【作者】 钱莘；

【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】 背景和目的肺癌是严重危害人类健康的常见恶性肿瘤之一,近年来,其发病率和病死率呈全球性上升趋势。目前,肺癌早期诊断技术的提高和以手术、化疗、放疗和靶向治疗为主的综合治疗方法取得了较大的进步,但总的来说,肺癌的治疗效果不尽人意,其总的5年生存率仍然低于15%,因此,肿瘤学家们正在积极寻求新的解决办法和手段。近年来,肿瘤干细胞在肿瘤形成、转移、复发、耐药等方面的作用逐渐引起了肿瘤学家们的关注,该领域的研究为肺癌的早期诊断和合理治疗提供了新的契机。肿瘤干细胞是一群稀少的未分化的肿瘤细胞,具有无限增殖和自我更新的能力,能够产生大量分化细胞的前体细胞,最终形成肿瘤。肿瘤干细胞首先是在急性髓细胞样白血病的研究中发现的,随后陆续在人的实体瘤如黑色素瘤、乳腺癌、脑瘤、前列腺癌、视网膜母细胞瘤、胰腺癌、肝母细胞瘤和结肠癌中发现了肿瘤干细胞的存在;在动物模型中胃癌干细胞、肺癌干细胞也陆续得到了确认。众多研究表明,肿瘤干细胞与正常干细胞具有相似的特性,正常干细胞可能是更为原始的前体肿瘤细胞,因其具有信号通路(如Notch,Wnt,Hh等)活化和不定向分化的特性。如果致癌突变发生在这些正常的干细胞上,很可能会促使这些正常的干细胞无限生长,最终形成肿瘤细胞。肺具有复杂的组织结构和独特的气血交换功能,肺内的细胞种类至少有40多种,其中基底细胞、克拉拉细胞(Clara cell)和2型肺泡细胞(alveolar cells type 2,AT2)均被认为是具有干细胞特性的肺前体细胞。在原癌基因K-ras诱导的小鼠肺腺癌模型中有一群共表达Clara细胞特异性抗原(Clara cell specific antigen,CCA)和肺泡表面活性蛋白C(surfactant protein C,SP-C)的细胞,位于终末细支气管肺泡连接区(bronchoalveolar duct junction,BADJ),这群CCA+SP-C+双阳性细胞(double positive cells, DPCs)被称为支气管肺泡干细胞(bronchoalveolar stem cells,BASCs),其表面标记是CD45-CD31-Sca-1+CD34+。在体外研究中发现,BASCs具有典型的自我更新和多向分化的潜能,被认为是远端肺上皮的干细胞。在正常情况下BASCs处于静止状态,而在K-ras基因活化引起的终末细支气管和肺泡上皮的不典型增生和腺瘤中,其数量明显增加,提示BASCs可能是小鼠肺腺癌的起源细胞。侧群细胞(Side populations,SP)是肺组织中另一群特殊类型的未分化细胞,其表达CD34、乳腺癌耐药蛋白1(breast cancer resistance protein1,BCRP1)等标记,同样具有干细胞相关特性。2007年Eramo等在人的肺癌组织中发现一群具有自我更新、多向分化能力的CD133+细胞,其同时具有肿瘤细胞的恶性特征,因此被命名为肺癌干细胞。如上所述,肺癌干细胞很可能来源于肺内正常干细胞,然而正常的肺干细胞如何发生恶性转化成为肺癌干细胞,其详细机制有待进一步研究。尽管目前关于肺癌发生的研究多集中在一些已知的基因和蛋白上,近年来,其它一些未知的调控因子如非编码RNA在肺癌中的作用也逐渐引起了人们的重视。微RNA(micro RNA,miRNAs)是一类约22个核苷酸长度的的调控性非编码小分子RNA,存在于多种生物体内,在细胞的生长发育、凋亡、干细胞分化和肿瘤发生中均发挥了重要作用。一些特异的miRNAs多位于恶性肿瘤的常见染色体缺失或易位区域,肿瘤高表达和低表达的miRNAs分别具有癌基因和抑癌基因的功能,表明miRNAs在肿瘤的形成中扮演了重要角色。越来越多的研究证明,miRNAs的异常表达与肺癌的形成、发生具有密切的关系。同时,近一步研究发现一些特异性的miRNAs表达于多种类型的干细胞内,提示miRNAs很可能也参与调控干细胞的自我更新和分化,这一观点已在果蝇的研究中得到证实。miRNAs具有使干细胞对环境刺激不敏感的特性,从而使干细胞越过因环境刺激形成的细胞停滞点进入持续循环的状态,而肿瘤细胞很可能就是利用了这一机制,通过无限增殖最终形成肿瘤细胞。因此,miRNAs表达谱的建立对于研究肺癌的诊断和治疗具有潜在的应用价值。鉴于miRNAs在参与恶性肿瘤发生、发展,并在维持干细胞自我更新及多向分化中所发挥的重要作用,我们推测:调控正常肺干细胞自我更新和分化的miRNAs发生异常突变,很可能是正常肺干细胞恶性转化形成肺癌干细胞的重要分子机制之一。目前,虽然在人、小鼠中各种组织的miRNAs表达谱都有所研究,但是关于肺干细胞的miRNAs表达谱至今尚未见报道。为了验证肺癌干细胞及其特征性miRNAs在肺癌发生中的重要作用,因此,我们拟从肺干细胞入手,首先,通过双重免疫荧光染色技术鉴定临床肺癌组织和鼠正常肺组织中DPCs的存在;以此为基础,分离、鉴定小鼠的DPCs细胞(即BASCs);流式细胞仪分选出BASCs;微阵列技术检测BASCs的miRNAs表达情况,并应用实时定量荧光PCR技术(quantitative real-time polymerase chain reaction,qRT-PCR)进行验证;生物信息学初步预测所选miRNAs的功能及其作用靶点,最后通过荧光素酶报告基因载体进行验证。本研究以临床肺癌组织发现具有干细胞表面分子标志的DPCs细胞为基础,进一步以肺干细胞特征性miRNAs为突破点研究小鼠正常肺干细胞发生恶性突变的分子机制,不仅鉴定了正常小鼠肺干细胞的miRNAs表达谱,而且为后续深入探讨miRNAs调控干细胞自我更新和分化及肺干细胞向肺癌干细胞转变的分子机制奠定了基础。方法1.双重免疫荧光染色法鉴定人肺癌组织内CCA+SP-C+细胞(DPCs):取人的正常肺组织、肺鳞癌、肺腺癌的癌组织和其各自对应的癌旁组织,冰冻切片,进行双重免疫荧光染色,通过激光扫描共聚焦显微镜观察肺内是否存在DPCs细胞。每例标本观察时镜下随机选择4～6个视野,计数100个肺细胞,得出每百个细胞内DPCs百分率,采用SPSS 11.0统计软件包对数据进行分析。2.小鼠BASCs的培养鉴定和分选:取成年鼠(小鼠、大鼠)、新生鼠(小鼠、大鼠)的正常肺组织,双重免疫荧光染色法鉴定BASCs。采用胶原酶和分散酶联合消化小鼠肺组织制备肺单细胞悬液,经免疫磁珠分选Sca-1+细胞;胶原蛋白预先包被培养板;无血清乳腺上皮细胞培养基培养Sca-1+细胞;双重免疫荧光染色CCA和SP-C鉴定BASCs ;通过流式细胞仪分选CD45-CD31-Sca-1+CD34+细胞(即BASCs) ,CD45-CD31-Sca-1-CD34-细胞作为对照。3.小鼠BASCs miRNAs表达谱的鉴定:将分选出的细胞常规提取RNA后,经YM-100 (Millipore)微离心过滤柱抽提小于300核苷酸长度的小RNA;微阵列法检测BASCs和其对照细胞的miRNAs表达谱,筛选出差异miRNAs;构建miRNAs特异性TaqMan MGB探针,qRT-PCR法验证所选差异miRNAs在两种细胞的表达;生物信息学初步预测被选miRNAs的功能及其作用靶点(WEE1基因);最后通过荧光素酶报告基因载体进行验证。