| 研究課題/領域番号 |
16K10137
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| 研究機関 | 久留米大学 |
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研究代表者 |
TEYE KWESI 久留米大学, 皮膚細胞生物学研究所, 助教 (30599303)
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| 研究分担者 |
橋本 隆 久留米大学, 皮膚細胞生物学研究所, 教授 (20129597) [辞退]
名嘉真 武国 久留米大学, 医学部, 教授 (50221453)
沼田 早苗 久留米大学, 医学部, 助教 (40599312)
石井 文人 久留米大学, 医学部, 准教授 (80330827)
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| 研究期間 (年度) |
2016-04-01 – 2019-03-31
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| キーワード | CD44 / Skin / Skin disease / Keratinocyte / Squamous cell carcinoma / Skin Cancer |
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| 研究実績の概要 |
The aim of the research is to determine the functions of specific CD44 proteins in human skin and epidermis and their relation to skin diseases, cancer and autoimmunity. We explored the expression of variant CD44 proteins in various skin conditions including Squamous cell carcinoma, basal cell carcinoma, Extramammary Paget’s disease, atopy dermatitis, psoriasis, and melanoma. We stained sections of formalin fixed paraffin embedded tissues with a specific monoclonal antibody recognizing variant exon 6 (CD44v6) by immunofluorescence analysis. The results indicated that CD44 expression was lower in non-melanoma skin cancer, patchy in Extramammary Paget’s disease and nearly not affected in psoriasis and atopy dermatitis. Our results suggest that CD44 variant expression may be used as molecular marker in various skin diseases. Further analyses are required to further understand how reduced expression of CD44 affect the pathogenesis of the skin cancer phenotype.
Using a keratinocytes cell line, we have been able to completely knock-out CD44 by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system through homology directed repair (HDR) and the cells expressed no CD44. These cells will be useful for further analysis of CD44 function in human keratinocytes and epidermis. Selection of a suitable keratinocyte is critical for the success of these studies.
Transfection of keratinocytes is challenging. By varying the transfection protocols of several transfection reagents, we have improved upon the transfection efficiency of keratinocytes with reduced toxicity.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
The research is still ongoing and progress is being made although there is still a lot of work to be done.
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| 今後の研究の推進方策 |
Using CRISPR technology, we are developing systems in which CD44 is completely disrupted. Additionally, because CD44 has many variants whose functions are not well understood, we will re-introduce each variant of CD44 into keratinocytes in which CD44 has been completely disrupted. Instead of many CD44 proteins, each cell will have a single type of CD44 protein. This will allow us to know with certainty the function of each CD44 molecule. The cells will be used for various analysis including RNA micro-array to identify novel pathways controlled by CD44, proliferation, differentiation and their possible role various skin diseases.
The positioning of CD44 in epidermis suggest that it could be targeted by auto-antibodies resulting in autoimmune disease of the skin. Using recombinant CD44 proteins, we will invest the occurrence of autoantibodies against CD44 proteins.
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| 次年度使用額が生じた理由 |
(Reasons)
The research is still ongoing and there are many reagents and cells that needed to be purchased in order to continue with the research.
(Usage Plan)
We are in the process of using CRISPR technology to knock-out CD44 in keratinocytes to determine cell behavior associated with loss of CD44. During knocking out CD44, selected CD44 variants obtained from human epidermis will be knocked-in into the cells to enable analysis of these variants in keratinocytes. The grant will be used to purchase reagents that are needed to perform gene expression analysis including reagents for Western blotting, immunofluorescence ELISA, etc. These include primary and secondary antibodies as well as substrates for detection of signals. RNA from keratinocyte with loss of CD44 and for those in which specific CD44 cDNA have been re-introduced will be used in Microarray analysis. Part of the grant will be used to pay for Micro-array analysis services. A small part of the grant will also be used to travel to present the results of the research at academic meetings.
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