【作者】 敖绪军；

【导师】 陈正堂；

【作者基本信息】 第三军医大学 ， 肿瘤学， 2008， 博士

【摘要】 肺癌是威胁人类健康的最常见恶性肿瘤,尽管不断有新的化疗药物和放疗手段出现,但肺癌总的5年生存率仍不足15%。肺癌细胞体外培养的实验表明,在1000～5000个细胞中仅有1个细胞能在软琼脂上形成克隆,提示不是每个肺癌细胞都具有驱动肺癌形成的能力。近年来的研究表明,肿瘤是一种干细胞疾病,在肿瘤组织中存在少量癌干细胞。现已经从多种肿瘤组织中成功分离出癌干细胞,包括白血病、黑色素瘤、脑瘤、乳腺癌、前列腺癌、胰腺癌和肠癌等。这些细胞具有无限的增殖能力、自我更新能力和分化能力,是肿瘤发生的根源。因此,探索癌干细胞的调控机制和寻找癌干细胞特异性分子,将为肿瘤的诊断和治疗提供有效的靶点。在调控干细胞自我更新、分化和增殖的研究中,一类调控性非编码微小RNA(microRNA,miRNA)的作用倍受人们关注。miRNA具有进化保守性、表达时空阶段性等特征,这些特征决定了它可能成为癌干细胞的特异性靶标。虽然肺癌干细胞是否存在特异性的miRNA目前尚未见文献报道,但与肺腺癌有关的miRNA中,let-7家族备受关注。let-7最早在线虫中发现,它调控细胞的分化和增殖的时序。let-7在肺癌组织中是低表达的,它能在转录后水平抑制RAS和HMGA2蛋白的表达,因此被认为是抑癌的“miRNA”。最近的文献显示let-7与癌干细胞关系密切,let-7通过改变乳腺癌干细胞自我更新和分化能力而抑制乳腺癌的发生。因此,深入阐明let-7在肺癌干细胞的功能,有望揭示let-7抑制肺癌的本质,并提供有效治疗肺癌的新方法。目的:检测从LLC细胞中分选的Sca-1+LLC细胞是否具有癌干细胞特性,并研究let-7在Sca-1+LLC细胞中的功能。方法:1. Sca-1+LLC细胞分离、培养和鉴定:免疫荧光检测Sca-1在LLC细胞的表达,以Sca-1作为磁珠分选的表面标志,从LLC细胞中分选Sca-1+LLC细胞和Sca-1-LLC细胞,流式细胞仪检测分选后细胞的纯度。为了鉴定Sca-1+LLC细胞是否具有癌干细胞特性,将分选的Sca-1+LLC细胞和Sca-1-LLC细胞分别培养于含有10% FBS的RPMI-1640培养基和含有碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)和胰岛素-转铁蛋白-硒(ITS)的无血清培养基。体外培养检测Sca-1+LLC细胞自我更新能力、分化能力以及对化疗药物的耐药能力,体内成瘤实验比较Sca-1+LLC细胞与Sca-1-LLC细胞致瘤能力的差异。2.检测let-7家族成员的表达:Trizol法分别从Sca-1+LLC细胞和Sca-1-LLC细胞中提取总RNA,使用TaqMan MicroRNA检测试剂盒检测let-7家族成员成熟体的表达,包括let-7a、let-7c、let-7d、let-7f和let-7g,通过与内参snoRNA202比较,计算检测基因的相对含量。3. let-7a慢病毒过表达载体构建、包装和滴度测定:通过PCR技术扩增pre-let-7a cDNA ,将扩增的片段插入载体pGCSIL-GFP中,形成重组的慢病毒载体pGCSIL-let-7a。pGCSIL-let-7a与辅助质粒pHelper 1.0和pHelper 2.0共转染入包装细胞293T细胞中,48小时后收集并浓缩病毒上清液。将病毒液进行系列稀释后感染293T细胞,通过实时定量PCR技术,检测各组293T细胞GFP含量来测定病毒的滴度。4. Lenti-let-7/Sca-1+LLC细胞分离、培养和鉴定:在感染复数为2、20和200时,将let-7a慢病毒颗粒和空载体病毒颗粒分别感染LLC细胞,通过在荧光显微镜下计数阳性细胞来测定各组的感染效率。利用磁珠分选技术将Sca-1+LLC细胞从感染的LLC细胞中分选出来,并分别命名为Lenti-let-7/Sca-1+LLC细胞和Lentivector/Sca-1+LLC细胞。体外培养检测两者在自我更新、增殖和耐药能力的差异,体内成瘤实验比较两者致瘤性的差异。5. let-7联合紫杉醇对lewis肺癌的治疗效应:在lewis肺癌体积达到100～200mm3时,将荷瘤小鼠分成五组。前三组分别单独给予let-7a慢病毒颗粒(1×108 TU/只)、空载体病毒颗粒(1×108 TU/只)和生理盐水(100μl)瘤内注射;第四组给予腹腔注射紫杉醇(l0 mg/kg,连续使用10d);第五组给予let-7a慢病毒颗粒(1×108 TU/只)和紫杉醇(l0 mg/kg,连续使用10d)联合治疗。在注射后连续测量各组肿瘤的体积,并在瘤内注射18 d后处死小鼠,计数各组肺转移节结数、瘤重和抑瘤率,免疫组化检测各组肿瘤K-ras蛋白的表达。结果:1.在荧光显微镜下可以见到少量LLC细胞Sca-1染色阳性,流式细胞仪检测Sca-1阳性细胞百分比为(10.23±1.65)%。磁珠分选后Sca-1阳性细胞百分比为(93.92±1.07)%。在体外无血清培养体系中,Sca-1+LLC细胞可以生长为非贴壁的、多细胞的细胞球,细胞球形成率为10%。而Sca-1-LLC细胞肿瘤细胞球形成率为1%。在含有血清的培养基中,Sca-1+LLC细胞可以分化为Sca-1-LLC细胞,而Sca-1-LLC细胞不能分化为Sca-1+LLC细胞。Sca-1+LLC细胞对于化疗药物具有更强的耐药性,这与它高表达ABCG2 mRNA有关。体内的成瘤实验表明,Sca-1+LLC细胞的致瘤能力是Sca-1-LLC细胞的25倍以上。2.实时定量PCR检测显示,let-7a、let-7d和let-7g在Sca-1+LLC细胞的表达水平有明显的下降。与Sca-1-LLC细胞相比,分别下降了1.32倍、1.27倍和1.45倍。而let-7c和let-7f的表达水平在两者之间没有显著性的差别。3.重组的慢病毒载体pGCSIL-let-7a通过PCR扩增、酶切以及测序,证明目的片段(MI0000557)成功克隆到慢病毒载体中。将pGCSIL-let-7a与辅助质粒共转染293T细胞,48h后收集并浓缩的病毒颗粒滴度可达到2×109TU/ml。4.在复感染指数(MOI)为20时,病毒感染LLC细胞的效率约为70%。病毒感染后,LLC细胞系中Sca-1阳性细胞的比例下降至5.47%。Lenti-let-7/Sca-1+LLC细胞肿瘤细胞球形成率为(0.01±0.005)%,Lentivector/Sca-1+LLC细胞肿瘤细胞球形成率为(0.09±0.015)%,两者有显著性差异(P<0.01)。