张荣红,廉芳,周孟,贾铁文,何志旭,刘杰麟
ZHANG Ronghong,LIAN Fang,ZHOU Meng,JIA Tiewen,HE Zhixu,LIU Jielin

• 1:贵州医科大学组织工程与干细胞实验中心
• 2:贵州医科大学免疫学教研室
• 3:贵州医科大学民族药与中药开发应用教育部工程研究中心国家苗药工程技术研究中心

摘要(Abstract)：

目的:制备抗RecQ解旋酶单克隆抗体(6H5抗体),鉴定其生物学特性并开展其对肿瘤细胞及肿瘤干细胞中RecQ解旋酶的检测。方法:在Balb/c小鼠腹腔接种杂交瘤细胞6H5后收集腹水,ELISA法鉴定腹水中6H5抗体,间接免疫荧光技术检测6H5抗体与乳腺癌细胞MDA-MB-435、人肾癌细胞GRC-1、人舌癌细胞Tca8113中BLM、RecQ4和RecQ5的结合;以6H5抗体作为一抗,免疫组织化学染色法分析Jurkat、MDA-MB-435肿瘤细胞及肿瘤干细胞中RecQ解旋酶的表达水平。结果:经鉴定,小鼠腹水中6H5抗体阳性,该抗体能识别不同肿瘤细胞株中的BLM、RecQ4和RecQ5解旋酶,还可敏锐检测Jurkat、MDA-MB-435肿瘤细胞与肿瘤干细胞中RecQ解旋酶的表达; RecQ解旋酶在Jurkat肿瘤干细胞中表达水平明显高于Jurkat肿瘤细胞,而在MDA-MB-435肿瘤干细胞表达水平则低于MDA-MB-435肿瘤细胞,差异有统计学意义(P <0. 05)。结论:RecQ家族解旋酶可以作为多种肿瘤细胞及肿瘤干细胞表面标志物的备选指标。
Objective: To prepare monoclonal antibodies(6H5) against RecQ helicase,to identify its biological characteristics and carry out detection of RecQ helicase in tumor stem cells. Methods:Inoculating hybridoma cell 6 H5 in abdomen of Balb/c rats and then collecting ascitic fluid; adopting ELISA assay to identify 6 H5 antibody in the fluid,and indirect immunofluorescence to detect the effects of 6 H5 antibody and breast cancer cell MDA-MB-435,GRC-1 of human renal cancer cells,BLM,RecQ4 and RecQ5 in Tca8113 of human tongue cancer cells. Immunohistochemical method was used to analyze RecQ helicase expression level in Jurkat,breast cancer cell MDA-MB-435 and its stem cells. Results: 6 H5 antibody in ascitic fluid of rats abdomen was positive,and such antibody could identify BLM,RecQ4 and RecQ5 helicase in different tumor cells,and can also detect the expression of RecQ helicase expression in Jurkat,MDA-MB-435 tumor cells and tumor stem cells. RecQ helicase expression level in Jurkat tumor stem cell was obviously higher than Jurkat stem cell,while its expression in MDA-MB-435 tumor cells was lower than MDA-MB-435 tumor stem cell,difference was statistically significant(P < 0. 05). Conclusion: RecQ family helicase can be used as an alternative marker for tumor cells and tumor stem cell surface markers.

关键词(KeyWords)： RecQ解旋酶;单克隆抗体;免疫荧光;免疫组织化学染色;肿瘤
RecQ helicase;monoclonal antibody;immunofluorescence;immunohistochemistry;tumor

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金资助项目(81360349);; 贵州省优秀科技教育人才省长专项资金资助项目[黔省专合字(2006)57];; 贵州省国际科技合作计划基金资助项目[黔科合外G字(2011)7012];; 贵州省应用基础研究计划重大专项子课题资助项目[黔科合J重大字(2015)2003]

作者(Author): 张荣红,廉芳,周孟,贾铁文,何志旭,刘杰麟
ZHANG Ronghong,LIAN Fang,ZHOU Meng,JIA Tiewen,HE Zhixu,LIU Jielin

