血管瘤血管内皮细胞体外分离纯化方法的优化(1)
[摘要]目的：寻找更高效的婴幼儿血管瘤内皮细胞培养方法，为婴幼儿血管瘤的研究提供基础的科研保证。方法：选取增生期婴幼儿血管瘤手术标本，采用组织块贴壁法与酶消化法结合进行原代培养；二代细胞进行流式分选，取CD31阳性的细胞继续培养。分别进行细胞形态学观察、细胞表面标记物免疫荧光鉴定、细胞表面标记物流式鉴定。结果：培养48～72h清晰可见组织块周围细胞游出，细胞核清晰，细胞呈长梭形及多边形，2周左右部分可形成“血管腔样”结构，20d可铺满平传代；细胞流式测定CD31阳性率达98.4%，VWF与CD31双阳率90.3%，免疫荧光染色CD31、VWF均呈阳性表达。结论：组织块与消化结合法经流式分选后可获得高纯度的内皮细胞，为下一步研究婴幼儿血管瘤增生消退机制奠定了基础。
[关键词]婴幼儿血管瘤；细胞培养；流式分选；组织块与酶消化法；血管瘤内皮细胞
[中图分类号]R732.2 [文献标志码]A [文章编号]1008-6455(2018)04-0061-03
Flow Separation was Combined with Tissue Block and Digestion to Train the Endothelial Cells of Hemangioma
LANG Cui-ling， SUN Bin， LEI Hong-zhao， MA Yu-chun， DONG Chang-xian
(Department of Hemangioma Surgery，Henan University Henan Provincial People's Hospital，Zhengzhou 450003，Henan，China)
Abstract： Objective To explore the endothelial cell culture method of more efficient infant hemangioma ，to provide the basic research guarantee for the research of hemangioma of infants. Methods In order to select the surgical specimens of the hemangioma of the hyperplasia， the primary culture was carried out by means of Tissue block adherent and enzyme digestion. Two generations of cells were selected for flow separation， and CD31 positive cells continued to be cultured.It were respectively carried out to observe the cell of morphology， immunofluorescence identification of cell surface marker， of fluid identification of cell surface marker. Results The cells in the tissue block around the tissue block could be clearly visible from 48-72 hours. The nuclei were clear， the cells were long fusiform and polygons， and the portion could form "blood vessel cavity" structure in about 2 weeks. The positive rate of CD31 was 98.4%， the VWF and CD31 were 90.3%， and the immunofluorescence staining CD31 and VWF were positive. Conclusion The high purity endothelial cells can be obtained after the flow separation of the means of tissue block and digestive association， which lays the foundation for the next study on the mechanism of proliferation and regression of hemangioma of infants.
Key words： infant hemangioma； cell culture ； flow separation；tissue block and enzyme digestion method； hemangioma endothelial cells
嬰幼儿血管瘤(Infantile hemangioma)是来源于内皮细胞的先天性良性肿瘤，好发于面颈部(60%)[1-2]，可造成皮肤色素沉着，瘢痕甚至畸形等，但其病因和发病机制目前并不清楚[3-4]，组织病理学证实血管瘤增生期可发现大量微血管内皮细胞增生[5-6]。婴幼儿血管瘤研究的标本大多数来源于病理组织，若进行分子生物学水平研究，则纯度不足。因此，要从细胞生物学、分子生物学研究血管瘤的发病机制、提纯目的细胞，婴幼儿血管瘤体外细胞培养是关键一步。本次实验采用组织块贴壁法与酶消化法结合，经流式细胞仪分选培养原代内皮细胞取得了较理想的结果，现将该方法报道如下。
1 材料和方法, 百拇医药(稂翠玲 孙斌 雷红召 马玉春 董长宪)
[关键词]婴幼儿血管瘤；细胞培养；流式分选；组织块与酶消化法；血管瘤内皮细胞
[中图分类号]R732.2 [文献标志码]A [文章编号]1008-6455(2018)04-0061-03
Flow Separation was Combined with Tissue Block and Digestion to Train the Endothelial Cells of Hemangioma
LANG Cui-ling， SUN Bin， LEI Hong-zhao， MA Yu-chun， DONG Chang-xian
(Department of Hemangioma Surgery，Henan University Henan Provincial People's Hospital，Zhengzhou 450003，Henan，China)
Abstract： Objective To explore the endothelial cell culture method of more efficient infant hemangioma ，to provide the basic research guarantee for the research of hemangioma of infants. Methods In order to select the surgical specimens of the hemangioma of the hyperplasia， the primary culture was carried out by means of Tissue block adherent and enzyme digestion. Two generations of cells were selected for flow separation， and CD31 positive cells continued to be cultured.It were respectively carried out to observe the cell of morphology， immunofluorescence identification of cell surface marker， of fluid identification of cell surface marker. Results The cells in the tissue block around the tissue block could be clearly visible from 48-72 hours. The nuclei were clear， the cells were long fusiform and polygons， and the portion could form "blood vessel cavity" structure in about 2 weeks. The positive rate of CD31 was 98.4%， the VWF and CD31 were 90.3%， and the immunofluorescence staining CD31 and VWF were positive. Conclusion The high purity endothelial cells can be obtained after the flow separation of the means of tissue block and digestive association， which lays the foundation for the next study on the mechanism of proliferation and regression of hemangioma of infants.
Key words： infant hemangioma； cell culture ； flow separation；tissue block and enzyme digestion method； hemangioma endothelial cells
嬰幼儿血管瘤(Infantile hemangioma)是来源于内皮细胞的先天性良性肿瘤，好发于面颈部(60%)[1-2]，可造成皮肤色素沉着，瘢痕甚至畸形等，但其病因和发病机制目前并不清楚[3-4]，组织病理学证实血管瘤增生期可发现大量微血管内皮细胞增生[5-6]。婴幼儿血管瘤研究的标本大多数来源于病理组织，若进行分子生物学水平研究，则纯度不足。因此，要从细胞生物学、分子生物学研究血管瘤的发病机制、提纯目的细胞，婴幼儿血管瘤体外细胞培养是关键一步。本次实验采用组织块贴壁法与酶消化法结合，经流式细胞仪分选培养原代内皮细胞取得了较理想的结果，现将该方法报道如下。
1 材料和方法, 百拇医药(稂翠玲 孙斌 雷红召 马玉春 董长宪)