| Endoglin基因沉默对卵巢癌微血管内皮细胞干扰效应的研究* |
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| 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 |
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| 作者姓名： | 徐燕 王丹 朱丽芳 曾卫 谢尧 梁志清 |
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| 作者单位： | 作者单位:第三军医大学西南医院妇产科(重庆市400038) |
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| 基金项目： | "十一五"全军科技计划青年基金资助 |
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| 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。
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| 关 键 词： | Endoglin ?siRNA ?质粒 内皮细胞 卵巢癌 |
| 收稿时间： | 2009-09-29 |
The Effect of RNAi-mediated Endoglin Gene Silencing in Endothelial Cells of Ovarian Carcinoma |
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| XU Yen,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 |
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| Authors: | XU Yen WANG Dan ZHU Lifang ZENG Wei XIE Yao LIANG Zhiqing |
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| Affiliation: | Department of Obstetrics and Gynecology, The Southwest Hospital of the Third Military Medical University, Chongqing 400038, China |
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| 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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| Keywords: | Endoglin siRNA |
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