DOI: 10.19367/j.cnki.1000-2707.2018.10.017

参考文献(References)：

• [1]曾倩,崔芳芳,宇传华,等.中国癌症发病、死亡现状与趋势分析[J].中国卫生统计,2016,33(2):321-323.
• [2]陈万青,梁智恒,岑惠珊,等.中国肿瘤登记现况及发展[J].中国医学前沿杂志,2016,8(7):1-5.
• [3]邹波.中国老龄化的现状与积极应对[J].中国民政,2017(20):42-44.
• [4]李蕾,乔安娜,汪琼.中国老龄化问题的现状分析及对策研究[J].河北能源职业技术学院学报,2012,12(3):24-26.
• [5]RAYNARD S,BUSSEN W,SUNG P. A double Holliday junction dissolvasome comprising BLM, topoisomerase IIIα,and BLAP75[J]. Journal of Biological Chemistry,2006,281(20):13861.
• [6]张荣红,贾铁文,廉芳,等.抗Rec Q解旋酶单克隆抗体的制备和鉴定及应用[J].细胞与分子免疫学杂志,2018,34(2):169-174.
• [7]鲁媛,葛章文,刘杰麟.乳腺癌干细胞BLM,Rec Q4蛋白表达变化及意义[J].山东医药,2017,57(4):17-20.
• [8]廉芳,葛章文,李红,等.无血清培养富集乳腺癌干细胞的初步研究[J].贵阳医学院学报,2014,39(1):12-17.
• [9]ZHANG J,ZHANG Y,CHENG L,et al. Enrichment and characterization of cancer stem-like cells in ultra-low concentration of serum and non-adhesive culture system[J]. American Journal of Translational Research,2018,10(5):1552.
• [10]刘哲亮,邬娇,王林纤,等.人肺鳞癌干细胞的体外分离培养和鉴定[J].中国组织工程研究,2015,19(28):4526-4530.
• [11]杨佳,方志红,吴建春,等.人肺腺癌细胞系中肿瘤干细胞的分离培养及鉴定[J].中国肿瘤临床,2016,43(3):89-94.
• [12]SINGH S K,HAWKINS C,CLARKE I D,et al. Identification of human brain tumour initiating cells[J]. Nature,2004,432(7015):396.
• [13]郭哲,向邦德,姜经航,等.高转移潜能肝癌HCCLM3细胞中侧群细胞的分离及其干细胞特性鉴定[J].中国肿瘤生物治疗杂志,2015,21(2):220-224.
• [14]FONG D,YEH A,NAFTALOVICH R,et al. Curcumin inhibits the side population(SP)phenotype of the rat C6glioma cell line:towards targeting of cancer stem cells with phytochemicals[J]. Cancer Letters,2010,293(1):65-72.
• [15]潘睿.中国慢性病前瞻性研究队列恶性肿瘤发病与死亡分析[D].南京:南京医科大学,2017.
• [16]崔畅畅,柯学,吕慧侠.肿瘤干细胞靶向治疗研究进展[J].药学进展,2016,40(1):20-29.
• [17]SKUBITZ A P N,TARAS E P,BOYLAN K L M,et al.Targeting CD133 in an in vivo ovarian cancer model reduces ovarian cancer progression[J]. Gynecologic Oncology,2013,130(3):579-587.
• [18]CHEN J,LIU Q,XIAO J,et al. Ep CAM-antibody-labeled noncytotoxic polymer vesicles for cancer stem cellstargeted delivery of anticancer drug and siRNA[J]. Biomacromolecules,2015,16(6):1695-1705.
• [19]LAWSON D A,BHAKTA N R,KESSENBROCK K,et al. Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells[J]. Nature,2015,526(7571):131.
• [20]YANG Y,FAN Y,QI Y,et al. Side population cells separated from A549 lung cancer cell line possess cancer stem cell-like properties and inhibition of autophagy potentiates the cytotoxic effect of cisplatin[J]. Oncology Reports,2015,34(2):929-935.
• [21]IACOPINO F,ANGELUCCI C,PIACENTINI R,et al.Isolation of cancer stem cells from three human glioblastoma cell lines:characterization of two selected clones[J].Plo S One,2014,9(8):e105166.

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