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肿瘤基质血管内皮细胞模型的建立及生物学特性研究

Establishment and Identif ication of Tumor Vascular Endothelial Cell Model

  • 摘要: 目的 建立肿瘤基质血管内皮细胞模型并探讨其生物学特性。方法 用人卵巢癌细胞SKOV3培养上清液诱导人内皮细胞ECV304,获得能稳定生长的ECV304-SKOV3细胞。观察其形态改变,检测其生长情况,免疫细胞化学测定其增殖细胞核抗原(PCNA)、bcl-2、基质金属蛋白酶-9(MMP-9)、粘合素(TN)表达变化,逆转录聚合酶链反应(RT-PCR)测定其端粒酶活性,并分析其核型、蛋白合成能力、对常用抗癌药物的反应性。结果 ECV304-SKOV3细胞出现畸形核、有微绒毛的腔隙,染色体众数略有增加。细胞群体倍增时间较亲代细胞BCV304缩短;分裂指数增高;表达PCNA、bcL-2、MMP-9增强(t≥2.4671,P<0.05),而TN减弱(t=2.2473,P<0.05);S期细胞数增加,G0/G1期细胞数减少(x2=923.313,P<0.01),增殖指数(PI)明显增高(x2=570.197,P<0.01)。对作用于S期或S期和M期的抗癌药物较ECV304敏感,且与SKOV3癌细胞一致(F≥24.14,P<0.01)。蛋白质合成能力增强(t=-12.2465,P<0.001)。端粒酶活性强于亲代细胞ECV304(t=-25.2990,P<0.001)。结论 DCV304-SKOV3细胞较亲代细胞DCV304增殖快,核型发生变化,存活能力强,功能活跃,某些血管形成因子表达增强,具有肿瘤基质血管内皮细胞的某些特性,可作为肿瘤基质血管内皮细胞模型用于进一步研究。

     

    Abstract: Objective  To establish the cell model of vascular endothelial cell in tumors and explore its biological features. Methods  The normal human umbilical vein endothelial cell ECV304 was cultured with the supernatant of human ovarian cancer cell SKOV3 for three and half months, and ECV304-SKOV3 cells with stable growth was acquired. Then the changes of their biological characteristics were determined by light microscopy, electron microscopy, karyotype analysis, MTT assay, and flow cytometry. The expression of proliferating cell nuclear antigen ( PCNA), bcl-2, tenascin ( TN), matrix metalloproteinase 9 (MMP-9), and telomerase activity was detected by immunocytochemistry and reverse transcription polymerase chain reaction (RT-PCR) respectively. The ability to synthesize proteins was determined. Results  Abnormal nuclei and compartments with microvilli were observed in ECV304-SKOV3 cells and their chromatosome modes increased slightly. As compared with parental cells, the population doubling time of ECV304-SKOV3 cells shortened, mitotic indices increased, the expression of PCNA, bcl-2 and MMP-9 enhanced (t ≥2. 4671, P<0. 01), while TN decreased ( t=2. 2473, P <0. 05), the cell number in S-phase increased, while in G0 >>/ G1>>-phase decreased significantly (χ2 = 923. 313, P < 0. 01) . ECV304-SK-OV3 cells were more sensitive than ECV304 to anticancer drugs with inhibition effect on S-phase or S-and M-phase, in accordance with SKOV3 cells ( F≥24. 14, P<0. 01) . The ability to synthesize proteins and telomerase activity enhanced ( t=-12. 2465, P<0. 001 ; t =-25. 2990, P<0. 001) . Conclusion  The result s suggest that ECV3042SKOV3 cells have some biological characteristics of vascular endothelial cells in tumors, including proliferating more quickly, surviving longer and more functional, expressing some angiogeneic factors st ronger, and can act as a cell model for developing drugs targeting the tumor vascular endothelial cells in the future.

     

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