体外诱导负载TGF-β1基因的兔关节滑膜细胞向软骨细胞分化

Construction of eucaryotic expression plasmid carrying the recombinant rabbit TGF β1 gene and serf-induction of rabbit articular synoviocytes into chondrocytes in vitro

目的探讨体外诱导负载TGF-β1基因的兔关节滑膜细胞向透明软骨细胞分化的可行性，为组织工程学修复关节软骨损伤提供体外实验依据。方法体外培养扩增B型滑膜细胞并纯化，构建pcDNA3．1-TGF-β1，用Lipofectamine 2000法转染，转染pcDNA3．1-TGF-β1组为实验组，转染pcDNA3．1（＋）空质粒组为对照组，未转染组为空白组。转染后48h行G418筛选，RT—PCR检测TGF-β1的瞬时表达，同时取阳性细胞爬片，行抗TGF-β1免疫组织化学染色；G418维持筛选，每2d取部分细胞计数，连续12d，绘制生长曲线，转染后3周，每周取部分细胞爬片行抗TGF-β1和抗Ⅱ型胶原免疫组织化学染色。图像分析采用Image—Pro Plus V6．0软件分析处理，实验数据采用SPSS 13．0行统计学处理，组间两两比较采用方差分析。结果实验组转染后转染率为18％，转染后生长曲线显示细胞早期生长活性下降，细胞数量在转染后第4天降低到3．6×10^4／ml，第3天RT—PCR检测到TGF-β1阳性片段，抗TGF-β1免疫组织化学染色见细胞内阳性颗粒，对照组及空白组均为阴性；细胞转染后第3周，实验组抗TGF-β1及抗Ⅱ型胶原免疫组织化学染色仍见细胞内阳性颗粒，对照组及空白组为阴性；图像分析结果表明，转染后第7天实验组抗TGF-β1的PU值为（19．04±1．26），对照组及空白组PU值分别为（4．07±0．65）和（3．23±0．56），差异有统计学意义（P〈0．05）；转染后14d实验组抗Ⅱ型胶原的PU值为（13．74±1．27），对照组及空白组PU值分别为（4．62±0．56）和（3．93±0．38），差异有统计学意义（P〈0．05）。结论脂质体法重组pcDNA3．1-TGF-β1基因可成功转染兔关节滑膜细胞，转染后细胞成功表达TGF-β1，并分泌Ⅱ型胶原蛋白，表现出透明软骨细胞样生理特征，滑膜细胞有望成为一种良好的软骨组织工程的种子细胞。

Objective To investigate the experimental methods of transfecting 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 3.6×10^4/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.