NICD1过表达P53及Lkb1联合缺失肺鳞癌小鼠模型的构建

发布时间：2018-05-03 22:21

本文选题：肺鳞癌 + Notch　；参考：《浙江大学》2016年博士论文

【摘要】：背景和目的：肺鳞癌是一种致死率很高的肺癌类型,在所有肺癌中,其发病率仅次于肺腺癌。因治疗手段有限,晚期肺鳞癌的5年生存率不到15%。人类肺鳞癌的发生发展过程漫长,一般认为：正常的支气管黏膜假复层柱状上皮细胞受慢性刺激和损伤,正常纤毛丧失,出现不同程度的支气管上皮增生,随后可能发生不典型增生,鳞状上皮化生和发育不全,最后部分进展为原位癌及浸润性癌。提示若能阻止或逆转肺鳞癌发生前任意一环节的良性病变,都将有可能阻止肺鳞癌的最终发生。目前肺鳞癌转基因小鼠已逐渐应用于人肺鳞癌的研究,已知的构建方法主要有四种：Lkbl敲除/KrasGI2D过表达,IKKa死亡激酶敲入,Lkbl/Pten同时敲除和Sox2过表达/Lkbl敲除。尽管这些小鼠能发生肺鳞癌,但却存在肺癌类型混杂、潜伏期长、成瘤率低、仅能出现肺鳞癌却缺失与人肺鳞癌发生前类似的阶段性病理改变过程等缺点。并且,尽管肺鳞癌的发生与许多基因的同时突变有关,但驱动正常肺组织进展为肺鳞癌的启动基因仍未明确。因此,本课题组选取了三个与人肺鳞癌密切相关的基因,构建出转基因小鼠模型,以期寻找肺鳞癌的启动基因,并试图通过在小鼠肺内模拟与人肺鳞癌阶段性病变类似的过程,帮助临床肺鳞癌的诊断、治疗和预防。另外,目前认为,cytokeratin 5(K5)来源细胞是一类具有干细胞特性的细胞群,并可能与肺癌的发生有关。我们应用Cre-loxP系统,分别研究了K5特异性来源的细胞与非特异性来源的细胞在相同损伤及基因缺陷的背景下,是否都能在小鼠肺内形成肿瘤及形成肿瘤的区别,从而探讨K5来源细胞是否具有潜在的肺癌干细胞功能。方法：根据2012年Cancer Genome Atlas Research Network(TCCA)发布的178例人肺鳞癌标本基因分析数据和本课题组小鼠体外气道干细胞球气液相体系培养的结果(未发表数据),我们选择了NICD1,Lkb1和P53三个基因,利用Cre-loxP系统,构建出K5-CrePR;NICD1;P53;Lkbl转基因小鼠模型。分别通过Ru486诱导K5细胞特异性Cre重组酶表达或直接应用非细胞特异的Adenovirus Cre重组酶的方法,引起Notch通路重要因子NICD1的过度激活,并同时沉默抑癌基因Lkbl和P53在小鼠体内的表达。此后对转基因小鼠予以聚多卡醇肺损伤刺激,并长期观察小鼠,当小鼠出现异常状态或体征后,处死小鼠留取肺组织标本。首先,用HE染色的方法,筛查肺部肿瘤的形成情况及初步判断肺部肿瘤的病理类型。随后,对于筛选得到的肺部肿瘤标本,用不同类型肺癌的特异性标记抗体进行免疫荧光染色,进一步判断肺部肿瘤的具体病理类型。最后,通过整理所有转基因小鼠的数据：1.总结各基因型小鼠的肺部病理特点,并分析各基因对肿瘤形成的作用；2.总结两种来源Cre重组酶对小鼠肺部肿瘤形成的异同,并分析K5来源细胞在肺部肿瘤形成中的作用。另外,我们还在第一部分实验中,初步探讨了K5来源细胞的干细胞功能：1.通过流式分选术及体外气道干细胞成球气液相体系培养的方法,观察Trop-2+ITGA6+气道干细胞(ABSCs)中是否富集K5来源干细胞。2.应用K5-CrePR-YFP转基因小鼠模型,通过K5-YFP+ve细胞的自身示踪,追踪K5来源细胞在博来霉素肺损伤小鼠肺组织中的动态变化。3.将K5-CrePR-YFP转基因小鼠作为供体鼠,通过Frop-2+ITGA6+ABSCs的异体移植实验,追踪供体鼠K5-YFP+ve干细胞在博来霉素肺损伤受体鼠肺组织中的动态变化。结果：成功构建了肺鳞癌转基因小鼠模型,并在小鼠体内观察到类似人肺鳞癌发生前的不同程度的上皮异型增生。所有肺鳞癌均发生在NICD1杂合突变型小鼠(NICD1纯合突变型小鼠均不能存活),提示NICD1可能作为肺鳞癌发生的驱动基因,在外在损伤刺激和P53及Lkbl共同缺陷的情况下,促使正常细胞转变为肺鳞癌细胞,从而参与肺鳞癌形成的过程。具体结果如下：(1)Ru486治疗组小鼠出现肺鳞癌,提示K5来源细胞可能具有肿瘤干细胞功能,在损伤刺激及基因缺陷共同作用下转变为肺鳞癌细胞。在所有小鼠中：①NICD1野生型小鼠,均状态如常。②NICD1纯合突变型小鼠,均于出生后早夭或在母体内流产。③NICD1杂合突变型小鼠,根据P53和Lkbl基因型的不同,表型明显不同：NICD1+/fl;P53fl/fl、NICD1+/fl; Lkb1fl/fl和NICD1+/fl; P53+/fl; Lkb1fl/fl小鼠如常。而NICD1+/fl; P53+/fl; LKB1+/fl、NICD1+/fl; P53fl/fl/; LKB1+/fl和NICD1+/fl; P53fl/fl; LKBfl/fl/三种基因型小鼠,肺自发鳞癌形成,三组的成瘤小鼠数目分别为1只、2只和2只,成瘤率分别为25.0%、28.6%和33.3%,潜伏期为5-12月。所有肺癌标本在免疫荧光染色后均强表达肺鳞癌标记物K5,而肺鳞癌标记物TTF1和cytokeratin 8(K8)表达缺失,经免疫学证实为肺鳞癌。(2) Adeno virus Cre (AdenoCre)治疗组小鼠出现肺鳞癌,且较Ru486治疗组小鼠病变更为严重,提示除K5来源细胞外,可能存在其它具有肿瘤干细胞功能的细胞群,在损伤刺激及基因缺陷共同作用下与K5来源细胞协同,共同转变为肺鳞癌细胞。与Ru486治疗组类似：①NICD1野生型小鼠,均状态如常。②NICD1纯合突变型小鼠,均于出生后早夭或在母体内流产。③NICD1杂合突变型小鼠,根据P53和Lkbl基因型的不同,表型明显不同：NICD1+/fl;P53fl/fl, NICDl+/fl; Lkb1fl/fl和NICDl+/fl; P53+/fl; Lkb1fl/fl小鼠如常。而NICD1+/fl; P53+/fl; LKB1+/fl, NICD1+/fl； P53fl/fl; LKB1+/fl和NICD1+/fl; P53fl/fl; LKB1fl/fl/三种基因型小鼠,肺部自发形成肺鳞癌,三组的成瘤小鼠均为1只,成瘤率均为100%,潜伏期为5-9月。所有肺癌标本在免疫荧光染色后均强表达肺鳞癌标记物K5,中等强度表达ProSPC,而肺鳞癌标记物TTF1和K8表达缺失,经免疫学证实为肺鳞癌。另外,我们还意外发现NICD1+/fl; P53fl/fl; LKB1fl/fl基因型小鼠在AdenoCre治疗后,而不是在Ru486治疗后,主气道上皮K5细胞活跃增生,黏液明显增多,正常纤毛丢失,Clara细胞消失。提示相同的损伤刺激和基因缺陷背景在近端大气道和远端肺组织可引起截然不同的病理改变：近端大气道的某些细胞(不包括K5来源细胞)可能过度分化为黏液分泌细胞,形成黏液增生性病变；而远端肺组织的某些细胞(包括K5来源细胞)可能转变为肿瘤细胞,参与肺鳞癌的形成。(3)Ru486治疗组与AdenoCre治疗组小鼠的主要区别有：①经AdenoCre治疗后的小鼠肺部肿瘤明显较Ru486治疗组小鼠肺部肿瘤多发,体积大,异型性更为明显。②经AdenoCre治疗后的小鼠明显较Ru486治疗组小鼠肺癌形成潜伏期短。