基于单细胞层层组装技术构建三维多细胞肿瘤球体模型

发布时间：2018-05-07 23:01

本文选题：层层组装 + 三维培养　；参考：《南方医科大学》2015年硕士论文

【摘要】：研究背景肿瘤是指机体细胞在各种致癌因素作用下发生基因突变,导致组织失去机体对其生长的正常调控,进而经过一系列不可控的异常克隆增殖所形成的异常新生组织。肿瘤包括良性和恶性两大类。肿瘤,特别是恶性肿瘤(癌症)严重危害人类的生命健康。而且随着人类寿命的不断增长,癌症的发病率和死亡率也逐渐上升,成为全世界最受关注的健康问题之一。肿瘤微环境是肿瘤细胞在其发生发展过程中所处的内环境,由肿瘤细胞本身、间质细胞、微血管、组织液及少量浸润细胞等共同组成。肿瘤的发生发展始终伴随着肿瘤细胞同其周围微环境的相互作用。相关研究已揭示,早在实体瘤血管发生之前,因肿瘤细胞迅速增殖而造成的缺氧环境可能导致肿瘤进展和更差的临床结局。而在血管形成后,肿瘤局部长期的缺血缺氧环境所造成的瘤体中央坏死也被看作是侵袭性癌的共同特征。体外模拟肿瘤细胞微环境,可以为更进一步研究肿瘤细胞癌变机理、增殖、分化及临床药物筛选提供更好的研究方法。迄今为止,在绝大多数研究中,肿瘤细胞的培养均是采用平面培养皿的二维(2D)培养模型和动物在体培养模型。在2D培养模型中,肿瘤细胞所处的环境显然与在体的实体瘤微环境截然不同,肿瘤细胞缺乏细胞外基质(extracellular matrix, ECM),无法同肿瘤微环境中的多种因素发生交互作用,导致细胞与细胞、细胞与细胞外基质之间相互联系所致的细胞形态改变、细胞生长、增殖与分化等变化被忽略。相比于2D培养模型,在三维(3D)培养条件下的肿瘤组织中存在不同表型的肿瘤细胞,包括增殖细胞、非增殖细胞以及坏死细胞等,这使培养的肿瘤细胞能够表现出更接近于在体肿瘤细胞的特性。另外,通过在裸鼠体内注射肿瘤细胞或者移植肿瘤组织的方法建立的动物肿瘤模型已经得到大多数学者的认可,这也是目前进行肿瘤研究的金标准。然而,动物肿瘤模型有一定的局限性,注射到裸鼠体内的肿瘤细胞会受到体内诸多因素的影响,体内的肿瘤细胞难免会发生不可控制的基因及相关蛋白表达的改变。因此,我们有必要探索一种更好的方法来替代2D培养系统和动物模型。3D体外组织的构建可以用来弥补二维培养模型的不足,为我们研究肿瘤及其相关机制提供了一种比动物模型更快捷、经济的可靠选择。随着近年来组织工程学的迅猛发展,基于3D培养的组织工程肿瘤技术进展迅速,呈现出多样化的发展趋势。然而,这其中的大部分方法都难以控制瘤体结构以及细胞与细胞之间、细胞与细胞外基质间的相互作用。例如,多细胞肿瘤悬浮球(multicellular tumor suspension, MCTs)由多种单个细胞组成,但是细胞与细胞之间无细胞外基质,因而预想的细胞与基质之间的相互作用缺失。另一方面,目前的3D模型大多依赖于天然的和人工合成的高分子聚合物。天然分子虽然具有与ECM相似的特性,能够尽可能的模拟肿瘤微环境,但由于其往往存在残存的信号分子及不定量的杂质,导致使用天然材料的实验无法做到完全可控和绝对严谨。人工聚合高分子材料虽然避免了上述缺陷,但由于其微纤维间的孔径往往能达到细胞直径的千倍级甚至万倍级,从而导致有效成分的过快扩散和迅速丢失。据报道,胶原凝胶硬度低,降解速度快,而且其许多特性受提取过程的影响,从而限制了它们的应用。因此,从该领域发展的角度来看,我们可以将从纳米级或微纳米级到宏观水平的各种因素均应考虑在体外构建的三维组织模型中。纳米级的细胞外基质存在诸多优点。首先,ECM本身就是由多种纳米级高分子纤维组织组成；其次,纳米级细胞外基质有较高的表面积和体积比,能够促使细胞之间的联系,并且促进生物活性分子的运输；再者,细胞膜和细胞迁移相关的细胞骨架均属于纳米级。因此,纳米级ECM可以促进细胞与细胞之间,细胞与细胞微环境之间的相互作用,在肿瘤发生发展、诊断及临床药物筛选的研究中具有明显的优势。寻找合适的生物材料,并通过便捷的方法构建三维培养肿瘤模型,以达到模拟体内真实肿瘤微环境的目的,是本研究的重点。明胶和海藻酸钠作为天然生物材料,无毒、可降解,并且具有良好的亲水性、细胞亲和性及细胞生物相容性。在组织工程支架中,明胶/海藻酸钠溶液作为一种软支架材料,可以与细胞混合,实现细胞的立体培养。这种支架材料的优势在于不但在表面而且在支架内部也可以含有细胞,从而为多种细胞的空间合理分布提供了可能。壳聚糖是甲壳素脱乙酞化产物,属于聚阳离子多糖(Pka=6.3)物质,壳聚糖中的N-乙酞基毗喃与细胞外基质中的糖胺聚糖(GAGs)组成类似,且具有生物活性、可生物降解。本研究选用A型明胶、海藻酸钠及中分子量壳聚糖,通过层层组装和聚合的方法,构建一种体外三维多细胞肿瘤球体模型来模拟肿瘤细胞微环境,为研究乳腺癌的癌变机理、癌细胞转移以及药物临床前评价等方面提供一种更加灵活简便的方法。上皮间质转化(Epithelial Mesenchymal Transition, EMT)是指本身具有极性的上皮细胞在某些因素的刺激下,上皮特性减少、间质特性增多、细胞基质粘附消失和细胞骨架重塑,从而导致上皮细胞极性消失,细胞运动能力增加,获得浸润性和迁移能力。在上皮来源的恶性肿瘤中,EMT是肿瘤细胞获得浸润和迁移能力的重要途径。尽管EMT的发生同肿瘤微环境密切相关,但肿瘤微环境诱导肿瘤细胞发生EMT的确切机制仍不清楚。本研究在已构建的三维肿瘤模型的平台上比较研究二维培养体系与三维培养体系中乳腺癌细胞EMT相关蛋白的表达差异,如CD47、N-Cadherin等。在体外三维培养体系下模拟体内乳腺癌N-Cadherin表达增多触发EMT促进肿瘤细胞浸润和迁移的临床特点,对我们构建的三维肿瘤模型进行验证。目的：1.构建用于基础研究以及药物筛选的体外组织工程化肿瘤三维模型,并对该模型的形貌、表面电荷、生物相容性等性质进行研究；2.体外所构建的三维人源性乳腺癌肿瘤模型的体外验证。方法：1.细胞培养购入人乳腺癌细胞株MDA-MB-231,复苏后传代,将其培养在含有10%胎牛血清,0.1%青霉素和链霉素的RPMI 1640培养基中,于37℃,含5%C02的培养箱中常规培养。每天换液,待细胞融合率达80%时,用含0.02% EDTA的0.25%的胰蛋白酶液消化并传代。待细胞量足够,取足量细胞用于层层组装。2.对单个细胞进行(明胶-海藻酸钠)3-壳聚糖的层层组装及多细胞肿瘤球体的形成设计合成与细胞相容性良好的纳米级细胞外基质成分,以促进细胞与细胞之间,细胞与细胞微环境之间的相互作用。该纳米级细胞外基质成分包括A型明胶、海藻酸钠,需分别带有正电荷和负电荷,因此细胞外基质成分依次层层包裹于细胞表面。细胞经胰酶消化后,计数1×107,于离心机中,1000rpm/min,离心5min,弃去上清液,用7ml 0.1% (w/v)明胶溶液轻轻重悬收集的细胞后,常温放置10min;接着于离心机中,1000rpm/min,离心5min,弃去明胶溶液,此时明胶层粘附于单个细胞表面。