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目的构建携带肿瘤转移相关基因原肌球蛋白2(TM2)的绿色荧光蛋白(GFP)真核表达载体,初步探讨其在肿瘤细胞稳定表达后的生物学功能。方法采用逆转录多聚酶链反应(RT- PCR)从人胃癌细胞株AGS中扩增全长的TM2-cDNA序列,采用基因重组技术构建带TM2的绿色荧光蛋白真核表达载体pIRES2-EGFP;通过脂质体转染方法将重组基因转入TM2低表达肝癌细胞株QGY-7703中。荧光显微镜观察及流式细胞仪检测重组pIRES2-EGFP-TM2的荧光表达;Western blot检测细胞转染后TM2的蛋白表达;研究TM2基因与肝癌细胞株QGY-7703体外侵袭、移动及裸鼠成瘤等生物学特性的关系。结果成功构建了携带TM2的绿色荧光蛋白真核表达载体;荧光显微镜观察显示,在转染重组质粒的QGY-7703细胞中均有绿色荧光的表达;流式细胞仪检测显示,QGY-7703- EGFP-TM2的荧光表达率为90%以上;Western blot检测证实,TM2在转染的QGY-7703细胞系中表达明显升高;转染了TM2的细胞株侵袭能力(45.6±9.9)显著高于其亲代细胞(21.6±3.3,P<0.05);转染TM2的细胞株迁移能力(41.4±11.8)显著强于其亲代细胞(16.7±3.7,P<0.05);实验组、空白对照组和载体对照组肿瘤平均体积分别为(241.5±95.1)mm3、(123.7±92.3)mm3和(100.6±85.4)mm3,实验组与空白对照组比较,差异有统计学意义(P<0.01),表明转染TM2能明显促进肿瘤的生长。结论TM2基因对促进肝癌细胞的生长和转移发挥重要作用。
Objective To construct a green fluorescent protein (GFP) eukaryotic expression vector carrying tropomyosin 2 (TM2), a tumor metastasis related gene, and to investigate its biological function after stable expression of tumor cells. Methods The full-length TM2-cDNA sequence of human gastric cancer cell line AGS was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The recombinant plasmid pIRES2-EGFP was constructed by gene recombination technique. The recombinant plasmid was transfected into TM2 low expression hepatoma cell line QGY-7703 by plastid transfection method. Fluorescence microscopy and flow cytometry were used to detect the expression of recombinant pIRES2-EGFP-TM2. The protein expression of TM2 was detected by Western blot. The invasion and migration of TM2 gene and hepatocellular carcinoma cell line QGY-7703 in vitro were studied. Other biological characteristics of the relationship. Results Eukaryotic expression vector carrying green fluorescent protein (TM2) carrying TM2 was constructed successfully. Fluorescence microscopy showed that green fluorescent protein was expressed in QGY-7703 cells transfected with recombinant plasmid. Flow cytometry showed that QGY-7703- EGFP The expression of TM2 was over 90%. The expression of TM2 in QGY-7703 cells transfected with TM2 was significantly higher than that of TM2 (45.6 ± 9.9) (21.6 ± 3.3, P <0.05). The migration ability of TM2 transfected cells (41.4 ± 11.8) was significantly stronger than that of their parental cells (16.7 ± 3.7, P <0.05). The experimental group, blank control group and vehicle control The mean volume of tumor was (241.5 ± 95.1) mm3, (123.7 ± 92.3) mm3 and (100.6 ± 85.4) mm3, respectively. The difference between the experimental group and the blank control group was statistically significant (P <0.01) Can significantly promote tumor growth. Conclusion TM2 gene plays an important role in promoting the growth and metastasis of hepatocellular carcinoma cells.