【作者】 吴俊；

【导师】 雷霆；

【作者基本信息】 华中科技大学 ， 外科学， 2012， 博士

【摘要】 目的原代培养出具有干细胞特性的胶质瘤干细胞并进行体内体外的鉴定，为胶质瘤干细胞靶向治疗实验奠定基础。方法术中留取高级别胶质瘤标本(术后病理证实WHOⅢ或以上胶质瘤)，组织胰酶消化法提取细胞，用悬浮神经球培养方法培养出胶质瘤干细胞(Glioblastomastem-like cell, GSC)；免疫荧光法、RT-PCR法检测GSCs表面标志物，在裸鼠(Bulb/cnude mice)颅内再次成瘤实验检测GSCs成瘤性。对数据采用SPSS17.0进行统计学分析。结果在悬浮神经球干细胞培养条件下，成功培养出成球生长的胶质瘤干细胞球(GSC-Sph)；RT-PCR、Western Blotting和免疫荧光检测提示原代培养的GSCs高表达CD133(3.02%~4.92%); Western Blotting检测提示GSCs在蛋白水平高表达Nestin；将原代培养的GSCs种植于裸鼠脑内，8周后成瘤，成功构建裸鼠原位移植瘤模型。结论通过留取人高级别胶质瘤原代悬浮神经球干细胞培养方法可以得到具有干细胞特性的GSCs。其高表达干细胞表面标志物CD133和Nestin。GSCs原位种植于鼠脑8周后成瘤，原代GSCs有成瘤性。目的构建针对细胞周期检测点激酶1和2(Checkpoint kinase1/2, Chk1/2)基因的短发夹RNA (shRNA)表达载体，包装成慢病毒，建立稳定转染的GSCs细胞株，为研究干扰Chk1或Chk2基因的表达对治疗脑胶质瘤的方法研究提供理论基础。方法从GenBank数据库中查找Chk1和Chk2基因的核苷酸序列，根据所查序列分别设计出能转录的短发夹样RNA (shRNA)的DNA序列，命名为Chk1-shRNA和Chk2-shRNA，同时设计1条阴性对照DNA序列，命名为negative-shRNA，设计的序列合成后与pLKO.1-TRC质粒载体连接，构建质粒，并包装慢病毒。重组表达慢病毒载体转染原代GSCs，用嘌呤霉素筛选后扩增获得稳定细胞株。逆转录酶-聚合酶连反应(RT-PCR)和免疫印迹(Western blot)分别在核酸水平及蛋白水平上检测Chk1和Chk2的表达情况。结果成功构建重组质粒后，利用限制性酶切位点和琼脂糖凝胶电泳鉴定shRNA片段成功插入到预定位点；送公司进行基因序列检测，与设计序列一致。利用嘌呤霉素抗性对转染慢病毒后的GSCs细胞筛选出稳定转染三种质粒的细胞，干扰Chk1组和干扰Chk2组细胞Chk1和Chk2的表达情况在mRNA水平和蛋白水平上显著低于阴性对照组。结论构建了干扰Chk1或Chk2基因的shRNA慢病毒载体，成功转染GSCs后可抑制Chk1或Chk2基因的表达，为下一步探讨Chk1和(或) Chk2基因在脑胶质瘤细胞中的生物学作用奠定基础。目的研究干扰Chk1/2基因表达对脑胶质瘤干细胞放疗抵抗性的影响，为胶质瘤的靶向基因治疗提供新的途径。方法从GenBank数据查找出Chk1和Chk2基因的DNA序列，并以此各设计1条短发夹基因RNA （Small hairpin RNA, shRNA）的DNA序列，构建慢病毒并转染GSCs进行传代扩增。用RT-PCR和Western blot方法分别在核酸和蛋白水平上测定Chk1及Chk2的表达。转染后的GSCs经X照射后，进行细胞周期和凋亡的检测。结果RT-PCR和Western blot提示稳定转染Chk1-shRNA和Chk2-shRNA细胞在蛋白和mRNA水平上Chk1和Chk2的表达被抑制。放疗后，细胞周期检测提示对照组60.28±1.28%阻滞在G2/M期，而Chk1-shRNA组细胞仅22.37±2.01%在G2/M期，与未放疗组细胞23.77±2.31%相差不大(P>0.05)；Chk1-shRNA组细胞放疗后的凋亡率较对照组和Chk2-shRNA组细胞高，而对照组与Chk2-shRNA组之间差异无显著性意义。结论慢病毒转染细胞可以有效的将干扰Chk1和干扰Chk2基因带到GSCs并发挥作用，干扰Chk1可以增加放疗对GSCs的敏感性，为胶质瘤的治疗提供新的治疗途径。更多还原