然后,用7m1预热的0.1%(w/v)海藻酸钠溶液轻轻重悬包裹有明胶层的细胞团,则带有阴离子的聚合电解质层粘附于明胶层上,常温孵育10min后,离心弃去海藻酸钠溶液；接着将等体积的A型明胶溶液(阳离子聚合电解质溶液)加入包裹有明胶和海藻酸钠的细胞团。此过程通过依次在细胞表面包裹带有相反电荷的聚合电解质三个循环。最后,用7m1预热的0.2%(w/v) PH 6.7的中分子量壳聚糖溶液(阳离子聚合电解质)使最外层均粘附有阴离子聚合电解质(海藻酸钠)的单个肿瘤细胞之间发生聚合反应,数分钟后,1000rpm/min,离心10min,多细胞肿瘤球形成,弃去壳聚糖溶液,用预热的含10%FBS的RPMI 1640培养基轻轻重悬,然后将多细胞肿瘤球转移至一个新的六孔板中,加入RPMI 1640培养基,置于5%CO2,37℃培养箱中培养,培养基每天更换。3.组织工程化人源性乳腺癌三维模型的表征及生物相容性研究组织工程化人源性乳腺癌三维模型构建成功后,一般情况下,需要对其表面形貌、所带电荷、生物相容性等性质进行研究。本实验中分别采用光学显微镜、激光粒度分析仪、扫描电镜、荧光显微镜、活死染色等仪器和技术对其性质进行测量和分析。4.体外所构建三维人源性乳腺癌肿瘤模型的体外验证实验分为三组,3Di,3Dm和2D,称肿瘤球中的细胞为3Dis；肿瘤球培养过程中,观察发现其培养皿底板有细胞迁移出来并贴壁生长,称迁移出来的细胞为3Dms；称二维培养的细胞为2D。分别培养四天后,提取总蛋白,Western blotting检测p-ERK, ERK, CD47蛋白的表达；取出多细胞肿瘤球,PBS洗三次,4%多聚甲醛固定24小时,30%蔗糖溶液固定1周后,冰冻切片,放于37℃干燥箱中烤24小时,免疫荧光检测E-Cadherin, N-Cadherin蛋白的表达。5.统计学分析以上实验均设有阴性对照实验,每次实验至少重复3次。统计分析采用SPSS13.0统计软件完成,结果用均数±标准差(Mean±SD)来表示。组间比较采用单因素方差分析(One-way ANOVA),方差齐性的组间比较采用Tukey法,方差不齐的组间比较采用Dunnett's T3法,p0.05认为差异有统计学意义。结果：1.成功构建体外组织工程化人源性乳腺癌三维模型。通过A型明胶与海藻酸钠静电作用层层包裹于细胞三个循环,得到单个肿瘤细胞层层组装的模型；然后加入中分子量壳聚糖溶液,使层层组装的单个细胞间发生聚合,形成多细胞肿瘤球,即组织工程化人源性乳腺癌肿瘤模型。2.光学显微镜下显示人源性乳腺癌三维模型呈团块状,内部细胞(3Dis)形态规则,呈圆形,轮廓清晰,连接紧密；随着细胞培养时间的延长,一些肿瘤细胞从肿瘤球体迁出,迁移出的肿瘤细胞(3Dmms)形态规则,轮廓清晰,贴壁生长,状态良好。3.用FITC标记明胶,在荧光显微镜下单纯(明胶-海藻酸钠)3层层组装组以及(明胶-海藻酸钠)3层层组装后用壳聚糖聚合组均显示：每个细胞周围均有一层绿色荧光。这说明层层组装的纳米膜成功包裹于单个细胞。4.激光粒度分析仪测试结果显示,细胞表面电荷随着带相反电荷聚合电解质的轮流加入,呈现曲折变化,表明细胞表面层层组装上了纳米材料。5.扫描电镜观察3D体外肿瘤模型的形态显示：多细胞肿瘤球具有三维立体结构,细胞生长良好,细胞表面成功包裹了纳米材料,保持了细胞外基质的完整性；细胞与细胞之间并非紧密连接,有较大孔隙,有利于水分,营养物质及氧气渗透,形成适于肿瘤细胞生长的三维立体环境。6.对多细胞肿瘤球进行活死染色,荧光显微镜下观察示,整个肿瘤球呈绿色(活细胞),几乎没有红色(死细胞)出现。证明纳米材料生物相容性良好,成功构建了便于基础研究以及药物筛选的三维人源性乳腺癌模型。7. Western blotting和免疫荧光验证了此基于单个细胞层层组装构建的三维多细胞肿瘤球体模型内的微环境可通过N-Cadherin的过表达来触发EMT。Western blotting结果显示3Dm和2D组p-ERK和ERK的表达均明显高于3Di组；而3Di组CD47表达明显高于3Dmm和2D组。免疫荧光结果显示2D组呈现E-Cadherin过表达,但无N-Cadherin表达；而3Di组呈现高表达的E-Cadherin和N-Cadherin; 3Dm组则呈现E-Cadherin高表达,N-Cadherin低表达。结论：在本研究中,我们用超薄基质材料层层包裹单个肿瘤细胞的方法构建了一个三维多细胞肿瘤球体模型来模拟在体实体瘤的微环境,并对该模型的形貌及表面电荷进行一系列表征,通过活死染色验证了模型中细胞的活力。还在该三维模型的平台上进一步比较研究了二维培养体系与三维培养体系中乳腺癌细胞EMT相关蛋白的表达差异,并且该模型在体外三维培养体系下很好地模拟了体内乳腺癌N-Cadherin表达增多触发EMT促进肿瘤细胞浸润和迁移的临床特点,对我们构建的三维肿瘤模型进行验证。
[Abstract]:Background tumor is a kind of malignant tumor, including two types of benign and malignant tumors, including benign and malignant tumors, especially malignant tumors (cancer). With the growth of human life, the incidence and mortality of cancer are increasing, and it has become one of the most concerned health problems in the world. The tumor microenvironment is the internal environment of the tumor cells in the process of its development, including the tumor cells, interstitial cells, microvessels, tissue fluids and less. The development of the tumor is always accompanied by the interaction between the tumor cells and the surrounding microenvironment. The related research has revealed that the hypoxia environment caused by the rapid proliferation of tumor cells may lead to tumor progression and worse clinical outcome before the tumor angiogenesis. The central necrosis of the tumor caused by the ischemic anoxia environment