结果1. CCA+SP-C+细胞(DPCs)存在于人肺腺癌组织中:通过双重免疫荧光染色法在人的肺腺癌组织、正常肺组织中首次发现了DPCs,而在鳞癌组织中未见DPCs;在人的肺癌组织中DPCs数量明显多于相对应的癌旁组织(P<0.05)。2.建立了小鼠BASCs的鉴定、分离和培养方法:在成年鼠和新生鼠的正常肺组织中均发现了BASCs,其主要定位于BADJ区;通过胶原酶和分散酶联合消化小鼠肺组织,平均每只成年小鼠可得到有核细胞总数为1.6～1.8×107;经磁珠分选后的细胞其表面分子Sca-1+表达的阳性细胞百分率明显高于未经分选的肺单细胞(87.3%±5.9%vs 9.6%±1.8%,P<0.05);在无血清乳腺上皮细胞培养基中Sca-1+细胞在第6天可形成BASCs克隆和其他未知的细胞克隆;在有血清的条件下BASCs可分化为AT2细胞;流式细胞仪检测CD45-CD31-Sca-1+CD34+细胞占肺细胞总数的0.7%～1.1%,根据此分子标记能够分选出纯度约98%的BASCs。3.建立了小鼠BASCs的miRNAs表达谱:从BASCs和对照细胞中分别提取小RNA220ng和720ng,微阵列法检测miRNAs的表达并比较差异;以对数(log2)为差异倍数,比较BASCs和其对照细胞,共发现116个miRNAs的表达具有显著性差异(P<0.01),其中有56个miRNAs在BASCs高表达,60个miRNAs在BASCs低表达。在这些差异miRNAs中选取了10个与细胞周期、干细胞分化、肿瘤发生相关的miRNAs,即:miR-142-3p,miR-451,miR-106a,miR-142-5p,miR-15b,miR-20a,miR-106b,miR-25,miR-486和miR-497;通过qRT-PCR法检测其在BASCs和对照细胞中的表达,结果显示在BASCs中miR-497高表达,而其余9个miRNAs即miR-142-3p,miR-451,miR-106a,miR-142-5p,miR-15b,miR-20a,miR-106b,miR-25,miR-486在BASCs中低表达;qRT-PCR的检测结果与miRNAs芯片检测结果一致。生物信息学初步分析所选差异miRNAs的作用靶点和功能。随后,我们选取了在BASCs高表达的miR-497,通过miRNAs靶基因预测软件(TargetScan、MiRbase、miRanda)预测其作用靶点,取所有3个软件均预测到的Wee1做为靶基因,最后通过荧光素酶报告基因载体验证预测靶基因的准确性(荧光素酶验证预测靶基因的实验尚在进行中)。结论本研究首次在人的肺腺癌组织中发现了DPCs,而且证实了DPCs在鼠肺中存在的广泛性;初步建立了小鼠DPCs(即BASCs)体外克隆培养体系;通过微阵列法鉴定了小鼠BASCs的miRNAs表达谱,与对照细胞相比,发现116个miRNAs的表达具有显著性差异(56个高表达,60个低表达);初步证实miR-497可能通过靶基因Wee1调控BASCs的自我更新和分化。本课题不仅为深入研究miRNAs调控小鼠肺干细胞的自我更新和分化建立了实验平台,而且为进一步探讨人肺干细胞、miRNAs在肺癌发生中的作用提供了借鉴,有望为肺癌的早期诊断、预后监测及靶向治疗提供新的分子靶点。更多还原

【Abstract】 Background and ObjectiveLung cancer is the most common cause of cancer-related mortality worldwide. The overall five-year survival rate for the patients with lung cancer is still below 15% although the great progress in the early diagnosis and chemotherapy, radiotherapy, immune therapy and target therapy has been made during last fifty years.Tumorigenic cancer progenitor cells, also designated as cancer stem cells or cancer-initiating cells, has been proposed to be involved in cancer initiation and progression to metastatic disease states and resistance to conventional therapies. The breakthrough on cancer stem cells provides a new avenue of research to explore the pathogenesis of tumor and improve the efficiency of treatment. Cancer stem cells are a rare population of undifferentiated tumorigenic cells responsible for initiation, maintenance and spreading of tumor. These cells display unlimited proliferation potential, ability of self-renew and capacity to generate a progeny of differentiated cells that constitute the major tumor population. Cancer stem cells have been first isolated and expanded from leukemia and subsequently been reported in several human solid tumors including melanoma, breast cancer, brain cancer, prostate cancer, retinal glioblastoma, pancreatic cancer, hepatoblastoma and colon carcinoma. The gastric cancer stem cells and the lung cancer stem cells were both identified in the animal models as well.In light of the cancer stem cells-based model, normal stem cells might be considered as proto-tumorigenic cells endowed with some properties typical of malignant cells, including the constitutive activation of survival pathways and the ability to proliferate indefinitely. Oncogenic mutations occurring in such a favorable background