Objective      To construct lentiviral vector-medicated RNA interference (RNAi) targeting rat chemokine CX3C ligand 1 (CX3CL1) and investigate its effect on the growth and chemotaxis effect of bone marrow derived mesenchymal stem cells (bMSCs). Methods      bMSCs were isolated from rat bone marrow

cultured and identified with flow cytometry. Three potential RNAi sites were screened based on the mRNA sequence of CX3CL1 gene and then synthesized. The 3 synthesized siRNAs were transferred into bMSCs

Western blotting was used to analyze the inhibitory effect of target gene so as to identify the optimal siRNA sequence. Then the short hair RNA (shRNA) oligonucleotide targeting the optimal siRNA sequence was designed

synthesized and inserted into CD513B-1 plasmid to form CD513B-1/CX3CL1 shRNA lentiviral vector. After identified by sequencing

CD513B-1/CX3CL1 shRNA plasmid was transfected into the 293T cells to produce lentiviral particles. The bMSCs were transfected with the lentiviral particles. After rat splenic macrophages were isolated and cultured

and the cells were cocultured with the transfected bMSCs. The expression of green fluorescent protein (GFP) in the transfected bMSCs was observed by inverted microscopy. Cell count kit-8 (CCK-8) assay was employed to test the growth of transfected bMSCs. The expression of CX3CL1 and proliferating cell nuclear antigen (PCNA) in the transfected bMSCs was detected by real-time PCR. The levels of the proteins in the transfected bMSCs and those of M-CSF and IL-8 in the splenic macrophages were detected by Western blotting respectively. Results       Rat bMSCs were successfully isolated from bone marrow

cultured and identified. The optimal siRNA sequence CTCTATGAGCAATTATTTA was confirmed. The result of sequencing revealed that lentiviral vector CD513B-1/CX3CL1 shRNA was constructed successfully. The transfected bMSCs significantly expressed GFP. The results of CCK8 assay showed that the growth of bMSCs were slower after CX3CL1 silencing

especially in 48 h after transfection (P0.01). The results of real-time PCR and Western blotting revealed that the lentiviral vector CD513B-1/CX3CL1 shRNA effectively silenced CX3CL1 and down-regulated the expression of PCNA in the bMSCs. The bMSCs after CX3CL1 silencing down-regulated the protein levels of MCSF and IL-8 in the splenic macrophages co-cultured with the MSCs. Conclusion      The lentiviral vectormedicated RNAi targeting CX3CL1 is constructed successfully

and it can effectively silence CX3CL1 gene

down-regulate PCNA expression，and prevent the growth in bMSCs. The bMSCs with silenced CX3CL1 gene can down-regulate the expression of M-CSF and IL-8 protein in the splenic macrophages co-cultured with the MSCs.