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背景:以正常血管内皮细胞替代肿瘤内皮细胞进行抗肿瘤血管生成研究是一种间接、模拟的研究方式,其局限性显而易见,而以肿瘤内皮细胞为研究对象可提供更为直接、有效、真实的证据。目的:建立胰腺癌内皮细胞的分离纯化、鉴定方法。方法:构建胰腺癌原位荷瘤裸鼠模型,应用免疫磁珠技术,以结合有CD31单克隆抗体的磁珠从胰腺原位移植瘤中分离纯化胰腺癌内皮细胞,通过光学显微镜观察、免疫荧光染色和乙酰化低密度脂蛋白(Ac-LDL)摄取实验从细胞形态、表面标记物和功能三个层面对所获细胞进行鉴定。结果:从裸鼠胰腺原位移植瘤中分离纯化得到的CD31阳性细胞在光学显微镜下呈典型“铺路石样”改变,CD34/VE-cadherin、CD31/VEGFR-2免疫荧光双染色均为阳性,95%以上的细胞DiI标记的Ac-LDL摄取实验结果呈阳性。结论:本研究成功建立了胰腺癌内皮细胞的分离纯化、鉴定方法,可用于胰腺癌肿瘤血管发生机制和抗肿瘤血管生成新药的研究。
BACKGROUND: The study of antitumor angiogenesis by substituting normal endothelial cells with tumor endothelial cells is an indirect and mimic research method. Its limitations are obvious. However, the study of tumor endothelial cells can provide more direct, effective and real evidence. Objective: To establish a method for the isolation, purification and identification of pancreatic carcinoma endothelial cells. Methods: The in situ tumor-bearing nude mice model of pancreatic cancer was established. By means of immunomagnetic beads technique, the pancreatic cancer endothelial cells were isolated and purified from the transplanted pancreatic tumors by magnetic beads combined with CD31 monoclonal antibody. The immunofluorescence Staining and Acetylation of Low Density Lipoprotein (Ac-LDL) Uptake Assay The cells were identified from three aspects of cell morphology, surface marker and function. Results: CD31 positive cells isolated and purified from orthotopic transplanted pancreatic tumors in nude mice showed a typical “paving-stone-like” change under light microscope. The immunofluorescence double staining of CD34 / VE-cadherin and CD31 / VEGFR- Positive, more than 95% of cells DiI-labeled Ac-LDL uptake test results were positive. Conclusion: This study successfully established a pancreatic cancer endothelial cell isolation and purification methods can be used for pancreatic cancer angiogenesis and anti-tumor angiogenesis of new drugs.