【作者】 孙红玉；

【导师】 刘厚奇；

【作者基本信息】 第二军医大学 ， 人体解剖与组织胚胎学， 2010， 博士

【摘要】 表皮样干细胞瘤化的发生和TGF-β1信号通路的关系以及极性相关分子的变化研究背景和目的近几十年来，癌变的体细胞突变理论占据癌研究的主导地位，认为癌是体细胞突变积累的结果。尽管该理论取得众多成果，但在这一理论指导下的肿瘤临床治疗并未能取得重大突破。最近研究表明，并不是所有的肿瘤细胞都有重建肿瘤的能力，只是具有干细胞特性的一小群细胞(即肿瘤干细胞)才能驱动肿瘤的发生、发展和复发。由于干细胞存活时间较长，可以积累较多的突变，有更多的突变机会成为肿瘤干细胞，所以有研究者认为，在已明确有干细胞存在的皮肤、胰腺和结肠等组织中，都极有可能存在由各自干细胞经突变转化而来的肿瘤干细胞。目前已在白血病、乳腺癌、脑胶质瘤、黑色素瘤和一些肿瘤细胞系中证实存在肿瘤干细胞。随着对肿瘤干细胞研究的不断深入，提出了肿瘤干细胞的学说，认为肿瘤是产生于积累多次突变的干细胞，这也和体细胞突变理论中的多次突变致癌相一致。干细胞在机体组织中的居住环境即壁龛(niche),在正常干细胞的维持及功能上发挥着关键作用。干细胞只有发生突变后才可能出现表型明显的变化，根据肿瘤干细胞学说，这个过程可解释为：首先壁龛中突变干细胞克隆扩增，亦即瘤前演进，然后逃离所在的壁龛形成显微镜下可见的斑块甚至更大的区域。伴随着此过程，细胞微环境的改变、致癌剂等可使干细胞获得额外突变，从而促使早期致瘤的演进。比如，结肠癌的发生被认为至少要经历五个过程，亦即瘤性克隆性斑块(patches),增生(hyperplasia),原位癌(carcinoma in situ),侵袭癌(invasive carcinoma)和转移癌(metastatic carcinoma)。最近肿瘤微环境的作用受到了越来越多的关注。就像干细胞壁龛在正常干细胞的维持及功能上发挥着关键的作用一样，瘤前及恶性细胞的微环境在肿瘤发生及演进过程中也起着重要作用。即肿瘤微环境具有两面性：一些情况下，它作为肿瘤促进者加强恶性上皮表型的影响，另外，可规范化上皮肿瘤细胞的表型(虽然基因型改变)。肿瘤干细胞具有和正常干细胞壁龛类似的微环境，正处于转化的细胞及其基质环境的表观遗传的相互作用可能也反映了两者间正常交流的失常。在这个过程中，异常的基质可促进转化的细胞生长和发生，同时上皮细胞也可促使异常基质的发生。例如，正常的人前列腺上皮细胞系BPH-1移植到带有人前列腺癌相关的成纤维细胞(CAFs)的裸鼠上时，不能形成肿瘤，而当SV40端粒永生化人前列腺上皮细胞系BPH-1移植到裸鼠时，较大的肿瘤就能形成。同时具有代表性的例子如，干细胞和黑色素瘤细胞间的相互作用也受到了大家的关注。两种细胞间可在分泌的可溶性细胞因子或者胞外基质的作用下，通过细胞间的直接接触、自分泌因子和旁分泌因子进行双向的微环境信息交流。Postovit及其同事用创新的三维细胞培养模型研究了胚胎干细胞的微环境对黑色素瘤细胞的表观调控的作用，发现胚胎干细胞的微环境可转分化黑色素瘤细胞为黑色素样细胞。进一步研究这个模型表明，即侵袭·性黑色素瘤的基质环境可诱导正常黑色素细胞转化为具有血管拟态生成的黑色素瘤样细胞。这些研究表明胚胎微环境对肿瘤细胞的表型特性有重要的作用，但是肿瘤微环境对正常细胞的影响还尚未充分探究。在本研究中我们建立了体外诱导表皮样干细胞恶性转化的共培养模型，运用了肿瘤发生的两步法，即首先用肿瘤启动剂DMBA处理表皮样干细胞然后再经黑色素瘤细胞诱导的方法。研究发现表皮样干细胞经两步诱导后，体外不但发生了表皮向间质样细胞的转型(epithelial-mesenchymal transition, EMT)而且具有了肿瘤细胞的相关生物学特性，体内可致裸鼠黑色素瘤，这为肿瘤微环境可以造成干细胞自我更新能力的失控提供了一定的依据。随后进一步探讨了表皮样干细胞发生瘤化时TGF-p信号通路及其下游分子p-Smad2和整合素alphaV(ITGAV)的关系，以探明肿瘤细胞对周围干细胞的影响在肿瘤发生中的重要性及其分子机制，旨在了解肿瘤的病因并寻找有效方法将肿瘤扼杀在发生早期。最后，从细胞极性分子的变化入手，研究极性分子PAR3、Tiam1和p-PKCζ在肿瘤发生发展中的作用。第一部分黑色素瘤细胞体外诱导人胚表皮样干细胞瘤化模型的建立方法：(1)实验用人胚表皮样干细胞(Human Embryo Epidermal Stem Cell, hEpSCs)’I生质鉴定：取传代培养至4-8代的hEpSCs,免疫荧光法检测β1-integrin、K19和p63的表达情况。(2)体外共培养模型的建立：一是hEpSCs和A375共培养体系，具体方法是将A375和hEpSCs分别种植于cell culture insertsPET膜的两侧，作为接触共培养组；将A375种植于放有cell culture inserts的六孔板上，然后PET膜内面种植hEpSCs,作为间接共培养组；将hEpSCs单独种在多孔膜一侧，做为阴性对照组。二是首先以二甲基苯葸(DMBA)作为肿瘤启动剂处理hEpSCs16小时，然后DMBA处理的hEpSCs和A375进行共培养，也是设立接触共培养组，间接共培养组和单独培养组。相差显微镜下每天观察hEpSCs的生长状态情况，并进行必要的拍照。(3)共培养后的hEpSCs裸鼠体内移植实验：裸鼠真表皮交接处接种共培养后的hEpSCs,4周后，处死裸鼠，用HE染色、Fontana Masson Staining、HLA的碱性磷酸酶染色鉴定诱导后的hEpSCs在裸鼠体内是否致瘤。(4)共培养后hEpSCs增值相关抗原和间质相关抗原的检测：免疫荧光法检测各共培养组hEpSCs N-cadherin和Vimentin的表达情况；并进一步用流式细胞术鉴定其表达Vimentin和N-cadherin的阳性率；western blotting法检测hEpSCsE一钙粘蛋白的表达情况。(5)共培养后hEpSCs转化能力的检测：双层软琼脂克隆形成实验鉴定共培养后hEpSCs是否具有恶性转化的能力，培养14-21天，观察细胞集落并计算细胞集落形成率(形成集落数/接种细胞数×100%)。结果：(1)传代培养至4-8代的hEpSCsβ1-integrin、K19和p63的表达呈阳性。