而且培养7 d后,Lenti-let-7/Sca-1+LLC细胞形成的肿瘤细胞球体积较Lentivector/Sca-1+LLC细胞小。Lenti-let-7/Sca-1+LLC细胞的耐药能力较Lentivector/Sca-1+LLC细胞下降,在各化疗药物中两者均有显著性差异(P<0.01)。体内成瘤实验,lentivector/Sca-1+LLC细胞形成肿瘤所需的细胞为2×104个,而lenti-let-7/Sca-1+LLC细胞需要5×105以上的细胞。5.生理盐水组和Lentivector组肿瘤生长速度最快,两者之间无显著性差异(P >0.05)。let-7a或紫杉醇单独治疗,肿瘤生长均出现明显抑制,在28 d抑瘤率分别为49.6%和70.1%,较生理盐水组均有显著性差异(P <0.01)。Lenti-let-7a联合紫杉醇治疗的抑瘤率为92.9%,较生理盐水组和let-7a组均有显著性差异(P <0.01),其中有一只小鼠(33%)在治疗12d后肿瘤消失。let-7病毒颗粒抑制肿瘤的效应与K-Ras蛋白的下降相关。结论1.在LLC细胞系中,Sca-1是癌干细胞的表面标志之一,Sca-1+LLC细胞具有癌干细胞的特性。2.在Sca-1+LLC细胞中let-7是低表达,let-7的低表达是维持Sca-1+LLC细胞干细胞特性的重要条件。3. let-7a慢病毒载体是肺癌基因治疗的一个新的途径,联合let-7和化疗药物有望成为治愈肺癌的有效手段。更多还原

【Abstract】 Lung cancer is the most common cause of cancer-related mortality worldwide. Although new chemotherapy agents and radiotherapy have improved survival and quality of life of patients, the overall five-year survival rate for such tumor is lower than 15%. In vitro data have shown that only 1 in 1,000 to 5,000 lung cancer cells forms colonies in soft agar assay, indicating that not every lung cancer cell was capable of tumor initiation. Evidence is accumulating that a subset of cancer cells within some tumors, contain a minor population of“cancer stem cell”(CSC). CSC have been isolated and expanded from leukemia, and several solid tumors, including melanoma, breast, brain, prostate, pancreatic and colon carcinomas. These cells display unlimited proliferation potential, ability to self-renew and capacity to generate a progeny of differentiated cells. Therefore, focusing research efforts on the CSC may drive important advances in our understanding of cancer biology and developing potential cures for these devastating diseases.MiRNAs are small (22–25 nucleotides in length) noncoding RNAs that can effectively reduce the translation of target mRNAs by binding to their 3-untranslated region (UTR). let-7 is originally identified in Caenorhabditis elegans as a regulator of developmental timing and cellular proliferation. The expression level of let-7 is reduced in human lung cancer, and it is show to be the anticancer miRNA that represses RAS and/or HMGA2 expression at the translational level. A recent study suggests that let-7 suppresses tumorigenesis via alteration of self-renewal and differentiation of breast cancer stem cells. Therefore, analyzing the relationship between lung cancer stem cell and let-7 may insight into let-7-mediated tumor suppression and also establish unique treatment for lung cancer.ObjectiveTo isolate and characterize of tumorigenic, stem-like Sca-1 positive cells in lewis lung carcinoma( LLC) cell line, and research the expression levels of let-7 to assess the function of let-7 in Sca-1+LLC cells.Methods 1. Immunofluorescence staining technology was used to detect the expression