in the tumor bureau is also seen as a common feature of invasive cancer. In vitro simulation of the tumor cell microenvironment can provide better research methods for further research on the mechanism, proliferation, differentiation and clinical drug screening of tumor cells. The culture of tumor cells is a two-dimensional (2D) culture model and an animal model in vivo. In the 2D culture model, the environment of the tumor cells is obviously different from the solid tumor microenvironment in the body. The tumor cells lack the extracellular matrix (extracellular matrix, ECM), which can not be associated with various factors in the microenvironment of the tumor. The interaction caused cell morphology changes, cell growth, proliferation and differentiation caused by the interaction between cells and cells, cells and extracellular matrix. Compared to the 2D culture model, tumor cells with different phenotypes, including proliferating cells and non proliferative cells, were found in the tumor tissues under the condition of three-dimensional (3D) culture. In addition, the animal tumor model established by injecting tumor cells in nude mice or transplanted tumor tissue in nude mice has been recognized by most scholars, which is also the gold standard for cancer research. The animal tumor model has some limitations. The tumor cells injected into the nude mice will be affected by many factors in the body. The tumor cells in the body are unavoidable to change the changes in the expression of the incontrollable genes and related proteins. Therefore, it is necessary to explore a better formula to replace the 2D culture system and the animal model.3D in vitro The construction of the organization can be used to compensate for the deficiency of the two-dimensional culture model. It provides a more efficient and reliable choice for the study of tumor and its related mechanisms. With the rapid development of the tissue engineering in recent years, the technology of tissue engineering tumor based on 3D has developed rapidly and presents a variety of development trends. Most of these methods are difficult to control the structure of the tumor body and the interaction between cells and cells, cells and extracellular matrix. For example, multicellular tumor suspension (MCTs) is composed of a variety of single cells, but the cells and cells have no extracellular matrix, thus preconceived cells and bases. On the other hand, most of the current 3D models depend on natural and synthetic polymers. Although natural molecules have similar characteristics to ECM, they can simulate tumor microenvironment as much as possible, but they often exist in the presence of residual signal molecules and indeterminate impurities, resulting in the use of natural materials. The artificial polymer materials can not be completely controlled and absolutely rigorous. Although the artificial polymer materials can avoid the above defects, the pore size of the