[Construction of mouse VCAM-1 expression vector and establishment of stably transfected MSC line C3H10T1/2].

ABSTRACT

This study was aimed to construct the mouse VCAM-1 expression vector, to establish the stably transfected MSC line and to investigate the effect of VCAM-1-modified mesenchymal stem cells (MSC) on the immunological characteristics of MSC. The cDNA of murine VCAM-1 gene was amplified by RT-PCR from the total RNA isolated from the mouse spleen; then the cDNA was inserted into the retrovirus vector PMSCVmigr-1; the recombinant plasmid was confirmed by restriction endonuclease experiments and sequencing, then designated as PMSCVmigr-1-mVCAM-1; the recombinant plasmid PMSCVmigr-1-mVCAM-1 was transfected into 293 cells by lipofecamin and the supernatant was collected to transfect MSC cell line (C3H10T1/2). Moreover, VCAM-1 expression on MSC was evaluated by FACS. Furthermore, the inhibitory effect of VCAM-1-MSC on lymphocytic transformation was tested by (3)H-TdR incorporation assay. The results indicated that the successful construction of recombinant retroviral expression plasmid of mouse VCAM-1 was confirmed by digesting and sequancing. After transfection of MSC with retroviral supernaptant, the high expression of VCAM-1 on MSC could be detected by flow cytometry. The MSC high expressing VCAM-1 could significantly inhibit the proliferation of Con A-inducing lymphocytes in dose-depentent marrer. It is concluded that recombinant retroviral encoding VCAM-1 (PMSCVmigr-1-mVCAM-1) has been successfully constructed and mouse VCAM-1 has been stably expressed in C3H10T1/2. MSC over-expressing VCAM-1 show more potent immunosuppressive effect on cellular immune reaction in vitro. Our data laid a foundation for the subsequent studying the effect of VCAM-1 transfecting into MSC on immune related disease study.