Evaluation of cellular wound healing using flow cytometry and expanded polytetrafluroethylene implants.
Evaluation of cellular wound healing using flow cytometry and expanded polytetrafluroethylene implants.
复制标题
DOI：
10.1111/j.1524-475x.2010.00587.x
复制
发表时间：
2010-05
期刊：
影响因子：
--
通讯作者：
Swanson PE
中科院分区：
文献类型：
--
作者：
Tsuji JM;Whitney JD;Tolentino EJ;Perrin ME;Swanson PE
在研究治疗干预的伤口愈合研究中，表征细胞反应非常重要。在一项招募有手术感染风险的患者的随机试验中，一个目标是使用流式细胞术和免疫组织化学以及基于标准苏木精和伊红的组织学对聚四氟乙烯植入物内的细胞进行表型分析。术后 8-9 天取出皮下植入物。为了获得与伤口愈合机制相关的单细胞，我们最初使用了小鼠皮肤消化方案。我们对此进行了优化，以提高细胞产量并分离出足够的细胞用于流式细胞术。这些修改将每个受试者回收的总细胞从平均 5.3×104 增加到 41×104，平均活力为 80%。免疫荧光染色测定对我们的样品进行了验证，这些样品的细胞样品数量比组织活检更少。对三十二个样品进行了染色。分离来自聚四氟乙烯管的细胞并用针对 CD3、CD20、CD31、CD34、CD68、CD133 和 VEGFR2 的荧光标记抗体进行阳性染色。流式细胞术数据与 IHC 数据相关，尤其是与 CD68 相关的数据。这种抗原在两种细胞分析方法中都是最普遍的。我们的研究结果表明，流式细胞术可以与聚四氟乙烯样品一起使用，作为记录和描述细胞伤口愈合反应的附加评估方法。
In wound healing studies that investigate therapeutic interventions, it is important to characterize cellular responses. In a randomized trial enrolling patients at risk for surgical infection, one goal is to phenotype cells within a polytetrafluoroethylene implant using flow cytometry and immunohistochemistry, together with standard hematoxylin and eosin based histology. Subcutaneous implants are removed 8–9 days postoperatively. To obtain single cells associated with the mechanism of wound healing, we initially used a mouse skin digestion protocol. We optimized this to increase cell yield and isolate sufficient cells for flow cytometry. The modifications increased the total cells recovered per subject from an average of 5.3×104 to 41×104 with an average viability of 80%.The immunoflourescent staining assay was verified for our samples, which have smaller cell sample numbers than tissue biopsies. Thirty-two samples were stained. Cells from the polytetrafluoroethylene tubes were isolated and stained positively with fluorescent-labeled antibodies to CD3, CD20, CD31, CD34, CD68, CD133 and VEGFR2. Flow cytometry data correlated with IHC data especially with respect to CD68. This antigen was the most prevalent in both cell analysis methods. Our findings demonstrate flow cytometry can be used with polytetrafluoroethylene samples as an additional evaluation method to document and describe cellular wound healing responses.