may turn the finely regulated growth potential of normal stem cells into the aberrant uncontrolled growth of cancer cells. The lung is an extremely complex, conditionally renewing organ composed of at least 40 differentiated cell type lineages. The candidate stem progenitor cells are the basal cells for mucosal gland development and renewal of the branched epithelium of the trachea, the Clara cells of the bronchiole, and the type-2 pneumocytes of the alveolus (AT2). The regenerative potential stem cells residing in the bronchoalveolar junction of adult lungs have been further identified and characterized in a mouse model of lung carcinogenesis as CD45-CD31-Sca-1+CD34+ cells expressing both cytoplasmic Clara cell specific antigen(CCA) and surfactant protein-C proteins(SP-C), which are specific markers for Clara cells and AT2 cells, respectively. Some reports have described lung stem cells as cells expressing antigens typical of undifferentiated cells, such as CD34 and breast cancer resistance protein1 (BCRP1). In 2007, a rare population cells displaying the membrane antigen CD133 were identified in human lung tumor tissues, which show the ability of unlimited proliferation and self-renew, furthermore, they can differentiate into cells constituting the major tumor population. However, whether lung cancer stem cells might derive from the mutated normal lung stem cells remains to be elucidated.Previously unknown markers, such as noncoding RNA gene products, may also lead insight into the biology of lung cancer, although known genes and proteins have already yielded plenty of information. Micro RNAs (miRNAs) are a class of naturally occurring small noncoding RNA molecules, which are found in diverse organisms, involved in various biological processes, including developmental timing, apoptosis, stem cell division, disease and cancer in animals and humans. In addition, some miRNAs may function as oncogenes or tumor suppressors. More than 50% of miRNAs genes are located in cancer-associated genomic regions or in fragile sites, suggesting that miRNAs may play a more important role in the pathogenesis of a limited range of human cancers than previously thought.Increasing evidences has suggested the potential involvement of altered regulation of miRNAs in the pathogenesis of lung cancer. These findings demonstrated that miRNA splay an essential role in lung cancer pathogenesis, and the miRNAs profiles may be potentially useful for lung cancer diagnosis and prognosis. Previous studies have presented that some miRNAs are differentially expressed in stem cells, suggesting a potential role in stem cells regulation, such as self-renewal. Recent results from Drosophila and mouse have shown that miRNAs are important regulators for stem cells self-renewal, differentiation and division. MiRNAs may be involved in the mechanism that makes stem cells insensitive to environmental stimuli that would normally halt most cells at the G1/S checkpoint. The implication is that the mechanism used by stem cells to overcome this checkpoint could, possibly be usurped by tumor cells.Given that miRNAs contributing remarkably