microfibers can often reach the 1000 or even ten thousand times of the diameter of the cells, which leads to the rapid diffusion and rapid loss of the effective components. It is reported that the hardness of the collagen gel is low and the degradation rate is fast. And many of its characteristics are affected by the extraction process, thus limiting their applications. Therefore, from the perspective of development in this field, we can consider the various factors from nanoscale or micro nanoscale to macro level in the three-dimensional tissue model constructed in vitro. There are many advantages of the nanoscale extracellular matrix. First, ECM Ben The body is made up of a variety of nanoscale polymer fibers. Secondly, the nanoscale extracellular matrix has a high surface area and volume ratio, which can promote the connection between cells and promote the transport of bioactive molecules. Furthermore, the cell membrane and cell migration related bone shelves are all nanoscale. Therefore, nanoscale ECM can be promoted. The interaction between cell and cell, cell and cell microenvironment has obvious advantages in the study of tumor development, diagnosis and clinical drug screening. Finding the appropriate biomaterials and constructing a three-dimensional tumor model through a convenient method can be used to simulate the real tumor microenvironment in the body. Gelatin and sodium alginate, as natural biomaterials, are non-toxic, biodegradable, and have good hydrophilic, cellular affinity and cellular biocompatibility. In tissue engineering scaffolds, gelatin / sodium alginate solution can be used as a kind of soft scaffold material and can be mixed with cells to realize cell stereoscopic culture. The potential is that it is possible not only on the surface but also in the inside of the scaffold, thus providing the possible spatial distribution of a variety of cells. Chitosan is the product of chitin deglyphthalide, a polycationic polysaccharide (Pka=6.3) substance, and the N- phthalide in chitosan is similar to the glycosaminoglycan (GAGs) in the extracellular matrix. In this study, A gelatin, sodium alginate and medium molecular weight chitosan were used in this study to construct a three-dimensional multi cell tumor cell model in vitro to simulate the microenvironment of tumor cells in vitro, to study the carcinogenesis, metastasis of cancer cells and the pre clinical evaluation of drugs. A more flexible and simple method. Epithelial Mesenchymal Transition (EMT) refers to the epithelial cells in which their polar epithelial cells are stimulated by some factors, the decrease of epithelial properties, the increase of interstitial properties, the disappearance of cell matrix adhesion and the remodeling of cytoskeleton, resulting in the disappearance of the epithelial cell polarity and the cell movement. EMT is an important approach to the invasion and migration of tumor cells in epithelial cancer cells. Although