③经AdenoCre治疗后的小鼠明显较Ru486治疗组小鼠成瘤率高。④相同基因背景的小鼠,经AdenoCre治疗后肺上皮增生和／或癌前病变的程度均较Ru486治疗组严重。⑤经AdenoCre治疗后Cre+; NICDl+/fl; P53fl/fl; LKB1fl/fl小鼠支气管黏液明显增多,纤毛减少,而经Ru486治疗组小鼠未见类似改变。另外,在第一部分实验中,我们发现K5来源细胞具有自我更新和增殖分化能力,具有干细胞潜能,具体结果如下：(1)流式分选培养及免疫荧光结果示：Trop-2+ITGA6+ABSCs富集K5来源干细胞。(2)自身示踪实验表明：K5来源细胞,而非K5+细胞在博来霉素肺损伤模型中增多,表现出向K8+祖细胞分化倾向,并可能参与损伤后肺新生血管及肺纤维化的形成。(3)异体移植实验表明：供体K5-YFP+ve细胞在博来霉素损伤受体鼠肺内明显增多,且表现出向K8+祖细胞分化倾向。结论：尽管目前已存在四种肺鳞癌转基因小鼠模型,但这些模型存在形成肿瘤类型混杂,成瘤时间长,成瘤率低,小鼠肺鳞癌与人肺鳞癌病理特征之间差别大等缺点,而我们成功构建的K5-CrePR; NICD1; P53; Lkbl肺鳞癌转基因小鼠模型在病理切片上呈现出气道上皮轻度异型增生至肺鳞癌的各阶段改变,更符合人肺鳞癌的发生发展过程。且具有形成肺癌类型单一,仅为肺鳞癌的优点。因此,可为临床人肺鳞癌的研究提供理想的工具鼠模型。另外,NICD1可能作为肺鳞癌发生的驱动基因在肺鳞癌的发生发展过程中起至关重要的作用,抑制Notch通路过度激活可能有助于预防肺鳞癌的发生。而K5来源细胞具有肿瘤干细胞的特征,抑制K5来源细胞的异常过度增生,可能为临床肺鳞癌的预防治疗提供新的思路。
[Abstract]:Background and objective: lung squamous cell carcinoma is a highly lethal type of lung cancer. In all lung cancers, the incidence of lung cancer is second only to that of lung adenocarcinoma. Because of limited treatment, the 5 year survival rate of advanced squamous cell carcinoma is less than 15%. human lung squamous cell carcinoma. It is generally believed that normal bronchial mucosa false stratified columnar epithelial cells are chronic. Irritation and injury, loss of normal cilia, the emergence of varying degrees of bronchial epithelial hyperplasia, and subsequent atypical hyperplasia, squamous metaplasia and dysplasia, and the last part of the carcinoma in situ and invasive carcinoma. It is suggested that lung squamous cell carcinoma may be prevented if it can prevent or reverse the benign lesions of any link before the occurrence of squamous carcinoma of the lung. At present, transgenic mice of lung squamous cell carcinoma have been gradually applied to the study of human lung squamous cell carcinoma. There are four known methods of Construction: Lkbl knockout /KrasGI2D overexpression, IKKa death kinase knocking, Lkbl/Pten knockout and Sox2 overexpression /Lkbl knockout. Although these mice can develop squamous cell carcinoma of the lung, there is a mixed type of lung cancer. However, the initiation of lung squamous cell carcinoma is still undefined. Therefore, three of them are selected in this group. The gene of human lung squamous cell carcinoma is closely related to the construction of a transgenic mouse model, in order to find the promoter gene of lung squamous cell carcinoma, and try to help the diagnosis, treatment and prevention of the clinical lung squamous cell carcinoma by simulating the similar process in the lung of the lung squamous cell carcinoma in the lungs of mice. In addition, the cytokeratin 5 (K5) source cell is a class of stem cells. Cell specific cell groups and may be associated with the occurrence of lung cancer. We used Cre-loxP system to investigate whether cells from K5 specific sources and non-specific sources can form tumors in mice and form tumors in the lungs of mice with the same damage and gene defects, so as to explore whether the K5 derived cells have a tumor or not. Methods: according to the genetic analysis data of 178 human lung squamous cell carcinoma specimens