(2) DMBA处理的hEpSCs接触共培养组，相差显微镜下观察到共培养4d时有克隆出现，7d时可见明显的克隆，而间接共培养组和hEpSCs单独培养组以及DMBA未处理的hEpSCs共培养体系均未见有克隆形成；共培养7d后的hEpSCs于正常培养基培养中贴壁后可见部分细胞变为梭形，这说明A375细胞诱导后hEpSCs接触抑制开始丧失。(3)裸鼠真表皮交接处接种实验HE染色和Fontana Masson Staining,HLA．1的免疫组化AP染色证明DMBA处理的hEpSCs接触共培养组诱导后部分细胞可在裸鼠体内致瘤而间接共培养组和对照组以及DMBA未处理的hEpSCs共培养体系均未成瘤。各组肿块的生长曲线图，可见DMBA处理的hEpSCs接触共培养组移植瘤块体积明显较其他组生长迅速。随后我们用黑色素瘤细胞株MV3和DMBA处理的hEpSCs,进行了同样的共培养体系，结果也只有接触共培养组hEpSCs体内移植可见黑色肿块。而人永生化细胞株HaCaT和DMBA处理的hEpSCs的共培养体系，各组却未见有明显变化。因此，下面所有的实验结果，我们主要是对DMBA处理的hEpSCs和A375细胞的共培养体系进行的研究。(4)免疫荧光法检测表明接触共培养组中hEpSCs N-cadherin和Vimentin的表达呈胞膜强阳性而间接共培养组中其呈阴性信号；western blotting法的结果进一步也证实了诱导后hEpSCs的E一钙粘蛋白表达量下降；流式细胞术也证实了诱导后hEpSCs表达N-cadherin和Vimentin阳性率分别为73．43%和99．87%而间接共培养组和对照组为阴性表达。(5)3次软琼脂克隆形成实验统计数据显示接触共培养组克隆形成率为5.5%，间接共培养组克隆形成率为O.9%，对照组克隆形成率为0.1%。5.5%细胞能在双层软琼脂生长，说明这部分细胞已获得恶性转化。结论：我们成功建立了体外诱导hEpSCs恶性转化的共培养模型。在共培养池两侧种植A375细胞和DMBA处理的hEpSCs共培养，体外hEpSCs不但发生了表皮向间质样细胞的转型而且具有了肿瘤细胞的相关生物学特性，并且裸鼠体内可致黑色素瘤，这为干细胞在肿瘤微环境中自我更新和分化能力失控提供了一定的依据。第二部分表皮样干细胞瘤化的发生和整合素aV介导的TGF-β1信号通路方法：(1)Elisa检测共培养体系中TGF-β1细胞因子的分泌水平：同样用DMBA处理的hEpSCs和A375，设立接触共培养组，问接共培养组和单独培养组。(2)检测表皮样干细胞EMT转型过程中TGF-βl的信号转导分子磷酸化Smad2 (p-Smad2)的表达情况：在6、12、24、48h，用western blotting法和和免疫细胞荧光法检测hEpSCs中p-Smad2的表达情况。(3)表皮样干细胞瘤化发生过程中ITGAV的表达情况：在1、3、5、7d用RT-PCR法检测接触共培养组hEpSCs中ITGAV和18S的mRNA表达情况，免疫荧光实验检测接触共培养组ld和3d时hEpSCs中ITGAV的表达。(4)不同ITGAV干扰效率细胞株的建立：首先构建pGCsilencerTM U6/Neo/GFP/ITGAV-RNAi质粒，然后转染hEpSCs,经G418筛选，挑选克隆至24孑L板继续筛选，后扩大培养，然后采用RT-PCR和Western-blotting的方法检测鉴定干扰效率。(5)干预后的hEpSCs建立共培养模型，然后体内移植观察成瘤能力：干预后的hEpSCs(NC，C1，D和F4)，一方面同第一部分的方法建立共培养模型，同样设立共培养组，问接共培养组和单独培养组，共培养后hEpSCs裸鼠体内移植，将裸鼠分为三组，分别以G-RNAiCtl,G-RNAiC1, G-RNAiD2和G-RNAiF4表示，每组有小鼠6只，每只裸鼠在左腿皮下接种2×105只细胞。自接种之日起，每天观察小鼠的生长状态以及肿瘤的变化情况。每2-3d，用直尺测量瘤块的长径与短径。肿瘤体积公式为4/3丌×(d/D)2×D/2，其中，d为瘤块的短径，D为瘤块的长径。肿瘤数据统计采用方差分析的方法，各组间比较采用Student-Newman-Keuls test。结果：(1)共培养体系中细胞因子的检测情况：TGF-β1的分泌水平只在接触共培养组有显著提高，这提示hEpSCs和黑色素瘤细胞的接触可导致TGF-β1的激活。(2)p-Smad2的表达情况：western blotting实验表明，接触共培养组hEpSCs由p-Smad2有瞬时表达现象，在共培养6h开始可检测到，于24h表达水平最高，48h时表达有所下降；免疫细胞荧光法实验可检测到在接触共培养48h，p-Smad2在hEpSCs细胞核中的阳性信号较其它两组有明显增强。p-Smad2表达水平的升高和p-Smad2的入核现象，这提示TGF-β信号可能参与了干细胞的瘤化。(3)表皮样干细胞瘤化发生过程中ITGAV的表达变化：RT-PCR和免疫细胞荧光实验证明接触共培养7d后，hEpSCs中ITGAV的表达明显增高，由此我们推测ITGAV可能在干细胞瘤化发生过程中可能起着重要的作用。(4)不同ITGAV干扰效率细胞株的建立：首先成功构建了pGCsilencerTM U6/Neo/GFP/ITGAV-RNAi质粒，转染hEpSCs经418筛选出A2，C1，D，F4和NC(阴性对照组)五个克隆，然后扩增。RT-PCR和Western-blotting的方法检测了这五个克隆由ITGAVmRNA的表达，结果表明我们获得了干扰效率分别为30%，50%，70%和90%左右的hEpSCs细胞株。(5)干预后的hEpSCs建立共培养模型，然后体内移植观察成瘤能力：干预后的hEpSCs同第一部分的方法建立共培养模型，共培养后hEpSCsi(即G-RNAiCtl, G-RNAiC1, G-RNAiD2和G-RNAiF4)裸鼠体内移植仍能形成黑色素瘤，但是随着干预水平的升高，肿瘤体积呈递减趋势。各组实验数据统计分析，G-RNAiCtl相比G-RNAiD2来说，P<0.05；G-RNAiCt1相比G-RNAiF4来说，P<0.01，这提示ITGAV可能是干细胞瘤化发生过程的关键分子。ITGAV表达降低，TGF-β1分泌水平也下降，P-Smad2的表达有减弱趋势，这提示干细胞瘤化过程中，ITGAV可介导TGF-β1信号的激活。结论：以上结果表明A375和hEpSCs接触共培养可促使TGF-β1分泌水平升高并且激活TGF-β1/Smad信号通路，ITGAV是表皮样干细胞瘤化发生的关键分子。