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目的:建立人子宫内膜癌(EC)微血管内皮细胞的原代分离培养方法并研究其体外免疫组化特征。方法:采用免疫磁珠法结合胶原酶消化法及筛网过滤法分离纯化EC微血管内皮细胞,采用含有20%胎牛血清的M199培养。通过光镜、细胞免疫荧光技术对所获得的微血管内皮细胞进行鉴定,流式细胞仪检测微血管内皮细胞的纯度。微血管内皮细胞的生长曲线通过MTT法测定。结果:微血管内皮细胞台盼蓝拒染率为90%,纯度95%,并能连续传代。原代细胞7～9d完全融合,传代培养后5～6d可传代1次。细胞单层生长,融合后呈铺路石样具有接触抑制现象。免疫荧光证实,传代细胞表达内皮细胞特异性vWF因子及细胞间黏附蛋白CD31。经MTT法测定得到的微血管内皮细胞体外生长曲线呈“S”型。结论:成功建立了人EC微血管内皮细胞的分离培养方法,所获得的细胞可用于EC的相关研究,为探讨EC的临床治疗方法提供了新的平台。
Objective: To establish a method for the primary isolation and culture of human endometrial carcinoma (EC) microvascular endothelial cells and study its immunohistochemical characteristics in vitro. Methods: EC microvascular endothelial cells were isolated and purified by immunomagnetic beads method combined with collagenase digestion and sieve filtration, and cultured in M199 with 20% fetal bovine serum. The microvascular endothelial cells were identified by light microscopy and cell immunofluorescence, and the purity of microvascular endothelial cells was detected by flow cytometry. Microvascular endothelial cell growth curve was determined by MTT assay. Results: The trypan blue exclusion rate of microvascular endothelial cells was 90% with a purity of 95% and could be passaged continuously. Primary cells 7 ~ 9d complete fusion, subculturing 5 ~ 6d after passage 1 times. Cell monolayer growth, after fusion was paving stone-like contact inhibition phenomenon. Immunofluorescence confirmed that the passaged cells express endothelial cell specific vWF and intercellular adhesion protein CD31. The in vitro growth curve of microvascular endothelial cells obtained by MTT assay showed “S” type. Conclusion: The method of isolation and culture of human EC microvascular endothelial cells has been successfully established. The obtained cells can be used in the related research of EC and provide a new platform for exploring the clinical treatment of EC.