[Self-induction of rabbit marrow stromal stem cells into chondrocytes by transfection with reconstructed PGL3-transforming growth factor beta1 gene in vitro].
OBJECTIVE
To explore an experimental method of transfecting the marrow stromal stem cells (MSCs) with the reconstructed PGL3-transforming growth factor-beta1 (TGF-beta1) gene and to evaluate the feasibility of self-induction of MSCs to the chondrocytes in vitro so as to provide a scientific and experimental basis for a further "gene enhanced tissue engineering" research.


METHODS
The rabbit MSCs was transfected with the reconstructed PGL3-TGF-beta1, gene by the Liposomes Method, the growth of the cells were observed, and the growth curve was drawn. The living activity of the transfected cells in the experimental group was evaluated by MTT, and the result was significantly different when compared with that in the control group. By the immunohistochemistry method (SABC), the antigens of TGF-beta1 and collagen II were examined at 2 and 7 days of the cell culture after transfection with PGL3-TGF-beta1 gene. The pictures of the immunohistochemistry slice were analyzed with the analysis instrument, and the statistical analysis was performed with the software of the SPSS 11.0, compared with the control group and the blank group.


RESULTS
Transfection of the cultured rabbit MSCs in vitro with the reconstructed PGL3-TGF-beta1 gene by the Liposomes Method achieved a success, with a detection of the Luceraferase activity. The result was significantly different from that in the control group (P < 0.01). Tested by MTT, the living activity of the transfected cells was proved to be significantly decreased (P < 0.01 vs. the control group). By the immunohistochemistry method (SABC) to study TGF beta1, positive particles were detected in the experimental group, but there were no positive particles in the control and the blank groups. There was a significant difference between the two groups of the experiment and the control group based on the analysis of the t-test (P < 0.01). By the immunohistochemistry method (SABC) to study collagen II, there were more positive particles in the transfected cells in the experimental group than in the control and the blank groups, and there was a significant difference between the experimental group and the two other groups based on the t-test (P < 0.01).


CONCLUSIONS
Transfection of the rabbit MSCs with the reconstructed PGL3-TGF-beta1 gene by the Liposomes Method is successful. There may be some damage to the cells when transfection is performed. The transfected BMS cells with PGL3-TGF-beta1 gene can express and excrete TGF-beta1 when cultured in vitro. The transfected MSCs that secret TGF-beta1 can be self-induced into the chondrocytes after being infected for 7 days when cultured in vitro.
Lei
Lei
L
Department of Orthopaedics, 1st Affiliated Hospital, Sun Yat-Sen University, Guangzhou Guangdong 510080, P R China.
Liao
Weiming
W
Sheng
Puyi
P
Huang
Gang
G
Jiang
Li
L
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology*; D059630:Mesenchymal Stem Cells / Q000166:cytology; D011817:Rabbits; D017154:Stromal Cells / Q000166:cytology*; D023822:Tissue Engineering / Q000379:methods*; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000235:genetics*
D053773:Transforming Growth Factor beta1