第三部分表皮样干细胞瘤化的发生和极性相关分子PAR3、Tiam1和p-PKCζ方法：(1)免疫荧光法(?) Western-blot检测极性形成相关分子PAR3、Tiam1和p-PKCζ在干细胞瘤化发生时的表达情况：免疫荧光实验将两种种属来源不同的抗体一起孵育，如p-PKCζ和ITGAV,p-PKCζ和Tiam1,p-PKCζ和PAR3。Western-blot实验抗体的稀释度均为1：1000，也可将两种种属来源不同的、分子大小不同的抗体一起孵育，如Tiam1和：ITGAV等。(2)免疫荧光法和Western-blot检测ITGAV干预后，极性形成相关分子PAR3、Tiam1和p-PKCζ在干细胞瘤化发生时的变化情况：免疫荧光实验和Western-blot实验，方法同上(1)。结果：(1)免疫荧光法和Western-blot检测极性形成相关分子PAR3、Tiam1和p-PKCζTiam1在干细胞瘤化发生时的表达：免疫荧光法检测显示极性形成相关分子PAR3、p-PKCζ,和在干细胞瘤化发生过程阳性信号明显减弱。Western-blot的检测结果也证实随着共培养时间的延长，PAR3、Tiam1和p-PKCζ蛋白的表达也下降。这说明伴随干细胞的瘤化，细胞的极性也逐渐被破坏。(2)免疫荧光法和Western-blot检测ITGAV干预后，极性形成相关分子PAR3、Tiam1和p-PKCζ在干细胞瘤化发生时的表达：激光共聚焦检测显示ITGAV干预后，极性形成相关分子PAR3和Tiam1在干细胞瘤化发生过程阳性信号有所减弱。Western-blot的检测结果也证实随着共培养时间的延长，PAR3和Tiam1蛋白的表达也下降。而p-PKCζ表达量没有明显变化，但是有明显的入核现象。这说明ITGAV干预后，伴随干细胞的瘤化，细胞的极性也逐渐被破坏，至于p-PKCζ的入核现象，很可能和极性分子变化的重组有关，是否也和TGF.β信号通路有关系，尚待进一步探讨.结论：本部分实验表明伴随干细胞的瘤化，极性形成相关分子PAR3、Tiam1和p-PKCζ的表达降低，亦即极性分子的破坏，这可作为干细胞瘤化发生的一个标志。创新点与结论：首先成功建立了体外诱导hEpSCs发生表皮向间质样细胞的转型及恶性转化的共培养模型，这为干细胞在肿瘤微环境中自我更新和分化能力失控提供了一定的依据。然后借助于干细胞瘤化的模型，研究干细胞与肿瘤发生之间的关系及机制，这也是本课题的创新点之一。研究发现，干细胞的瘤化是通过整合素alphaV介导的TGF-β1信号通路激活的机制，这提示整合素alphaV是致瘤的关键分子，为肿瘤的靶点治疗提供了有力证据。最后，从细胞极性分子的变化入手，研究极性分子在肿瘤发生发展中的作用，这是课题的另一创新点。结果表明极性形成相关分子PAR3、Tiam1和p-PKCζ的低表达可作为干细胞瘤化发生的一个标志。更多还原

【Abstract】 Direct contact by melanoma cells causes malignant transformation of human epithelial-like stem cells via alpha V integrin activation of TGF-β1 signalingBackground and objectiveThe somatic mutation theory of cancer has been the prevailing paradigm in cancer research for decades and states that malignant transformation is the result of accumulation of multiple mutations in normal differentiated cells. Recent studies have demonstrated that not all tumor cells have the capacity to perpetuate the tumor, and that only a subset of tumor cells with stem cell-like properties, called cancer stem cells (CSCs), is capable of driving tumor initiation, progression, and recurrence. The current CSC hypothesis suggests CSCs, which arise from normal stem cells (SCs) by mutation of genes that make the stem cells cancerous, is consistent with the classical model of multi-step carcinogenesis, as SCs have longer life spans than their differentiated progeny and could accumulate multiple mutations over many years.Somatic SCs reside in a specialized physical local known as a’niche’.The microenvironment in the niche is essential for the maintenance and functioning of normal SCs. According to the CSC hypothesis, the first step of tumor formation is the clonal expansion of a mutation-harboring SC within a niche, known as pretumor progression. With additional mutations, these cells may expand beyond the confines of an individual niche into microscopic or larger sized patches/field. For instance, colon tumors are thought to progress through at least five stages:pretumor