issued by Cancer Genome Atlas Research Network (TCCA) in 2012 and the results (unpublished data) of this group of mice in vitro, we chose the three genes of NICD1, Lkb1 and P53, using Cre-lox. P system, K5-CrePR; NICD1; P53; Lkbl transgenic mice model. The expression of K5 cell specific Cre recombinant enzyme was induced by Ru486 or the method of using non cell specific Adenovirus Cre recombinant enzyme to induce the excessive activation of NICD1 in Notch pathway, and the expression of silencing tumor suppressor gene and in mice in the same time. After that, the transgenic mice were stimulated by polycol lung injury, and the mice were observed for a long time. After the abnormal state or signs of the mice, the mice were sacrificed to leave the lung tissue specimens. First, the HE staining method was used to screen the formation of lung tumor and the pathological type of lung tumor. Then, the lung tumor was screened out. The specific marker antibody of different types of lung cancer was used to carry out immunofluorescence staining to further determine the specific pathological types of lung cancer. Finally, the data of all transgenic mice were collated. 1. the pathological characteristics of the lung were summarized, and the effect of each gene on the formation of the tumor was analyzed. 2. the two sources of Cre weight were summarized. The effects of the enzyme on the formation of lung tumor in mice were analyzed and the role of K5 derived cells in the formation of lung cancer was analyzed. In addition, in the first part of the experiment, we preliminarily discussed the stem cell function of the K5 derived cells: 1. the method of flow sorting and the culture of the gas liquid phase system of the extracorporeal airway stem cells was used to observe the Trop-2+ITGA6+ gas. Whether or not the stem cells (ABSCs) enriched K5 derived stem cells,.2. used K5-CrePR-YFP transgenic mice, and traced the dynamic changes of K5 source cells in the lung tissue of mice with bleomycin lung injury by the self tracing of K5-YFP+ve cells and.3. used K5-CrePR-YFP transgenic mice as donor mice by Frop-2+ITGA6+ABSCs allograft transplantation. The dynamic changes of the donor mouse K5-YFP+ve stem cells in the lung tissue of the bleomycin lung injury rat were traced. Results: a transgenic mouse model of lung squamous cell carcinoma was successfully constructed and the epithelial dysplasia of different degrees before the carcinogenesis of human lung squamous cell carcinoma was observed in the mice. All lung squamous cell carcinomas occurred in the NICD1 heterozygous mutant mice (N The ICD1 homozygous mutant mice could not survive), suggesting that NICD1 may be the driving gene of lung squamous cell carcinoma. In the case of external injury stimulation and common defects of P53 and Lkbl, the normal cells change into lung squamous cell carcinoma cells and participate in the formation of lung squamous cell carcinoma. The specific results are as follows: (1) lung squamous cell carcinoma in the Ru486 group of mice is presented. The K5 source cells may have the function