title: [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.
authors.affiliation: The Third Xiangya Hospital, Changsha 410013, Hunan Province, China; Department of Cell Biology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China; Affiliated Hospital of Academy of Military Medical Sciences,Beijing 100071,China.
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Cell Biology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
authors.forename: Heng
authors.initials: H
authors.lastname: Zhu
authors.affiliation: Department of Cell Biology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China; Department of Pediatrics, General Hospital of Tianjin Medical University, Tianjin 300052, China.
authors.forename: Ya-Nan
authors.initials: YN
authors.lastname: Chu
authors.affiliation: Department of Cell Biology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China; Department of Pediatrics, General Hospital of Tianjin Medical University, Tianjin 300052, China.
authors.forename: Fen-Fen
authors.initials: FF
authors.lastname: Xu
authors.affiliation: Department of Cell Biology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
authors.forename: Yuan-Lin
authors.initials: YL
authors.lastname: Liu
authors.affiliation: Department of Cell Biology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China; Training Base for Postgraduates, Shenyang Military Command General Hospital and Liaoning Medical College, Shenyang 110016, Liaoning Province, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Tang
authors.affiliation: Department of Cell Biology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China; Training Base for Postgraduates, Shenyang Military Command General Hospital and Liaoning Medical College, Shenyang 110016, Liaoning Province, China.
authors.forename: Xi-Mei
authors.initials: XM
authors.lastname: Li
authors.affiliation: The Third Xiangya Hospital, Changsha 410013, Hunan Province, China; Affiliated Hospital of Academy of Military Medical Sciences, Beijing 100071, China. E-mail: huliangding@sohu.com.
authors.forename: Liang-Ding
authors.initials: LD
authors.lastname: Hu
authors.affiliation: Department of Cell Biology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China. E-mail: zhangyi612@hotmail.com.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary; D005822:Genetic Vectors; D059630:Mesenchymal Stem Cells / Q000378:metabolism; D051379:Mice; D012190:Retroviridae; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D019010:Vascular Cell Adhesion Molecule-1