patches/fields, hyperplasia, carcinoma in situ, invasive carcinoma and metastasis.The microenvironment has major effects on the development and progression of cancer. In the classic two-stage tumorigenesis model, the microenvironment has been proposed to play a part in the promotion phase. Several groups have demonstrated the involvement of microenvironmental factors in tumor progression and proposed that the microenvironment may promote cancer progression through distinct signaling mechanisms. For example, Olumi reported that carcinoma-associated fibroblasts can direct tumor progression of initiated prostate epithelial cells by SV40-T.Recent attention has been given to the interactions between stem cells and melanoma cells. Indeed, the bi-directional microenvironmental communication may be propagated among cells through at least three different mechanisms:direct cell-cell contact, autocrine and paracrine signaling driven by soluble secreted factors, and modeling (or re-modeling) of the extracellular matrix. Postovit and colleagues studied the epigenetic influence of the microenvironment of human embryonic stem cells on the reprogramming of aggressive melanoma cells using an innovative 3D culture model. They demonstrated redifferentiation of the melanoma cells to a melanocyte-like phenotype after exposure to the microenvironment of human embryonic stem cells. Further studies with this model revealed that the aggressive melanoma-conditioned matrix microenvironment induced transdifferentiation of normal melanocytes into melanoma-like cells exhibiting a vasculogenic phenotype. These studies demonstrated that the embryonic microenvironment may have a significant influence on the phenotypic characteristics of aggressive cancer cells; however, the effects of a tumorigenic microenvironment on normal stem cells have not been sufficiently explored.In this study, we established a novel in vitro 3D co-culture model system in order to induce malignant transformation of human epithelial-like stem cells (hEpSCs) in a microenvironment created by the A375 melanoma celi line. Using a 2-step protocol (exposure to the mutagen 7,12-dimethylbenz[a]anthracene (DMBA) and subsequent direct contact with A375 melanoma cells), we showed that hEpSCs could be converted to melanoma cells via a mechanism involving integrin alpha V-mediated activation of TGF-β1 signaling.Part I hEpSCs undergo maligant transformation and epithelial-mesenchymal transition when treated with DMBA and then cultured in contact with A375 melanoma cells.Methods:(1) To characterize the cells:Using immunofluorescence methods to detectβ1-integrin, K19 and p63 in the 4-8 passage HEMSC. (2) Establish in vitro co-culture model of A375 and hEpSCs:hEpSCs were treated