[1]李晓琴,方银花,任泂,等.体外辐射模拟P53依赖的小鼠肠干细胞损伤研究[J].第三军医大学学报,2017,39(09):865-871.
 Li Xiaoqin,Fang Yinhua,Ren Jiong,et al.Simulation of P53-dependent cell death in murine intestinal stem cells in vitro by X-ray radiation[J].J Third Mil Med Univ,2017,39(09):865-871.
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体外辐射模拟P53依赖的小鼠肠干细胞损伤研究(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
39卷
期数:
2017年第09期
页码:
865-871
栏目:
基础医学
出版日期:
2017-05-15

文章信息/Info

Title:
Simulation of P53-dependent cell death in murine intestinal stem cells in vitro by X-ray radiation
作者:
李晓琴方银花任泂唐才智冉新泽粟永萍王锋超程红缨
第三军医大学:护理学院基础护理学教研室,军事预防医学院全军复合伤研究所,创伤、烧伤与复合伤国家重点实验
Author(s):
Li Xiaoqin Fang Yinhua Ren Jiong Tang Caizhi Ran Xinze Su Yongping Wang Fengchao Cheng Hongying

Department of Fundamental Nursing, College of Nursing, State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, College of Military Preventive Medicine, Third Military Medical University, Chongqing, 400038, China

关键词:
辐射损伤隐窝肠干细胞P53
Keywords:
radiation injury crypt intestinal stem cells P53
分类号:
R-332; R332.45; R818.74
文献标志码:
A
摘要:

目的      建立6 Gy小鼠肠干细胞体外辐射模型,模拟在体P53依赖的肠干细胞辐射损伤修复过程。方法     分离C57BL/6(野生型)、P53-/-小鼠小肠隐窝,分别于基质胶内接种进行3D培养,24 h后予X线6 Gy照射。比较两组隐窝克隆出芽过程和照后6 d克隆存活率。对野生型隐窝克隆用RT-PCR检测照后24 h内不同时间点P53靶基因Bax、Bbc3、Mdm2、P21、Bcl2l1以及肠干细胞特征性基因Lgr5、Olfm4、Ascl2的表达。结果      C57BL/6和P53-/-小鼠小肠隐窝于接种后48 h出芽,在接种后96~120 h形成类器官样结构。6 Gy辐照可诱导C57BL/6隐窝细胞24 h内大量死亡,完全抑制克隆出芽,仅(43.84±5.73)%的克隆存活;而对P53-/-小鼠隐窝出芽时间无影响,且有(83.69±5.89)%隐窝克隆存活,显著高于对照组(P<0.01)。在野生型隐窝克隆中,P53下游靶基因的表达可被辐射迅速激活,并在24 h内维持高水平,而干细胞特征性基因在12 h迅速下降,24 h到达最低水平。结论     6 Gy照射小鼠小肠隐窝能较好地模拟在体P53依赖的促凋亡因素对肠干细胞的损伤。

Abstract:

Objective      To establish an in vitro irradiation model in murine intestinal stem cells (ISCs) with 6 Gy X-ray radiation in order to mimic the repair process of radiation-induced in vivo P53-dependent ISC death. Methods      The small intestinal crypts were isolated from wide type (WT) and P53-/- mice, and then cultured in a matrigel 3D system for 24 h. Then the crypt clones were irradiated by X-ray at a dose of 6 Gy. The growth and death of crypt clones were observed in 6 d after irradiation. The expression levels of P53 targeting genes, Bax, Bbc3, Mdm2, P21 and Bcl2l1, and ISCs specific genes Lgr5, Olfm4 and Ascl2 in the WT crypt clones at different time points within 24 h after irradiation were measured by quantitative real-time PCR (RT-PCR). Results     The crypts clones derived from WT and P53-/- mice could bud in 48 h after embedding in matrigel and form the organoids in 96 to 120 h. Irradiation of 6 Gy X-ray resulted in massive death of crypt clones from WT mice within 24 h, and total blocking in the budding of crypt clone, with only (43.84±5.73)% clones survival. But it had no effect on the budding of the crypt clones from P53-/- mice, and led to (83.69±5.89)% clones survival, significantly higher than that in the WT crypt colons (P<0.01). In the WT crypt clone, the P53 downstream genes were activated immediately after irradiation, and maintained in high levels within 24 h, while the levels of ISCs specific gens were decreased rapidly within 12 h, and reached the lowest in 24 h after irradiation exposure. Conclusion     Intestinal crypt 3D culture model exposure to  6 Gy irradiation can well mimic P53dependent massive crypt clone death and regeneration in vivo.