of tumor stem cells and change into lung squamous cell carcinoma cells under the joint action of injury stimulation and gene defect. In all mice, the NICD1 wild type mice are all state as usual. (2) the NICD1 homozygous mutant mice were all aborted early or in the mother body. 3. NICD1 heterozygous mutant mice, according to P53 and Lk The phenotype of BL genotypes was distinct: NICD1+/fl; P53fl/fl, NICD1+/fl; Lkb1fl/fl and NICD1+/fl; P53+/fl; Lkb1fl/fl mice as usual. And NICD1+/fl; P53+/fl; LKB1+/fl, NICD1+/fl; three genotypes, spontaneous squamous cell carcinomas of the lungs, and the number of three groups of tumorigenic mice 1, 2 and 2 were 25%, 28.6% and 33.3%, respectively, and the incubation period was 5-12 months. All lung cancer specimens strongly expressed lung squamous cell carcinoma markers K5 after immunofluorescence staining, while lung squamous cell carcinoma markers TTF1 and cytokeratin 8 (K8) were expressed as lung squamous carcinoma. (2) Adeno virus Cre (AdenoCre) treatment group showed lung scale. Cancer, and more serious than that of the Ru486 treatment group, suggests that there may be other cell groups with tumor stem cell functions except the K5 source cells, which together with K5 derived cells under the joint action of injury stimulation and gene defects, together with the lung squamous cell carcinoma cells. It is similar to the Ru486 treatment group: (1) the NICD1 wild type mice are all as usual. (2) NICD1 homozygous mutant mice were aborted early or in the mother body after birth. 3. The phenotype of NICD1 heterozygous mutant mice was obviously different according to the P53 and Lkbl genotypes: NICD1+/fl; P53fl/fl, NICDl+/fl; Lkb1fl/fl and NICDl+/fl; P53+/fl; Lkb1fl/fl mice. LKB1+/fl and NICD1+/fl; P53fl/fl; LKB1fl/fl/ three genotypes mice, lung squamous cell carcinomas were spontaneously formed in the lungs, and 1 mice in the three group were all tumor forming mice, the tumor formation rate was 100% and the incubation period was 5-9 months. All lung cancer specimens strongly expressed K5 of lung squamous cell carcinoma markers after immunofluorescence staining, the medium intensity expressed ProSPC, and the markers of lung squamous cell carcinoma TTF1 and K8 table. In addition, we also found that NICD1+/fl; P53fl/fl; LKB1fl/fl genotype mice were treated with AdenoCre, but not after Ru486 treatment, K5 cells in the main airway epithelium were proliferated, mucus was markedly increased, normal cilium lost, and Clara cells disappeared. The same injury stimulation and gene defect back were suggested. The near terminal large airway and distal lung tissue can cause completely different pathological changes: some cells in the proximal large airway (not including K5 source cells) may excessively differentiate into mucous secretory cells to form mucous proliferative lesions; and some cells in the distal lung tissue (including K5 derived cells) may be transformed into tumor cells and participate in the lung scale. The formation of cancer. (3) the main differences between the Ru486 treatment group and the AdenoCre treatment group were as follows: (1) the lung tumor of the mice after the AdenoCre treatment was obviously more frequent than the Ru486 treatment group, the volume was large and the heteromorphy was more obvious. (2) the mice after the AdenoCre treatment were obviously shorter than the Ru486 treatment group. 