with 0.1μg/ml DMBA (Sigma-Aldrich) for 16 h prior to the co-culture with A375 cells. Three groups of cultures were studied:1) Contact Group:hEpSCs or hEpSCs treated with DMBA+A375 melanoma cells in contact co-culture by seeding on the bottom and top sides of porous membrane of a cell culture insert, respectively; 2) No Contact Group:hEpSCs or hEpSCs treated with DMBA+A375 melanoma cells co-cultured without contact (separated by cell culture insert) but allowing hEpSCs to be exposed to the A375 conditioned media; and,3) Control (control cultures containing hEpSCs or hEpSCs treated with DMBA only). (3) To assess the tumorigenesis of transformed hEpSCs, we established a xenograft model.hEpSCs at passage 6, which were treated with DMBA or not treated with DMBA and then were induced by A375 for 7days, were suspended in DMEM before implantation to facilitate establishment. (4) Transformational ability of hEpSCs after co-culture:Using immunofluorescence methods and flow Cytometry analysis to detect N-cahherin and Vimentin in the in 7d of co-cultured hEpSCs. In 3,5,7d, E-cahherin protein were assayed by western blotting respectively.Results:To assess the tumorigenesis of hEpSCs or hEpSCs treated with DMBA in the three groups, we established a xenograft model. Fourteeen days after receiving hEpSCs or hEpSCs treated with DMBA in the three groups, nodules appeared in nude mice and only hEpSCs treated with DMBA in the contact group fcrmed black nodules and therefore, the cells we studied in the following experiments were hEpSCs treated with DMBA and A375 cells. The growth kinetics of hEpSCs treated with DMBA in the contact group xenografts in nude mice (n=6 for each experimental group) showed the proliferous ability of transformed hEpSCs in vivo. HE staining of the subcutaneous tissue showed significant differences among 3 experimental groups. Fontana-Masson staining for the melanin revealed hyper-pigmentation in the contact group, but not in the other two groups. However, hEpSCs without treated with DMBA in the three groups of cultures presented no pigmentation in the contact group. HLA-1 immunohistochemistry confirmed the human origin of the cells with hyper-pigmentation. These data indicated that transformed hEpSCs were able to form melanoma in vivo.Transformed hEpSCs colonies were observed at d7 in hEpSCs treated with DMBA with direct contact to A375 cells, but not in the non-contact group or hEpSCs treated with DMBA cultured alone. The "pile-up" appearance of hEpSCs colonies in the contact group prompted us to examine their anchorage-independent growth in soft agar. We carefully wiped A375 cells on the bottom of porous membrane of the cell culture using a cotton swab, digested hEpSCs on the top sides of the