{"title":"敲除HaCaT细胞中的TP53突变体可增强其迁移活性","authors":"P. Kozhin, D. Romashin, A. Rusanov, NG Luzgina","doi":"10.24075/brsmu.2022.070","DOIUrl":null,"url":null,"abstract":"The HaCaT cell line represents the spontaneously immortalized non-carcinogenic human keratinocytes that are used as a model for studying the function of normal human keratinocytes. There are two TP53 alleles in the HaCaT cell genome, which comprise two gain-of-function (GOF) mutations acquired through spontaneous immortalization (mutTP53). Mutations result in the increased proliferation rate and violation of the stratification program. The study was aimed to assess the effects of the mutTP53 gene knockout on the HaCaT keratinocytes capability of proliferation and migration in the in vitro model of epidermal injury and regeneration (scratch test), and on the ability to form stratified epithelium in the organotypic epidermal model. To perform the scratch-test, cells were cultured until monolayer was formed, then the standardized injury was created. The organotypic model was obtained by growing keratinocytes in the polycarbonate membrane inserts with the pore size of 0.4 μm at the interface between the phases (air-liquid). It has been shown that the mutant TP53 gene knockout results in the increased migration capability of the HaCaT keratinocytes: in the HaCaT with the mutTP53 knockout, the defect closure occurred faster than in the appropriate group of the WT HaCaT (p < 0.05), on day three the defect size was 12% ± 3% and 66% ± 5% of the initial size. There is evidence that mutant TP53 in the HaCaT cells is a negative regulator of the laminin 5 expression (LAMC2 expression was 9.96 ± 1.92 times higher in the cells with the mutTP53 knockout, p < 0.05), however, this does not promote normalization of the program of epithelial differentiation and stratification followed by formation of the stratum corneum in the organotypic model.","PeriodicalId":9344,"journal":{"name":"Bulletin of Russian State Medical University","volume":" ","pages":""},"PeriodicalIF":0.2000,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Knockout of mutant TP53 in the HaCaT cells enhances their migratory activity\",\"authors\":\"P. Kozhin, D. Romashin, A. Rusanov, NG Luzgina\",\"doi\":\"10.24075/brsmu.2022.070\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"The HaCaT cell line represents the spontaneously immortalized non-carcinogenic human keratinocytes that are used as a model for studying the function of normal human keratinocytes. There are two TP53 alleles in the HaCaT cell genome, which comprise two gain-of-function (GOF) mutations acquired through spontaneous immortalization (mutTP53). Mutations result in the increased proliferation rate and violation of the stratification program. The study was aimed to assess the effects of the mutTP53 gene knockout on the HaCaT keratinocytes capability of proliferation and migration in the in vitro model of epidermal injury and regeneration (scratch test), and on the ability to form stratified epithelium in the organotypic epidermal model. To perform the scratch-test, cells were cultured until monolayer was formed, then the standardized injury was created. The organotypic model was obtained by growing keratinocytes in the polycarbonate membrane inserts with the pore size of 0.4 μm at the interface between the phases (air-liquid). It has been shown that the mutant TP53 gene knockout results in the increased migration capability of the HaCaT keratinocytes: in the HaCaT with the mutTP53 knockout, the defect closure occurred faster than in the appropriate group of the WT HaCaT (p < 0.05), on day three the defect size was 12% ± 3% and 66% ± 5% of the initial size. There is evidence that mutant TP53 in the HaCaT cells is a negative regulator of the laminin 5 expression (LAMC2 expression was 9.96 ± 1.92 times higher in the cells with the mutTP53 knockout, p < 0.05), however, this does not promote normalization of the program of epithelial differentiation and stratification followed by formation of the stratum corneum in the organotypic model.\",\"PeriodicalId\":9344,\"journal\":{\"name\":\"Bulletin of Russian State Medical University\",\"volume\":\" \",\"pages\":\"\"},\"PeriodicalIF\":0.2000,\"publicationDate\":\"2022-12-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Bulletin of Russian State Medical University\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.24075/brsmu.2022.070\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"MEDICINE, GENERAL & INTERNAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Bulletin of Russian State Medical University","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.24075/brsmu.2022.070","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"MEDICINE, GENERAL & INTERNAL","Score":null,"Total":0}
Knockout of mutant TP53 in the HaCaT cells enhances their migratory activity
The HaCaT cell line represents the spontaneously immortalized non-carcinogenic human keratinocytes that are used as a model for studying the function of normal human keratinocytes. There are two TP53 alleles in the HaCaT cell genome, which comprise two gain-of-function (GOF) mutations acquired through spontaneous immortalization (mutTP53). Mutations result in the increased proliferation rate and violation of the stratification program. The study was aimed to assess the effects of the mutTP53 gene knockout on the HaCaT keratinocytes capability of proliferation and migration in the in vitro model of epidermal injury and regeneration (scratch test), and on the ability to form stratified epithelium in the organotypic epidermal model. To perform the scratch-test, cells were cultured until monolayer was formed, then the standardized injury was created. The organotypic model was obtained by growing keratinocytes in the polycarbonate membrane inserts with the pore size of 0.4 μm at the interface between the phases (air-liquid). It has been shown that the mutant TP53 gene knockout results in the increased migration capability of the HaCaT keratinocytes: in the HaCaT with the mutTP53 knockout, the defect closure occurred faster than in the appropriate group of the WT HaCaT (p < 0.05), on day three the defect size was 12% ± 3% and 66% ± 5% of the initial size. There is evidence that mutant TP53 in the HaCaT cells is a negative regulator of the laminin 5 expression (LAMC2 expression was 9.96 ± 1.92 times higher in the cells with the mutTP53 knockout, p < 0.05), however, this does not promote normalization of the program of epithelial differentiation and stratification followed by formation of the stratum corneum in the organotypic model.
期刊介绍:
Bulletin of Russian State Medical University (Bulletin of RSMU, ISSN Print 2500–1094, ISSN Online 2542–1204) is a peer-reviewed medical journal of Pirogov Russian National Research Medical University (Moscow, Russia). The original language of the journal is Russian (Vestnik Rossiyskogo Gosudarstvennogo Meditsinskogo Universiteta, Vestnik RGMU, ISSN Print 2070–7320, ISSN Online 2070–7339). Founded in 1994, it is issued once every two months publishing articles on clinical medicine and medical and biological sciences, first of all oncology, neurobiology, allergy and immunology, medical genetics, medical microbiology and infectious diseases. Every issue is thematic. Deadlines for manuscript submission are announced in advance. The number of publications on topics in spite of the issue topic is limited. The journal accepts only original articles submitted by their authors, including articles that present methods and techniques, clinical cases and opinions. Authors must guarantee that their work has not been previously published elsewhere in whole or in part and in other languages and is not under consideration by another scientific journal. The journal publishes only one review per issue; the review is ordered by the editors.