3. AdenoC The mice after re treatment were significantly higher than those in the Ru486 treatment group. 4. The extent of lung epithelial hyperplasia and / or precancerous lesion after AdenoCre treatment in mice with the same gene background was more serious than that in the Ru486 treatment group. 5. After AdenoCre treatment, Cre+; NICDl+/fl; P53fl/fl; LKB1fl/fl rat bronchial mucus was significantly increased and cilia decreased, and Ru was reduced to Ru. In the 486 treatment group, no similar changes were found in the mice. In the first part of the experiment, we found that the K5 source cells have the ability of self renewal and proliferation and differentiation and have the potential of stem cells. The specific results are as follows: (1) flow cytometry and immunofluorescence results show that Trop-2+ITGA6+ ABSCs enriching K5 derived stem cells. (2) self tracer experiment showed that K 5 source cells, not K5+ cells in the bleomycin lung injury model, showed a tendency to differentiate into K8+ progenitor cells, and may participate in the formation of pulmonary neovascularization and pulmonary fibrosis after injury. (3) the allograft experiment showed that the donor K5-YFP+ve cells were significantly increased in the lungs of the injured mice in the bleomycin injury and displayed to the K8+ progenitor cells. Conclusion: Although there are four kinds of transgenic mice model of lung squamous cell carcinoma, these models have the disadvantages of forming tumor type mixed, long tumorigenesis, low tumor rate, large difference between lung squamous cell carcinoma and human lung squamous cell carcinoma, and we successfully constructed K5-CrePR; NICD1; P53; Lkbl transgenic mice model of lung squamous cell carcinoma The pathological sections show a slight dysplasia of the airway epithelium to the squamous cell carcinoma of the lung, which is more consistent with the development of human squamous cell carcinoma, and has the advantage of forming a single lung cancer type and only a squamous carcinoma of the lung. Therefore, it can provide an ideal tool mouse model for the study of human lung squamous cell carcinoma. In addition, NICD1 may be used as a squamous cell carcinoma of the lung. The induced gene plays a vital role in the development and development of lung squamous cell carcinoma. Inhibition of overactivation of Notch pathway may help prevent the occurrence of squamous cell carcinoma of the lung. K5 derived cells have the characteristics of tumor stem cells and inhibit abnormal hyperproliferation of K5 derived cells, which may provide a new idea for the prevention and treatment of lung squamous cell carcinoma.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R-332;R734.2

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