cell culture insert with 0.25% trypsin and seeded these cells in the three groups separately in soft agar at the density of 1000 cells per well. After incubation for 3 weeks, in contrast to that no colonies were found in control cells, nearly 5% of hEpSCs in the contact group formed large colonies, indicating that hEpSCs with direct contact to A375 cells had underwent the transformation. Additionally, we monitored the growth curve for hEpSCs in the three groups and showed that hEpSCs with direct contact to A375 cells had the enhanced proliferation prominently in vitro. Reduction of E-cadherin and concomitant production of N-cadherin are features of epithelial-mesenchymal transition (EMT). In the hEpSCs treated with DMBA and then induced by A375 cells, expression of N-cadherin and Vimentin was significantly increased, whereas expression of E-cadherin was decreased in the same experiment group. As is known, down-regulation of E-cadherin is the hallmark molecular change that occurs during EMT. The number of cells positive for N-cadherin and Vimentin in the contact group was significantly increased. Since N-cadherin and Vimentin are mesenchymal markers, these results clearly indicated that transformed hEpSCs possess mesenchymal characteristics and EMT. However, hEpSCs without treated with DMBA in the three groups of cultures presented no phenotype changes and transformation.Part II Malignant transformation of human epithelial-like stem cells via alpha V integrin activation of TGF-β1 signalingMethods:(1) The aforementioned observations indicate that malignant transformation of the hEpSCs required direct contact with melanoma. We speculate that direct contact between melanoma cells and hEpSCs may stimulate certain paracrine signals. To identify these signals, in vitro co-cultures of A375 cells and hEpSCs were established and their conditioned media were measured for the levels of various cytokines and growth factors by ELISA. (2) To detect phospho-Smad2 (p-Smad2), a downstream signal transduction factor of TGF-β1 in A375 and hEpSCs contact co-culture group and hEpSCs alone culture group were made as in Part One. In 0,6,12, 24,48h, p-Smad2 in hEpSCs were detected by western blotting; while p-Smad2 by immunocytochemistry method in the meantime. (3) hEpSCs (5×106) at passage 6, which were treated with DMBA and then were induced by A375 for 7days, were suspended in DMEM before implantation(100μL) to facilitate establishment. (4) To further test the accumulation of ITGAV in the transformed hEpSCs, mRNA expression of ITGAV was assessed by RT-PCR. To explore.the role of ITGAV in the process of tumorigenesis, we established stable siRNA expression vectors, pGCsilencerTM U6/NEGative (NCRNAi) and pGCsilencerTM