to both development of normal stem cells and cancer pathogenesis, we proposed that mutations must occur to turn BASCs to lung cancer cells, which is the one of important origin of lung adenocarcinoma. We therefore aimed to set up a reliable, in-house miRNAs microarray platform for mouse lung stem cells research. Although the expression profiling of miRNAs in human and mouse organs has been detected by some groups, the expression in the lung stem cells has yet to be defined.Therefore, we first carefully looked at the existence of DPCs in the lung both from adult, neonatal mice and the patients with non-small cell lung carcinoma by immunofluorescence staining. Secondly, we propagated BASCs in vitro and isolated them from mouse lung tissues by FACS. Subsequently, miRNAs from BASCs were labeled and then hybridized to microarray gene chips and ten candidate miRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). The predicted target of miRNA was proved by the luciferase report gene system. The present study attempts to define miRNAs profiles of BASCs, thereby leading a new insight into both the regulation of stem cell self-renewal and the mechanisms for the turn from BASCs to lung cancer stem cells.Methods1. CCA and SP-C staining in human lung cancer tissues by immunofluorescence: Adjacent serial 8μm sections of lung from human adenocarcinoma, squamous carcinoma and para-cancerous tissues respectively, all were stained for anti-CCA and anti-SP-C in order to detect the CCA and SP-C double-positive cells(DPCs). Finally, the expression was observed by co-focal laser scanning microscopy in lung tissues. Each tissue was observed 4 to 6 visual fields randomly and the percentage of DPCs in every 100 lung cells was counted. All the data were analyzed by the SPSS 11.0.2. Isolation and characterization of BASCs: The Sca-1+ cells were sorted from the pulmonary single cell suspension by magnetic labeling cell sorting with anti-Sca-1 microbeads following enzymatic digestion of lung tissue with dispase and collagenase in combination. The Sca-1+ cells were seeded into tissue culture flasks pre-coated with collagen I in serum-free culture system for BASCs cell colony, which was identified by the dual-color immunofluorescent staining for anti-CCA and anti-SP-C. The cell colony of BASCs was differentiated under the 10% fetal calf serum (FCS) on day 6. Finally, the pure CD45-CD31-Sca-1+CD34+ cells (BASCs) and CD45-CD31-Sca-1-CD34-cells (controls) were both isolated by the flow cytometry.3. Identification of the miRNAs profile from BASCs: Total RNA were isolated using Trizol reagent according to the manufacturer’s instructions. Small RNA were size-fractionated (<300nt) by YM-100 Microcon centrifugal filter (Millipore). RNA quality control, labeling, hybridization, scanning and data analysis were performed by LC Sciences. The results of microarray chip were further confirmed using qRT-PCR. The targets of miRNAs were predicted using public web-based prediction tools, such as TargetScan、MiRbase、miRanda and verified the target gene by the luciferase report gene system.Results1. Existence of DPCs in human lung