徐 燕, 王 丹, 朱丽芳, 曾 卫, 谢 尧, 梁志清. Endoglin基因沉默对卵巢癌微血管内皮细胞干扰效应的研究*[J]. 中国肿瘤临床, 2010, 37(10): 541-545. DOI: 10.3969/j.issn.1000-8179.2010.10.001
引用本文: 徐 燕, 王 丹, 朱丽芳, 曾 卫, 谢 尧, 梁志清. Endoglin基因沉默对卵巢癌微血管内皮细胞干扰效应的研究*[J]. 中国肿瘤临床, 2010, 37(10): 541-545. DOI: 10.3969/j.issn.1000-8179.2010.10.001
XU Yan, WANG Dan, ZHU Lifang, ZENG Wei, XIE Yao, LIANG Zhiqing. The Effect of RNAi-mediated Endoglin Gene Silencing in Endothelial Cells of Ovarian Carcinoma[J]. CHINESE JOURNAL OF CLINICAL ONCOLOGY, 2010, 37(10): 541-545. DOI: 10.3969/j.issn.1000-8179.2010.10.001
Citation: XU Yan, WANG Dan, ZHU Lifang, ZENG Wei, XIE Yao, LIANG Zhiqing. The Effect of RNAi-mediated Endoglin Gene Silencing in Endothelial Cells of Ovarian Carcinoma[J]. CHINESE JOURNAL OF CLINICAL ONCOLOGY, 2010, 37(10): 541-545. DOI: 10.3969/j.issn.1000-8179.2010.10.001

Endoglin基因沉默对卵巢癌微血管内皮细胞干扰效应的研究*

The Effect of RNAi-mediated Endoglin Gene Silencing in Endothelial Cells of Ovarian Carcinoma

  • 摘要: 目的:构建Endoglin 基因siRNA的表达载体,转染至原代培养的卵巢癌微血管内皮细胞(ovarian carcinoma-derived microvascular endothelial cells,ODMECs),并观察其对ODMECs 的干扰效应,为探讨使用siRNA 抗卵巢癌血管生成的可行性提供实验基础。方法:根据siRNA 表达载体的设计原则,设计并合成两个靶基因序列,寡核苷酸退火后插入到pGenesil-1 载体中,并对重组质粒进行测序鉴定,证实Endoglin 真核表达载体构建成功,插入片段测序结果与合成的寡核苷酸序列一致。将重组真核表达质粒pGenesil-ENG 1、pGenesil-ENG 2 采用脂质体法转染原代ODMECs 细胞,应用半定量RT-PCR 观察转染后48h 重组质粒对Endoglin mRNA 表达的影响;MTT 法检测转染后ODMECs 细胞增殖的变化;并且通过光镜观察转染pGenesil-ENG 1 后,对ODMECs 体外二维管腔样结构形成的影响。结果:将构建成功的Endoglin 基因siRNA 表达载体pGenesil-ENG 1 和pGenesil-ENG 2 转染至OD?MECs细胞,48h 后同阴性质粒pGenesil-H 和未转染组相比,二者均可引起靶基因表达水平的显著性下降。转染了pGenesil-ENG质粒的ODMECs ,其生长较未转染组和阴性质粒pGenesil-H 明显受到抑制(P<0.01);此外,转染了pGenesil-ENG 1 质粒的ODMECs体外二维血管腔样结构的形成较对照组亦有明显减少(P<0.01)。 结论:成功构建了Endoglin 基因siRNA 表达载体,转染至ODMECs 后可下调Endoglin 在靶细胞中的表达,并且前者能够抑制ODMECs 的增殖和体外二维管腔样结构的形成。为进一步研究采用RAN干扰技术进行卵巢癌血管生成的治疗奠定了基础,同时也为抗卵巢癌血管生成的治疗摸索了有效的基因靶点。

     

    Abstract: Objective:To construct an endoglin siRNA expression vector and observe it's silencing and anti-angiogene -sis effects on endothelial cells of ovarian carcinoma. Methods:According to the endoglin cDNA sequence in GenBank, two target sequences were designed and synthesized. After annealing, oligonucleotides were inserted into pGenesil- 1 vector. The recombinant plasmid was identified by DNA sequencing. pGenesil-ENG 1 and pGenesil-ENG2 were transfected into ovarian carcinoma-derived microvascular endothelial cells (ODMECs) with Lipofectin. After 48hours, expression levels of endoglin mRNA was assayed by RT-PCR. MTT assay was used to observe the proliferation of transfected and untransfect-ed control ODMECs. The formation of two dimensional tubular structures of ODMECs was observed in vitro by light micros-copy after transfection of pGenesil-ENG1. Results: Sequence analysis of the inserted fragment validated the successful construction of the eukaryotic expression plasmid of endoglin. After 48h, ODMECs transfected with the recombinant plas -mid showed decreased expression of the endoglin mRNA and significant suppression of cell growth (P<0.01). Transfected ODMECs also showed significant reduction in the invitro two-dimensional formation of vessel-like structures (P<0.01). Conclusions: The endoglin siRNA expression vector has been successfully constructed. It effectively downregulated the expres-sion of endoglin in ODMECs, thereby inhibiting proliferation and two-dimensional lumen-like structure formation.

     

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