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目的 探讨电穿孔法将ptsA5 8H质粒体外转染人胚肌腱细胞 (HETC)的条件 ,转化人胚肌腱细胞 (THETC)体外培养的生长特性。方法 体外分离培养HETC ,电穿孔法导入 ptsA5 8H质粒 ,并经潮霉素B筛选 ,获得阳性克隆 ,扩增后建立THETC系。体外传代培养、冻存、复苏THETC ,并分别绘制HETC和THETC在不同培养条件的生长曲线。结果 通过潮霉素B 0～ 10 0 0mg/L终浓度梯度的培养 ,选择 2 0 0mg/L作为筛选浓度。电穿孔法击穿电压选择 96 0 μF、40 0V。THETC增殖能力强 ,可长期连续传代 ,冻存、复苏不改变其生长特性 ,体外培养控制温度可改变其生长特性 ,且其生长存在血清营养依赖性。结论 将 ptsA5 8H质粒转入人胚肌腱细胞可使肌腱细胞在体外长期连续传代 ,为组织工程化肌腱的研究提供标准细胞。
Objective To investigate the in vitro transfection of ptsA5 8H plasmid into human embryonic tenocytes (HETC) by electroporation and to investigate the growth characteristics of human embryonic tenocytes (THETC) cultured in vitro. Methods HETC was isolated and cultured in vitro. The ptsA5 8H plasmid was induced by electroporation. After screening by hygromycin B, the positive clones were obtained and THETC lines were established after amplification. In vitro culture, cryopreservation, resuscitation THETC, and were drawn HETC and THETC growth curve in different culture conditions. Results The final concentration of 0 ~ 100 mg / L hygromycin B was used for culture, and 200 mg / L was selected as the screening concentration. Electroporation breakdown voltage selection 96 0 μF, 40 0V. THETC has strong proliferative ability and can be continuously passaged, frozen and resuscitated for a long time without changing its growth characteristics. The growth temperature can be changed by controlling the temperature in vitro, and its growth has serum nutrition dependence. Conclusion Transfection of ptsA5 8H plasmid into human embryo tendon cells can give long-term continuous passage of tendon cells in vitro and provide standard cells for the study of tissue-engineered tendons.