Construction of eucaryotic expression plasmid carrying the recombinant rabbit TGF β1 gene and self-induction of rabbit articular synoviocytes into chondrocytes in vitro / 中华创伤杂志

ABSTRACT

Objective To investigate the experimental methods of transferring the synoviocytes with the reconstructed pcDNA3.1-TGF-β1 gene by the liposomes and study the feasibility of self-induction of synoviocytes to the chondrocytes in vitro so as to provide a scientific and experimental basis for the further gene enhanced tissue engineering research in articular cartilage repair.Methods Synoviocytes were cultivate in vitro and purified to construct the eucaryotic expression plasmid carrying the recombinant rabbit TGF-β1 gene.By means of Lipofectamine 2000,the synoviocytes were transfected with pcDNA3.1-TGF-β1 (experimental group) and with pcDNA3.1 ( + ) blank plasmid (control group).The synoviocytes free from transfection was set as blank group.After 48 hours of transfection,the cells were screened by G418.Representative sections from among the positive clones were used for RT-PCR assay of instant expression of TGF-β1.The other sections were used for immunohistochemical analysis with antibodies to TGF β1.Screening of cells by G418 was continued for 12 days of cell counting and drawing the growth curve.The antigens of TGF-β1 and collagen Ⅱ were examined every week three weeks after transfection.All images were processed by using analysis instrument (Image-Pro Plus V6.0).Statistical analysis was conducted with SPSS 13.0 software package.The difference between groups was tested by using variance ( ANOVA) analysis.Results The transfection efficiency in experimental group was 18% ,with temporary decreased living activity of the transfected cells shown by growth curve.The cell population decreased to 3.6 × 104/ml four days after transfection.After 72 hours of transfection,the positive fragment of TGF-β1 was detected by RT-PCR assay,and immunohistochemical staining of antibiotics to TGF-β1 showed positive particles only in the experimental group.After three weeks of tranfection,the immunohistochemical analysis with antibodies to TGF-β1 and type Ⅱ collagen showed that there were positive particles in the transfected cells in the experimental group,with no positive particle in the control and blank groups.According to the results of Image-Pro Plus V6.0,PU value of anti-TGF-β1 was (19.04±1.26) seven days after transfection,while that of control and blank groups were (4.07 ± 0.65)and (3.23 ±0.56) respectively,with statistical difference (P<0.05).PU value of anti-type Ⅱ collagen in experimental group,control group and blank group was (13.74 ± 1.27),(4.62 ±0.56) and (3.93 ± 0.38) 14 days after transfection,with statistical difference ( P < 0.05).Conclusions Transfection of the rabbit articular synoviocytes with the reconstructed pcDNA3.1-TGF-β1 gene can be successfully accomplished by using Lipofectamine 2000 method.The transfected synoviocytes can express TGF-β1 and excrete type Ⅱ collagen,have chondrogenic potential when transfected by pcDNA3.1-TGF-β1 gene and can be used as candidate cells for repair of articular cartilage.