Chinese Journal of Tissue Engineering Research ›› 2014, Vol. 18 ›› Issue (29): 4636-4641.doi: 10.3969/j.issn.2095-4344.2014.29.008
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Shao Jia-jia1, Yu Yin2, Jiang Tao3
Revised:
2014-06-26
Online:
2014-07-09
Published:
2014-07-09
Contact:
Yu Yin, M.D., Attending physician, Department of Neurosurgery, the Third Affiliated Hospital (China-Japan Union Hospital of Jilin University), Changchun 130033, Jilin Province, China
About author:
Shao Jia-jia, Master, School of Food Production Technology and Biotechnology, Changchun Vocational Institute of Technology, Changchun 130033, Jilin Province, China
CLC Number:
Shao Jia-jia, Yu Yin, Jiang Tao. Construction of pcDNA3-Endo eukaryon expression plasmid and angiogenesis inhibition in vitro[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(29): 4636-4641.
2.1 RT-PCR产物的琼脂糖凝胶电泳 电泳结果显示在680 bp附近出现条带,与已知目的基因大小相符(图1A)。 2.2 重组质粒pcDNA3-Endo的酶切鉴定 结果显示在 5 400 bp及680 bp附近各出现一个条带(图1B),与单体pcDNA3和内皮抑素大小相符,说明内皮抑素基因正确插入pcDNA3质粒中,pcDNA3-Endo重组质粒构建成功。 2.3 RT-PCR结果 在680 bp附件出现条带,与内皮抑素基因大小相符(图2A),说明pcDNA3-Endo质粒转染骨髓间充质干细胞后内皮抑素基因能被有效转录。 2.4 Western Blot结果 经显色在Mr 20 000处出现条带,与单体内皮抑素的相对分子质量相符,而转染空载质粒的细胞收集上清液后检测未见条带出现(图2B),说明转染了重组真核表达质粒pcDNA3-Endo的骨髓间充质干细胞能够分泌出内皮抑素蛋白。 2.5 内皮抑素基因对血管内皮细胞增殖的影响 转染了外源pcDNA3-Endo质粒组的ECV-304细胞增殖明显受到抑制,较空载pcDNA3组、脂质体对照组及空白对照组相比差异有显著性意义(P < 0.01,表1,图3)。"
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