Construction of mouse VCAM-1 expression vector and establishment of stably transfected MSC line C3H10T1/2.
10.7534/j.issn.1009-2137.2014.05.041
- Author:
Hui CHEN
1
,
2
,
3
;
Heng ZHU
4
;
Ya-Nan CHU
5
;
Fen-Fen XU
5
;
Yuan-Lin LIU
4
;
Bo TANG
5
;
Xi-Mei LI
5
;
Liang-Ding HU
1
;
Yi ZHANG
6
Author Information
1. The Third Xiangya Hospital, Changsha 410013, Hunan Province, China
2. Department of Cell Biology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China
3. Affiliated Hospital of Academy of Military Medical Sciences,Beijing 100071,China.
4. Department of Cell Biology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
5. Department of Cell Biology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China
6. Department of Cell Biology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China. E-mail: zhangyi612@hotmail.com.
- Publication Type:Journal Article
- MeSH:
Animals;
Cell Line;
DNA, Complementary;
Genetic Vectors;
Mesenchymal Stromal Cells;
metabolism;
Mice;
Retroviridae;
Reverse Transcriptase Polymerase Chain Reaction;
Transfection;
Vascular Cell Adhesion Molecule-1;
genetics
- From:
Journal of Experimental Hematology
2014;22(5):1396-1401
- CountryChina
- Language:Chinese
-
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.