U6/ITGAV (ITGAV RNAi). (5) To investigate the functional consequences of ITGAV interference in the process of tumorigenesis, the growth kinetics of transformed hEpSCs with ITGAV interference in vivo were compared to those of hEpSCs over 4 weeks. (6) Since ITGAV and TGF-β1 were involved in the transformation of hEpSCs, we next set out to resolve the relationship between ITGAV and TGF-β1. We measured the levels of TGF-β1 in the media of hEpSCs before and after stable knockdown of 90% ITGAV.Results:(1) In some cases, certain released factors (for example, MMP-1 or Gro) were higher in the contact group than in the remaining two groups. Notably, TGF-β1 was 3-fold higher in the contact culture group than the hEpSCs alone or no-contact co-culture group, suggesting a synergistic interaction between A375 and hEpSCs. This cooperative induction of TGF-β1 was apparent as early as the third day of co-culture. To determine whether TGF-β1 signal pathways in hEpSCs are activated in the contact co-culture group, we measured the phospho-Smad2 (p-Smad2), a downstream signal transduction factor of TGF-β1. Importantly, subsequent co-culture of these hEpSCs and cancer cells continued to allow accumulation of p-Smad2 in hEpSCs in the contact co-culture group that were comparable to those observed in the no-contact group and control group. Meanwhile, immunocytochemistry revealed that p-Smad2 could be detected in the nuclei of 30-50% transformed hEpSCs, suggesting that direct contact between melanoma cells and hEpSCs activates the TGF-β1/Smad signaling pathways. (2) mRNA expression of ITGAV was increased in contact co-culture. To explore the role of ITGAV in the process of tumorigenesis, we established stable siRNA expression vectors, pGCsilencerTM U6/NEGative (NCRNAi) and pGCsilencerTM U6/ITGAV (ITGAV RNAi). After transfection of NCRNAi and ITGAV RNAi in cultured hEpSCs, colonies (NC, A2, C1, D2 and F4) were selected by neomycin (200ug/mL). Western blots revealed that ITGAV RNAi treatment decreased total cellular ITGAV protein levels. Histograms analysis showed the average level of ITGAV interference was 50%,30%,70% and 90% for A2, Cl, D2 and F4, respectively. (3) ITGAV interference inhibited the tumor growth. More importantly, ITGAV with interference efficiency of 90% and 70% of hEpSCs, their tumor volume displayed a significant decrease. These data suggested that ITGAV may contribute to the tumorigenesis. ELISA analysis revealed that stable knockdown of 90% ITGAV did not result in accumulated levels of TGF-β1 in the contact groups. Western blots of P-Smad2 in hEpSCs after the difference knockdown ITGAV levels in contact co-culture group at 3d also showed a weakening of TGF-(31 signaling. Taken together, these observations