of the putative stem cell marker Sca-1 in lewis lung carcinoma. Then Sca-1+LLC cells and Sca-1-LLC cells were sorted by magnetic activated cell sorting from lewis lung carcinoma cell line, and the purity was analyzed by flow cytometry. After isolation, Sca-1+LLC cells and Sca-1-LLC cells were cultured in 10% FBS RPMI-1640 culture medium or serum-free medium supplemented with bFGF, EGF, and ITS. To test the hypothesis that Sca-1+LLC cells were enriched for tumor-initiating cells, the ability of self-renewal, differentiation and resistant to drugs were evaluated in vitro. In vivo, Sca-1+LLC and Sca-1-LLC cells were inoculated into C57BL/6 mice at different injection dose to assess the tumorigenic distinction.2. Total RNA was extracted respectively from Sca-1+LLC cells and Sca-1-LLC cells using Trizol reagent. Then the TaqMan?MicroRNA Assays were used to detect and accurately quantify mature let-7 family, including of let-7a, let-7c, let-7d, let-7f, and let-7g. The relative amount of gene transcripts was normalized to snoRNA202.3. In order to generate the recombinant lentiviruses which carrying pre-let-7a gene, the pre-let-7a cDNA was amplified by PCR technique and subcloned into the lentiviral vector, pGCSIL-GFP, to generate the lentiviral expression vector, pGCSIL-let-7a. Recombinant lentiviruses were produced by 293T cells following the co-transfection of pGCSIL-let-7a with packaging plasmids pHelper 1.0 and pHelper 2.0. The viral supernatant was collected 48 h after transfection, and viral titers determined by transducing 293T cells at serial dilutions and analyzing GFP expression by real-time PCR.4. The LLC cells were infected by lentiviral expression vector and the control vector at the MOI of 2, 20, or 200, and the infectivity was determined by counting the cells that showed fluorescence under microscopic examination. Then the Sca-1+LLC cells were sorted by magnetic activated cell sorting and respectively named Lenti-let-7/Sca-1+LLC cells and Lentivector/Sca-1+LLC cells. The difference between Lenti-let-7/Sca-1+LLC cells and Lentivector/Sca-1+LLC cells were evaluated by self-renewal assays, proliferation assays, chemoresistance assays and tumorigenicity assays.5. To assess the effectiveness of in vivo gene therapy, some groups received let-7a lentiviral vector or control vector intratumoral injection when the volume of lewis lung carcinoma reached to 100～200mm3, and other groups received let-7a lentiviral vector in combination with paclitaxel. Tumor size was followed in the case of treatment with lentiviral vector, and the mice were sacrificed after 18 d to assess tumor formation rate, weight, lung metastasis, and the expression of K-Ras protein.Results1. Under the fluorescence microscope, a small proportion LLC cells had sparse light