γ射线照射对小肠类器官生长及小肠干细胞生物学特性的影响研究  

Effect ofγ-ray radiation on growth of intestinal organoids and biological properties of intestinal stem cells

作　　者：李小宇 王勇懿 段敏 叶雨萌 李杨 左红艳 LI Xiaoyu;WANG Yongyi;DUAN Min;YE Yumeng;LI Yang;ZUO Hongyan(Institute of Radiation Medicine,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;Troops 32026 of PLA,Kaifeng,Henan 475004,China)

机构地区：[1]军事科学院军事医学研究院辐射医学研究所,北京100850 [2]解放军32026部队,河南开封475004

出　　处：《军事医学》2023年第9期650-656,共7页Military Medical Sciences

摘　　要：目的探讨γ射线照射对小肠类器官生长及Lgr5^(+)隐窝基底柱状细胞、Hopx^(+)储备干细胞和Clu^(+)修复性干细胞生物学特性的影响。方法体外分离C57BL/6小鼠小肠隐窝,并行小肠类器官培养。将传代后第1天小肠类器官随机分为假照射(C)组、5和10 Gyγ射线照射组,分别在照射后1和6 h收集类器官,制作冰冻切片,用γH2AX免疫荧光染色和原位末端标记法检测类器官DNA损伤和凋亡。分别在照射后第1、3、5天观察类器官生长状态,通过形态学分析类器官的出芽率和表面积;用CellTiter 3D细胞化学发光法检测类器官细胞活力;免疫荧光染色观察类器官ki67、Apoa4和Clu表达;实时荧光定量PCR(qRT‐PCR)检测APOA4、CLU、LGR5和HOPX的mRNA表达。结果与C组比较,照射后第1、3、5天,5 Gy组和10 Gy组类器官出芽率显著减少(均P<0.001),细胞活力显著降低(均P<0.05),照射后第3、5天,5 Gy组和10 Gy组类器官表面积显著减小(均P<0.05),上述指标两组间差异无统计学意义。照射后1 h,5 Gy组和10 Gy组γH2AX阳性细胞显著增多(均P<0.001),照射后6 h,5 Gy组和10 Gy组凋亡细胞显著增多(均P<0.001),且10 Gy组显著多于5 Gy组(P<0.001);照射后第1、3、5天,5 Gy组和10 Gy组ki67^(+)细胞减少,Apoa4^(+)细胞、Clu^(+)干细胞增多;且APOA4和HOPX mRNA显著增高(均P<0.05),LGR5 mRNA显著降低(均P<0.05),CLU mRNA主要在照射后第1天显著增高(P<0.001),上述变化10 Gy组较5 Gy组更为显著(均P<0.05)。结论γ射线照射显著抑制小鼠小肠类器官生长,导致DNA损伤和细胞凋亡;Lgr5^(+)隐窝基底柱状细胞对电离辐射敏感,Clu^(+)修复性干细胞和Hopx^(+)储备干细胞对电离辐射较为耐受。Objective To investigate the effect ofγ‐ray radiation on the growth of intestinal organoids and biological properties of Lgr5^(+)crypt‐base columnar cells(Lgr5^(+)CBCs),Hopx^(+)reserve stem cells(Hopx^(+)rSCs)and Clu^(+)revival stem cells(Clu^(+)revSCs).Methods Intestinal crypts of C57BL/6 mice were isolated in vitro and randomly divided into the control group with sham radiation and radiation groups radiated with 5 and 10 Gyγ‐ray respectively.Organoids were collected 1 and 6 h after radiation and made into frozen sections,while DNA damage and apoptosis of organoids were detected by immunofluorescence staining ofγH2AX and TdT mediated nick end labeling.The growth state of organoids was observed 1,3 and 5 days after radiation,followed by morphological analysis of the bud ratio and surface area.The cell viability of organoids was examined by CellTiter 3D cell viability assay kit.The expressions of ki67,Apoa4 and Clu in organoids were observed via immunofluorescence staining.The mRNA expressions of APOA4,CLU,LGR5 and HOPX were assessed by real‐time fluorescence quantitative PCR(qRT‐PCR).Results Compared with group C,the bud ratio and cell viability of organoids in 5 Gy and 10 Gy groups were significantly reduced one,three and five days after radiation(P<0.001,P<0.05),and the surface area of organoids was significantly decreased three and five days after radiation(P<0.05).DNA damage was detected 1 h after radiation and apoptosis was evident 6 h after radiation in 5 Gy and 10 Gy groups(P<0.001),especially in the 10 Gy group(P<0.001).Fewer ki67^(+)proliferation cells but more Apoa4^(+)mature intestinal epithelial cells and Clu^(+)stem cells were observed in 5 Gy and 10 Gy groups one,three and five days after radiation,the expressions of APOA4 and HOPX mRNA were significantly higher(P<0.05),but LGR5 mRNA in organoids was significantly lower than in group C(P<0.05).CLU mRNA increased significantly one day after radiation(P<0.001).These changes were more significant in the 10 Gy group than in the 5 Gy group(P

关 键 词：小肠 类器官 电离辐射 小肠干细胞 

分 类 号：R818.74[医药卫生—放射医学] R816.5[医药卫生—临床医学]