adenocarcinoma tissues: The DPCs were first discovered in the human lung adenocarcinoma. However, the DPCs were not found in lung squamous cancer. Furthermore, the number of DPCs in tumor sites was much higher than that in paratumor tissues (P<0.05).2. Successfully isolated and characterized BASCs from mouse lung: One lung of normal adult mouse could yield 1.6~1.8×107 nucleated cells in the enzyme digestion procedure. The percentage of positive cells for Sca-1 markers was much higher than the unsorted mouse pulmonary cells (87.3%±5.9% vs 9.6%±1.8%, P<0.05). By day 6, the Sca-1+ cells formed into BASCs and other unknown cell colonies identified by immunostaining for anti-CCA and anti-SP-C. Under the condition with 10% FCS, the BASCs differentiated into AT 2 cells in which SP-C expression was maintained in the majority of the colony cells up to day 10, but the cell colony expressing SP-C disappeared by day 11. No CCA could be detected in the colonies during the 10-day culture period. In order to obtain the pure BASCs, we firstly measured CD45-CD31- Sca-1+ CD34+ cells which represents 0.7%~1.1% of the total number of pulmonary cells. Finally, 2×106 BASCs (CD45-CD31-Sca-1+CD34+) and 4×106 control cells (CD45-CD31-Sca-1-CD34-) were isolated from mouse pulmonary cells suspension by flow cytometry sorting.3. Identification of the miRNAs profile from BASCs: 220ng and 720ng small RNA were both extracted from BASCs and controls respectively. The miRNAs differentially expressed between BASCs and controls were performed by using mouse miRNAs array probes which included 568 mature mouse miRNAs (Chip ID miMouse 10.0 version; LC Science). Overall, 196 miRNAs of 568 arrayed miRNAs were found in normal mouse BASCs and 261 miRNAs of 568 arrayed miRNAs were found in control. The microarray identified 56 up-regulated miRNAs and 60 down-regulted miRNAs in BASCs when compared with control cells (P<0.01). Among the 116 miRNAs, miR-142-3p, miR-451, miR-106a, miR-142-5p, miR-15b, miR-20a, miR-106b, miR-25, miR-486and miR-497 were chosen from the microarray results and validated by TaqMan qRT-PCR. MiR-497 was the only up-regulated in BASCs compared to control cells, and the left 9 were down-regulated. The target gene Wee1 for miR-497 was predicted in the TargetScan、MiRbase、and miRanda, which was then verified by the luciferase report gene system( the data was not presented).ConclusionIn summary, the present findings not only validated the DPCs existing in normal lung tissues widely, but also firstly identified the DPCs in human lung adenocarcinoma. The miRNAs expression profile of mouse BASCs was first reported and miR-142-3p, miR-451, miR-106a, miR-142-5p, miR-15b, miR-20a, miR-106b, miR-25, miR-486, miR-497 might play important roles in the regulation of BASCs. The presently established platform would provide implications of the further research of miRNAs and lung stem cells in the pathogenesis of lung cancer. The present findings and consequent research based on this platform would provide new molecular targets which are in favor of the early detection, prognosis monitoring and targeted therapy for lung cancer.更多还原