参考文献/References:

[1]Chen W, Zheng R, Baade P D, et al. Cancer statistics in China, 2015[J].CA Cancer J Clin, 2016, 66 (2):115-132.DOI:10.3322/caac.21338.
[2]HauerJensen M, Denham J W, Andreyev H J, et al. Radiation enteropathy—pathogenesis, treatment and prevention[J].Nat Rev Gastroenterol Hepatol, 2014,11(8):470-479.DOI: 10.1038/nrgastro.2014.46.
[3]Sato T, Vries R G, Snippert H J, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche[J].Nature,2009,459(7244):262-265.DOI: 10.1038/nature07935.
[4]Wang X, Wei L, Cramer J M,et al. Pharmacologically blocking p53-dependent apoptosis protects intestinal stem cells and mice from radiation[J]. Sci Rep, 2015,5:8566.DOI: 10.1038/srep08566.
[5]Wilson J W,Pritchard D M,Hickman J A,et al.Radiation-induced p53 and p21 WAF-1/CIP1 expression in the murine intestinal epithelium:apoptosis and cell cycle arrest[J]. Am J Pathol,1998,153(3):899-909.DOI:10.1016/S0002-9440(10)656313.
[6]Komarova E A, Kondratov R V, Wang K, et al. Dual effect of p53 on radiation sensitivity in vivo: p53 promotes hematopoietic injury, but protects from gastro-intestinal syndrome in mice [J].Oncogene,2004,23(19):3265-3271.DOI: 10.1038/sj.onc.1207494.
[7]Barker N. Adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration[J]. Nat Rev Mol Cell Biol,2014,15(1): 19-33.DOI: 10.1038/nrm3721.
[8]Sato T, van Es J H, Snippert H J, et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts[J].Nature,2011,469(7330):415-418.DOI: 10.1038/nature 09637.
[9]Date S, Sato T. Mini-gut organoids: reconstitution of the stem cell niche[J].Annu Rev Cell Dev Biol, 2015,31:269-289.DOI: 10.1146/annurev-cellbio-100814-125218.
[10]Wang F, Cheng J, Liu D,et al.P53-participated cellular and molecular responses to irradiation are cell differentiation-determined in murine intestinal epithelium[J].Arch Biochem Biophys, 2014, 542 (10):21-27.DOI: 10.1016/j.abb.2013.11.012.
[11]Komarova E A, Kondratov R V, Wang K,et al. Dual effect of p53 on radiation sensitivity in vivo: p53 promotes hematopoietic injury, but protects from gastro-intestinal syndrome in mice[J]. Oncogene,2004,23(19):3265-3271.DOI: 10.1038/sj.onc.1207494.
[12]Johnke R M, Sattler J A, Allison R R.Radioprotective agents for radiation therapy: future trends[J]. Future Oncol, 2014,10(15):2345-2357.DOI: 10.2217/fon.14.175.
[13]Wei L,Leibowitz B J, Wang X, et al. Inhibition of CDK4/6 protects against radiationinduced intestinal injury in mice[J].J Clin Invest,2016,126 (11):4076-4087.DOI: 10.1172/JCI88410.
[14]Barker N, van Es J H, Kuipers J, et al. Identification of stem cells in small intestine and colon by marker gene Lgr5[J]. Nature,2007,449(7165):1003-1007.DOI:10.1038/nature06196.
[15]van der Flier L G, van Gijn M E, Hatzis P, et al. Trans-cription factor achaete scutelike 2 controls intestinal stem cell fate[J]. Cell,2009,136(5):903-912. DOI:10.1016/j.cell.2009.01.031.
[16]van der Flier L G, Haegebarth A, Stange D E, et al. OLFM4 is a robust marker for stem cells in human intestine and marks a subset of colorectal cancer cells[J]. Gastro-enterology, 2009,137(1): 15-17.DOI: 10.1053/j.gastro.2009.05.035.
[17]Hua G, Thin T H, Feldman R, et al. Crypt base columnar stem cells in small intestines of mice are radioresistant[J]. Gastroenterology,2012,143(5):1266-1276.DOI: 10.1053/j. gastro.2012.07.106.

更新日期/Last Update: 2017-05-05