bolaamphiphile with a tetraphenylethene unit attached and two pyridiniumsaltterminated alkyl groups) marker, lenti-virus group carried GFP, were injected into local ischemic area, and then laser Doppler was used to detect the blood flow of the models’ ischemic hindlimb on the operation day and the 14 th day after operation. Then tissue sections treated with immunofluorescence staining were subjected to observe angiogenesis of the ischemic region and survival amount of transplanted cells.Results: The results of DAPI staining, Di I-Ac-LDL endocytosis, qPCR and flow cytometry showed that the cells obtained from EBM-2 culture medium were in accordance with the characteristics of endothelial progenitor cells. Fluorescence microscopy showed that the transfection efficiency of siRNA and the transduction efficiency of Lenti-Virus were above 90%. RT-q PCR prompted that the expression of gene ifi204 inhibited by specific siRNA was decreased more than 50%, and increased more than 100% by lenti-virus. Flow cytometry and immunofluorescence staining showed that the expression of EPCs’ specific molecular markers(including CD31, VE-Cadherin, vWF, etc.) was significantly decreased in siRNA group, and was increased in lenti-virus group. In the function experiment, the angiogenesis of siRNA group was significantly lower than that of the normal group, and the lenti-virus group was higher than the normal group. The proliferation ability of siRNA group was significantly lower than that of the normal group, and the lenti-virus group was higher than the normal group. C57BL/6 mouse hindlimb ischemic models established successfully, the laser Doppler was used to measure hindlimb ischemia situation on the operation day and the 14 th day. The results displayed that the blood flow was obstructed completely, and the ifi204 overexpression EPCs treatment group had a higher blood flow recovery than the normal EPCs group, and the hindlimb blood flow recovery of low expression ifi204 EPCs treatment group was significantly lower than that of normal EPC group. Immunofluorescence staining of frozen sections showed that the survival rate of transplanted cells in mice treated with Ifi204 overexpression EPCs was higher than that in the normal EPCs group, and the survival rate of transplanted cells in EPCs treated mice with low expression of Ifi204 was lower than that in the normal EPCs group.Conclusion: Interferon induced protein Ifi204 has the ability to regulate EPCs proliferation, differentiation and angiogenesis. And reducing Ifi204 expression can significantly inhibit EPCs proliferation, differentiation, angiogenesis ability. In vivo experiments confirmed that increasing expression of Ifi204 can enhance the capacity of EPCs to treat ischemic diseases. |