of red fluorescence, and flow cytometry shown that (10.23±1.65) % of LLC cells expressed the membrane antigen Sca-1. After isolation by Magnetic Cell Sorting, the Sca-1 positive fraction was raised to (93.92±1.07) %. Under the serum-free medium, Sca-1+LLC cells could grow as nonadherent, multicellular spheres, and had clonogenic frequencies of 10%. Whereas, the clonogenic frequencies was only 1% in Sca-1 - LLC cells. Under serum-cultured conditions, Sca-1+LLC cells could differentiate into Sca-1-LLC cells but Sca-1-LLC cells could not differentiate into Sca-1+LLC cells. Sca-1+LLC cells exhibited higher resistance to chemotherapeutic drugs than Sca-1-LLC cells.The drug-resistance machanism may be relate to the over expression of ABCG2 mRNA. In vivo, the tumor formation ability of Sca-1+LLC cells were 25 times higher than Sca-1-LLC cells.2. Real-time PCR analysis showed that let-7a, let-7d and let-7g were markedly reduced by 1.32-fold, 1.27-fold, and 1.45-fold relative to control, respectively. The changes in expression levels of let-7c and let-7f were not significantly different from controls.3. The sequence of cloned DNA was in conformity with the sequence deposited in GeneBank (MI0000557) by PCR, enzyme digestion and sequencing. The recombinant lentiviruses which carrying pre-let-7a gene could be product by co-transfection pGCSIL-let-7a to 293T cells, and the titer of virus reached to 2×109TU/ml.4. The infectivity of the lentiviral vector was approximately 70% when infected at the MOI of 20. After infection, the percent of Sca-1+LLC cells in LLC cell line was decreased to 5.47%. Comparing with the lentivector/Sca-1+LLC cells, the lenti-let-7/Sca-1+LLC cells had lower clone formation efficiency, and more sensitiveness to chemotherapeutic drugs. lentivector/Sca-1+LLC cells generated a tumor with 2×104cells compared with at least 5×10~5 needed for lenti-let-7/Sca-1+LLC cells.5. A single intratumoral injection of the let-7a lentiviral vector can significantly reduce the volume of primary tumor and the number of lung metastasis. Moreover, let-7a lentiviral vector can completely regressed tumors in combination with paclitaxel in 33% of animals.These inhibition was correlated with a decrease in K-Ras protein.Conclusion1. Sca-1 is one of the markers for cancer stem cells in Lewis lung carcinoma cell line, and Sca-1+LLC cells exhibit characteristics of a tumor-initiating, cancer stem cell phenotype.2. let-7 expression is markedly reduced in Sca-1+LLC cells compared with Sca-1-LLC cells, and maintenance of an undifferentiated state of Sca-1+LLC cells require reduced let-7 levels.3. The let-7a lentiviral vector offers a powerful new strategy for lung cancer gene therapy. Combination of let-7 and chemotherapy may be an effective way to cure lung cancer.更多还原