【Abstract】 Objective To obtain glioma stem-like cells (GSCs) primary and identify the stem cellcharacteristics in vivo and in vitro, establish the foundation for glioma targeted therapy.Methods Specimens of high grade glioma were obtained from operations, the grade wasconfirmed by pathological examination, WHOⅢ or above. The specimens were digested bytrypsin for extraction cells, suspension neurosphere culture to cultivate glioma stem-likecells. Immunofluorescence assay and RT-PCR assay for detection of GSCs surface markers.GSCs were planted into brain of nude mice to detect the tumor formation of GSCs. Thedata were analyzed by SPSS17.0.Results The GSCs were successfully cultured in the suspension neural stem cell cultureconditions, CD133(11.02%~33.55%) were expressed highly in the primary GSCsaccording to the RT-PCR, western blotting and immune fluorescent. Nestin was alsoexpressed highly in protein level. The GSCs were planted into brain of nude mice, the brainbearing tumor after8weeks, the model of brain tumor was built successfully.Conclusions The GSCs could be obtained primary from high grade glioma specimens withthe way of suspension neurosphere culture and the GSCs exhibited the properties of thestem-like cell. The GSCs cell line was tumorigenic. Objective Construct the lentiviral expression vectors of shRNA interference specific toChk1or Chk2gene, then to screen the stably transfected cells of GSCs. And study on theinterference Chk1or Chk2gene to GSCs.Methods The DNA sequences of Chk1or Chk2were obtained from the Genbank and theshRNA were designed and synthesized, and one scrambled shRNA sequences served asnegative control. The shRNA were inserted into plasmids of pLKO.1. The recombinantlentiviral vectors were transfected into GSCs, and the stably transfected GSCs wereobtained after being screened by puromycin. Reverse transcriptase-polymerase chainreaction (RT-PCR) and Western blotting were adopted to detect the inhibitory effect onChk1or Chk2at the mRNA level and the protein level respectively.Results The expressions of Chk1or Chk2were significantly down-regulated by shRNA oflentiviral vectors in the stably transfected Chk1-shRNA and Chk2-shRNA GSCs. Theresults were tested by Western blotting and RT-PCR.Conclusions The expression of Chk1or Chk2in the GSCs can be significantlydown-regulated by RNA interference (RNAi) which mediated by the shRNA of lentiviralexpressing vectors. The stably transfected GSCs were obtained for further study. Objective Study on lentivirus transfected the interfered Chk1/Chk2gene to primarygloblastoma stem-like cells (GSCs) for therapy of radiosensitization. it would be a new methodcan be chosed to treatment of glioma.Methods The DNA sequences of Chk1or Chk2gene were obtained from the Genbank andshRNA were designed and synthesized. The shRNA lentiviral vectors were constructed andtransfected into GSCs. Reverse transcriptase-polymerase chain reactions (RT-PCR) andWestern blotting were used to detect the inhibitory effect on Chk1or Chk2at the mRNAlevel and the protein level in the GSCs. X-ray treated these GSCs, then cell cycle andapoptosis were detected.Results After radiation, most of group radiation and group radiation＆interfere Chk2wereblocked in G2/M; The rate of apoptosis after radiation: Group interfere Chk1higher thangroup control and group interfere Chk2.Conclusions Lentiviral transfection interfere with Chk1and Chk2could down-regulateChk1and Chk2genes expression, which could improve the sensitivity of GSCs inradiotherapy. It may be a good method in the treatment of glioblastoma.更多还原