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

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 microvascularendothelialcells,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-ENG1、pGenesil-ENG2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EndoglinmRNA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-ENG1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-ENG1和pGenesil-ENG2转染至ODMECs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-ENG1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。

Objective： To construct an endoglin siRNA expression vector and observe it＇s silencing and anti-angiogenesis 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, pGenesiI-ENG1 and pGenesiI-ENG2 were transfected into ovarian carcinoma-derived microvascular endothelial cells （ODMECs）＇with Lipofectin. After 48 hours, expression levels of endoglin mRNA was assayed by RT-PCR. MTT assay was used to observe the proliferation of transfected and untransfected control ODMECs. The formation of two-dimensional tubular structures of ODMECs was observed in vitro by light microscopy after transfection of pGenesiI-ENGl. 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 plasmid showed decreased expression of the endoglin mRNA and significant suppression of cell growth （P〈0.01）. Transfected ODMECs also showed significant reduction in the in vitro two-dimensional formation of vessel-like structures （P〈0.01）. Conclusions： The endoglin siRNA expression vector has been successfully constructed. It effectively downregulated the expression of endoglin in ODMECs, thereby inhibiting proliferation and two-dimensional lumen-like structure formation.