the occurrence of EMT is closely related to the tumor microenvironment, the exact mechanism of the tumor cells to induce EMT is still unclear. On the platform of the model, the difference in the expression of EMT related proteins in breast cancer cells in the two-dimensional culture system and the three-dimensional culture system, such as CD47, N-Cadherin and so on, is used to simulate the clinical characteristics of the increase of N-Cadherin expression in breast cancer in vivo to trigger the EMT to promote the invasion and migration of the tumor cells, and to build the three-dimensional swelling of the tumor cells. The tumor model was verified. Objective: 1. to construct an in vitro three-dimensional model of tissue engineered tumor for basic research and drug screening, and to study the morphology, surface charge, biocompatibility and other properties of the model. 2. in vitro validation of the three-dimensional human breast cancer model in vitro. Method: 1. cell culture buyers. The breast cancer cell line, MDA-MB-231, was transmitted after resuscitation, and was cultured in the RPMI 1640 medium containing 10% fetal bovine serum, 0.1% penicillin and streptomycin. It was cultured in a incubator containing 5%C02 at 37 degrees C. A daily liquid was changed, when the cell fusion rate was 80%, it was digested and passaged with 0.25% trypsin containing 0.02% EDTA. Sufficient cells are used to assemble layers of.2. to assemble a single cell (gelatin - sodium alginate) 3- chitosan and the formation of a multicellular tumor sphere to synthesize the nanoscale extracellular matrix components with good cellular compatibility to promote the interaction between cells and cells and the microcellular environment. The nanoscale cells The external matrix components include A gelatin, sodium alginate, with positive and negative charges, so the extracellular matrix components are sequentially coated on the surface of the cell. After trypsin digestion, the cells are counted 1 x 107, in centrifuge, 1000rpm/min, centrifuge 5min, supernatant, and 7ml 0.1% (w/v) gelatin solution. At the temperature of 10min, then in the centrifuge, 1000rpm/min, centrifuge 5min, discarded gelatin solution, gelatin layer adhered to the surface of a single cell at this time. Then, with 7m1 preheated 0.1% (w/v) sodium alginate solution to gently overhang the cell mass of the gelatin layer, then the polyelectrolyte layer with anions adhered to the gelatin layer, after incubating 10min at normal temperature. The solution of sodium alginate was abandoned; then the equal volume A gelatin solution (cationic polyelectrolyte solution) was added to the cell masses wrapped with gelatin and sodium alginate. This process was carried out by three cycles of polyelectrolytes with opposite charges on the cell surface. Finally, the molecular weight of the middle molecular weight of 0.2% (w/v) PH 6.7 was preheated with 7m1. The glucose solution (cationic polyelectrolyte) polymerized