靶向性重组PF4裸质粒抗血管生成活性的研究

摘要

构建重组真核表达载体pcDNA3.1（+）-PF4^47-70-RGD，并检测靶向性重组血小板因子四（PF4）在真核细胞内的表达水平，探讨其体外抗血管生成活性。设计构建了靶向性重组PF4真核表达载体pcDNA3.1（+）-PF4^47-70-RGD，用脂质体法将其转染至中华仓鼠卵巢细胞系（CHO），用G418筛选稳系，采用RT-PCR和Western blot检测表达产物，MTT法测定CHO稳转体系培养上清对脐静脉内皮细胞（HUVEC）增殖的影响。成功构建出重组真核表达载体pcDNA3.1（+）-PF4^47-70-RGD，并获得稳转CHO细胞系，从基因水平和蛋白水平均可检测到目的基因的表达。MTT法显示，CHO稳系培养上清对HUVEC生长抑制率是50.8%，与对照组比，血管内皮细胞的增殖能力显著减小。靶向性重组PF4具有较强的体外抑制血管生成活性，该重组肽能够抑制HUVEC增殖。

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	王听月
	王喆
	黄鹤

关键词：血小板因子四； RGD肽；抗血管生成；肿瘤治疗；基因治疗

Abstract：

To construct an recombinant eukaryotic expression vector of pcDNA3.1(+)-PF4^47-70-RGD, detect expression of targeting recombinant PF4 in eukaryotic cell and explore its biologic activity in vitro. A targeting recombinant PF4(platelet factor-4) eukaryotic expression vector was designed and constructed by cloning PF4^47-70-RGD cDNA into the pcDNA3.1(+) vector, and was transferred by lipofectamine into CHO cell line. Then G418 was used to select table-transfected CHO cell line with PF4^47-70-RGD gene. The mRNA and protein expression level of recombinant peptides were analyzed via RT-PCR and Western blot. MTT assay was used to observe the growth inhibition effect of expressing products of table-transfected CHO cell line on HUVEC(Human Umbilical Vein Endothelial Cells). The recombinant eukaryotic expression vector of pcDNA3.1(+)-PF4^47-70-RGD is successfully constructed and stabilized expressed CHO cell lines is acquired. Target gene expression is detected both at the genetic and protein level. MTT assay shows that growth inhibitory rate of expressing products of table-transfected CHO cell to HUVEC is 50.8%. Compared with the control group, the growth and tubular formation of HUVEC are significantly inhibited in the experimental group. The targeting recombinant PF4 peptides have antiangiogenic activity, and they inhibit the proliferation of HUVEC in vitro.

Keywords： platelet factor four； RGD peptide； anti-angiogenesis； cancer therapy； gene therapy

Received 2015-05-06;

Fund:

国家自然科学基金面上项目（31470967）。

Corresponding Authors: 黄鹤,E-mail:huang@tju.edu.cn。 Email: huang@tju.edu.cn

About author: 王听月(1988-)女,硕士研究生,现从事生物制药方向研究。

引用本文:

王听月, 王喆, 黄鹤.靶向性重组PF4裸质粒抗血管生成活性的研究[J]. 化学工业与工程, 2017,34(3): 88-94

Wang Tingyue, Wang Zhe, Huang He.Anti-Angiogenic Activity of Naked Plasmid Encoding Targeting Recombinant Platelet Factor 4[J]. Chemcial Industry and Engineering, 2017,34(3): 88-94

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