Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing
Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing
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DOI：
10.3791/59608
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发表时间：
2019-09-01
影响因子：
1.2
通讯作者：
Flockerzi, Veit
中科院分区：
文献类型：
--
作者：
Belkacemi, Anouar;Laschke, Matthias W.;Flockerzi, Veit
受损的皮肤伤口愈合是患有糖尿病的患者和老年人的主要关注点，并且需要有效的治疗。适当的体外和体内方法对于鉴定用于药物治疗的新靶分子以改善皮肤伤口愈合过程是必不可少的。我们在两项独立的测定中确定了电压门控钙通道的β 3亚基（Cav β 3）作为影响伤口愈合的潜在靶分子，即，体外划痕迁移试验和体内背侧皮褶室模型。使用从野生型（WT）和Cav β 3缺陷型小鼠（Cav β 3 KO）急性分离的原代小鼠胚胎成纤维细胞（MEF）或从用siRNA处理以下调编码Cav β 3的Cacnb3基因的表达的WT小鼠急性分离的成纤维细胞。在汇合的细胞单层上施加划痕，然后在规定的时间点拍摄显微图像，以闭合差距，直至迁移细胞完全重新填充差距。分析这些图像，并确定每种条件下的细胞迁移率。在体内测定中，我们在WT和Cav β 3 KO小鼠上植入背部皮褶室，施加直径为2 mm的限定圆形伤口，用盖玻片覆盖伤口以保护其免受感染和干燥，并监测随时间的宏观伤口闭合。Cacnb3基因缺陷小鼠的伤口闭合速度明显更快。由于体内和体外测定的结果相关性良好，体外测定可用于在通过体内伤口愈合模型验证体外命中之前进行高通量筛选。我们在这里所展示的野生型和Cav β 3缺陷小鼠或细胞也可能适用于Cav β 3以外的特定分子。
Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the beta 3 subunit of voltage-gated calcium channels (Cav beta 3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav beta 3-deficient mice (Cav beta 3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav beta 3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav beta 3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav beta 3-deficient mice or cells might also be applicable for specific molecules other than Cav beta 3.