胶质瘤相关癌基因1特异性阻断剂对结直肠癌肿瘤干细胞分化的影响及对Hedgehog信号通路的调控作用  

Effect of glioma associated oncogene 1 specific blocker on differentiation of colorectal cancer stem cells and regulation of Hedgehog signaling pathway

作　　者：徐纪中[1] 王贵宪[1] 袁维堂[1] 周全博[1] 胡晟云 Xu Jizhong;Wang Guixian;Yuan Weitang;Zhou Quanbo;Hu Shengyun(Department of Colorectal Surgery,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]郑州大学第一附属医院结直肠外科,450052

出　　处：《中华实验外科杂志》2020年第2期220-223,共4页Chinese Journal of Experimental Surgery

摘　　要：目的观察胶质瘤相关癌基因1特异性阻断剂(GANT-61)对结直肠癌肿瘤干细胞(CRC-CSCs)分化及对Hedgehog信号通路的调控作用的影响。方法磁珠分选法获得CD133+CRC-CSCs,用含不同浓度(0、10、20、40μmol/L)的GANT-61的干细胞培养液干预,设为对照组、低、中、高剂量组,测定干预不同时刻细胞活性、CRC-CSCs球形成率、CD133+占比、核转录因子1(Gli1)、Hh受体蛋白(Ptch1)、c-Myc、细胞角蛋白20(CK20)mRNA和蛋白表达。多样本计量资料比较采用单因素方差分析,两两样本比较采用SNK-q检验。结果与对照组比较,GANT-61各剂量组不同时刻CCK-8试验吸光度(A)值均降低(24 h:t=2.445、4.985、6.903;48 h:t=2.859、5.270、7.488;72 h:t=3.324、8.053、12.042,P<0.05),且随GANT-61干预剂量升高而降低(F=13.382、16.842、41.053,P<0.05),差异有统计学意义;对照组、低、中剂量组CCK-8试验A值随干预时间延长升高,且干预不同时刻CCK-8试验A值比较差异均有统计学意义(F=44.598、31.844,P<0.05),而高剂量组随干预时间延长无明显变化(F=1.585,P>0.05),差异无统计学意义。对照组、GANT-61低、中、高剂量组CRC-CSCs球形成率分别为(13.57±1.55)%、(11.22±1.01)%、(5.46±0.67)%、(1.60±0.30)%,CD133+占比分别为(96.20±3.07)%、(81.34±5.84)%、(62.01±5.03)%、(45.38±4.21)%;与对照组比较,GANT-61各剂量组CRC-CSCs球形成率及CD133+占比均降低(CRC-CSCs球形成率:t=2.840、10.739、16.954,P<0.05;CD133+占比:t=5.036、12.974、21.809,P<0.05),且随GANT-61干预剂量升高而降低,各剂量组组间比较差异均有统计学意义(CRC-CSCs球形成率:F=225.500,P<0.05;CD133+占比:F=62.988,P<0.05)。与对照组比较,GANT-61各剂量组Gli1、Ptch1、c-Myc、CK20 mRNA和蛋白相对表达量均降低(mRNA:低剂量组t=2.577、2.659、2.524、4.031;中剂量组t=5.271、5.587、6.163、6.500;高剂量组t=7.442、8.976、8.750、9.632,P<0.05;蛋白:低剂量组t=3.008、2.517、2.385、2.981;中剂量组t=5.664、8.9Objective To investigate the effect of small molecule drug glioma associated oncogene 1 specific blocker(GANT-61)on the differentiation of colorectal cancer stem cells(CRC-CSCs)and the regulation of Hedgehog signaling pathway.Methods CD133+CRC-CSCs were obtained by magnetic bead sorting.The cells were treated with GANT-61 stem cell culture medium containing different concentrations(0,10,20,40μmol/L)respectively.The cells were divided into control group,and low,medium and high dose groups.The cell activity,the spherical formation rate of CRC-CSCs,the proportion of CD133+,the mRNA and protein expression levels of nuclear transcription factor 1(Gli1),Hedgehog receptor protein(Ptch1),c-Myc and cytokeratin 20(CK20)were determined.Results Compared with the control group,absorbance(A)value of cell counting kit-8(CCK-8)test for CRC-CSCs in each dose group of GANT-61 decreased at different time points(24 h t=2.445,4.985,6.903;48 h t=2.859,5.270,7.488;72 h:t=3.324,8.053,12.042,P<0.05),and decreased with the increase of GANT-61 intervention dose(F=13.382,16.842,41.053,P<0.05).The A value of CCK-8 test in control group,low-dose group and middle-dose group increased with the prolongation of intervention time,and there were significant differences in A values of CCK-8 test among different intervening time points(F=44.598,31.844,P<0.05),while there was no significant change in high-dose group with the prolongation of intervention time(F=1.585,P>0.05).The spherical formation rates of CRC-CSCS in the control group and GANT-61 low,medium and high dose groups were(13.57±1.55)%,(11.22±1.01)%,(5.46±0.67)%,(1.60±0.30)%,and the proportions of CD133+were(96.20±3.07)%,(81.34±5.84)%,(62.01±5.03)%,(45.38±4.21)%,respectively.Compared with the control group,spherical formation rate and the proportion of CD133+of CRC-CSCs in each dose group of GANT-61 decreased at different time points(for the spherical formation rates of CRC-CSCS:t=2.840,10.739,16.954,P<0.05;for the proportions of CD133+:t=5.036,12.974,21.809,P<0.05),and decreased w

关 键 词：胶质瘤相关癌基因1特异性阻断剂 结直肠癌肿瘤干细胞 分化 HEDGEHOG信号通路 

分 类 号：R735[医药卫生—肿瘤]