胰腺癌内皮细胞的分离纯化和鉴定

Isolation,Purification and Identification of Pancreatic Cancer Endothelial Cells

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： Investigation of antiangiogenic cancer therapy using normal vascular endothelial cells instead of the actual tumor endothelial cells is a kind of indirect, mimic research with many obvious limitations, while studies using tumor endothelial cells could provide more direct, efficient, and truthful evidences. Aims： To establish a protocol for the isolation, purification, and identification of pancreatic cancer endothelial cells in vitro. Methods： Orthotopic model of pancreatic cancer was constructed in nude mice, and immunomagnetic method using magnetic beads conjugated with CD31 monoclonal antibodies was used to isolate and purify pancreatic cancer endothelial cells from the orthotopic pancreatic cancer xenografts. Isolated cells were identified by morphology under optical microscope, immunofluorescence staining of endothelial cell-specific markers, and uptake of acetylated LDL （Ac-LDL）. Results： CD31-positive cells isolated from orthotopic pancreatic cancer xenografts in nude mice were in a typical appearance of paving stones under optical microscope and were positive for double immunofluorescence staining of CD34/VE-cadherin and CD31/VEGFR-2. Uptake of DiI labeled Ac-LDL was positive in over 95% of the isolated cells. Conclusions： A protocol for the isolation, purification, and identification of pancreatic cancer endothelial cells in vitro has been successfully established, which can be used in studies on tumor angiogenesis and antiangiogenic therapy of pancreatic cancer.