the single tumor cells with the most outer layer adhered to the anionic polyelectrolyte (sodium alginate). After a few minutes, 1000rpm/min, centrifugation, 10min, multicellular tumor balls were formed, the chitosan solution was abandoned, and the preheated 10%FBS containing RPMI 1640 medium was gently suspended and then more thin. The cell tumor ball was transferred to a new six hole plate, the RPMI 1640 medium was added, and the culture medium was placed in the 5%CO2,37 centigrade incubator. The culture medium changed every day to replace the.3. tissue engineering human derived breast cancer three-dimensional model and the biocompatibility study. Study on the properties of surface morphology, charge and biocompatibility. In this experiment, optical microscopy, laser particle size analyzer, scanning electron microscopy, fluorescence microscopy, live death staining and other instruments and techniques were used to measure and analyze the properties of.4. in vitro. The three groups, 3Di, 3Dm and 2D, called the cells in the tumor ball 3Dis. In the process of tumor ball culture, it was observed that the cell plates were migrated and adhered to the wall in the culture plate. The cells migrated were 3Dms, and the two dimensional cultured cells were cultured for four days, respectively, to extract the total protein, and the Western blotting detected the tables of p-ERK, ERK, CD47 protein. Three times of PBS washing, 4% polyoxymethylene fixed for 24 hours, 30% sucrose solution for 24 hours, 30% sucrose solution fixed for 1 weeks, frozen section for 24 hours at 37 centigrade drying box, immunofluorescence detection E-Cadherin, and the expression of N-Cadherin protein in the above experiments all had negative control experiments, each experiment repeated 3 times at least. The analysis was completed with SPSS13.0 statistical software, and the results were expressed with mean standard deviation (Mean + SD). Single factor analysis of variance (One-way ANOVA) was used among groups. Tukey method was used to compare the homogeneity of variance. Dunnett's T3 method was used for the comparison of variance between groups. The difference was statistically significant. Results: 1. a successful construction of an in vitro group was made. A three-dimensional model of human derived human breast cancer is built. Through the three cycles of A gelatin and sodium alginate electrostatic action, a model of a single tumor cell is assembled by a single tumor cell. Then a medium molecular weight chitosan solution is added to make the aggregation of the individual cells of the layers assembled to form a multicellular tumor ball, that is, tissue engineering. Human breast cancer tumor model.2. under optical microscope showed that the three-dimensional model of human breast cancer was lump shaped and the internal cell (3Dis) morphology.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R73-35

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