A modified fluorimetric host cell reactivation assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts.
A modified fluorimetric host cell reactivation assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts.
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DOI：
10.1186/1472-6750-10-46
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发表时间：
2010-06-22
影响因子：
3.5
通讯作者：
Bergemann J
中科院分区：
文献类型：
--
作者：
Burger K;Matt K;Kieser N;Gebhard D;Bergemann J
宿主细胞再活化试验（HCRA）被广泛用于鉴定影响细胞修复能力的环境和物质，然而，它受到所使用的转染程序和检测方法的灵敏度的限制。原代皮肤细胞特别难以分离，因此需要灵敏的方法来检测由于细胞类型或个体间差异或由不同物质诱导的变化而引起的任何变化。检测皮肤细胞修复能力的灵敏且可重复的方法将在两个不同方面有用：一方面，以积极的方式识别影响修复能力的物质（这些物质可能是化妆品的有希望的成分），另一方面，排除物质对修复能力的负面影响（这可以作为替代或至少减少动物试验的进一步措施）。本论文基于双波长绿色荧光蛋白（GFP）和DsRed报告基因技术，建立了一种快速、灵敏的检测原代角质形成细胞、黑素细胞和成纤维细胞DNA修复能力的方法，以检测不同物质及其对DNA修复能力的影响。对于质粒恢复的检测，我们使用了FACS技术，与光度计技术相比，该技术具有高度灵敏度，并且允许基于单细胞的分析。该测定和研究修复能力的有用性通过DNA修复在成纤维细胞中被环孢菌素A抑制的证据来证明。本文描述的方法确定了不同类型的人类皮肤细胞的DNA修复能力。所述转染方案适用于黑素细胞、角质形成细胞和成纤维细胞的转染，达到了适用于通过FACS技术检测恢复的质粒的功效。因此，不同细胞类型的修复能力可以相互比较。所描述的测定也是高度灵活的，并且可以使用该方法的修改来确定其他修复机制的活性。
The Host Cell Reactivation Assay (HCRA) is widely used to identify circumstances and substances affecting the repair capacity of cells, however, it is restricted by the transfection procedure used and the sensitivity of the detection method. Primary skin cells are particularly difficult to transfect, and therefore sensitive methods are needed to detect any variations due to the cell-type or inter-individual differences or changes induced by diverse substances. A sensitive and repeatable method to detect the repair capacity of skin cells would be useful in two different aspects: On the one hand, to identify substances influencing the repair capacity in a positive manner (these substances could be promising ingredients for cosmetic products) and on the other hand, to exclude the negative effects of substances on the repair capacity (this could serve as one step further towards replacing or at least reducing animal testing). In this paper, we present a rapid and sensitive assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts based on two wave-length Green Fluorescent Protein (GFP) and DsRed reporter technology in order to test different substances and their potential to influence the DNA repair capacity. For the detection of plasmid restoration, we used FACS technology, which, in comparison to luminometer technology, is highly sensitive and allows single cell based analysis. The usefulness of this assay and studying the repair capacity is demonstrated by the evidence that DNA repair is repressed by Cyclosporin A in fibroblasts. The methodology described in this paper determines the DNA repair capacity in different types of human skin cells. The described transfection protocol is suitable for the transfection of melanocytes, keratinocytes and fibroblasts, reaching efficacies suitable for the detection of the restored plasmids by FACS technology. Therefore the repair capacity of different cell types can be compared with each other. The described assay is also highly flexible, and the activity of other repair mechanisms can be determined using modifications of this method.
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影响因子：
17.3
作者：
Kendall, JM;Badminton, MN
通讯作者：
Badminton, MN
影响因子：
2.9
作者：
Roguev, A;Russev, G
通讯作者：
Russev, G
影响因子：
6.5
作者：
Smit, N. P. M.;Van Rossum, H. H.;Van Pelt, J.
通讯作者：
Van Pelt, J.
影响因子：
9.2
作者：
Hein, R;Tsien, RY
通讯作者：
Tsien, RY
DOI：
10.1073/pnas.90.4.1614
发表时间：
1993-02-15
影响因子：
11.1
作者：
WEI, QY;MATANOSKI, GM;GROSSMAN, L
通讯作者：
GROSSMAN, L