suggested that ITGAV mediate the local activation of TGF-β1/Smad signaling pathways.PartⅢTransformed hEpSCs demonstrate impaired polarityMethods:(1) To determine whether polarity in transformed hEpSCs has changed, we performed intracellular expression studies for integrin alpha V (ITGAV), Par polarity proteins and Tiam1 by triplicate immunostaining and Western blot. (2) To determine whether polarity in transformed hEpSCs after stable knockdown of 90% ITGAV has changed, we performed intracellular expression studies for integrin alpha V (ITGAV), Par polarity proteins and Tiaml by triplicate immunostaining and Western blot.Results:(1) ITGAV was expressed in the cytoplasm; immunoreactive signals were significantly increased in transformed hEpSCs. Expression of endogenous Par3 protein and TIAM1 were consistently decreased at the leading edge and cytoplasm of hEpSCs, but not at leading edges of transformed hEpSCs. Endogenous p-aPKCζwere asymmetrically enriched throughout the hEpSCs in transformed hEpSCs, expression of endogenous p-aPKCζwas decreased in the nucleus. Western blot analysis of ITGAV, TIAM1, p-aPKCζand PAR3 confirmed these findings, suggesting that polarity of transformed HEpSCs had been disrupted. (2) Expression of endogenous Par3 protein and TIAM1 were also consistently decreased at the leading edge and cytoplasm of hEpSCs after stable knockdown, but not at leading edges of transformed hEpSCs. Endogenous p-aPKCζwere asymmetrically enriched throughout the hEpSCs in transformed hEpSCs after stable knockdown, expression of endogenous p-aPKCζwas decreased in the nucleus. Western blot analysis of ITGAV, TIAM1, p-aPKCζand PAR3 confirmed these findings, suggesting that polarity of transformed HEpSCs had been disrupted. We proved that TIAM1, p-aPKCζand PAR3 may be the hallmark of malignant transformation of hEpSCs.Conclusions: (1) We successfully established a model in vitro for inducing malignant transformation of epithelial-like stem cells with melanoma cell. The data have proved that epidermal-like stem cell could obtain the associated characteristics of tumor cell by the inducement of A375.This suggests that self-renewing and differentiation of stem cell in the tumor microenvironment be out of control.(2) Since DMBA treated hEpSCs by the inducement of A375 showed tumorigenesis in vivo, this data suggests hEpSCs have obtained neoplastic transformation.(3) These data suggest that the microenvironment of metastatic melanoma cells could lead to cancer formation of normal stem cell and imply that stem cells must be the targets of carcinogenic exposure. They also help to better understand the origins of malignant stem cells and their responses to to be critica to the therapy of malignancies.更多还原