利用一种简易的3D共培养体系探究乳腺癌相关成纤维细胞在MDA-MB-231细胞上皮—间充质转换中的作用
发布时间：2019-02-26 10:21
【摘要】：研究背景:乳腺癌是威胁女性健康最常见的一种上皮肿瘤。研究表明乳腺癌相关成纤维细胞(CAF)可以刺激乳腺癌细胞的侵蚀和转移。然而,由于缺乏合适的模型,CAF对于肿瘤的发展和转移所起到的作用仍然是不确切的。在这里我们建立了一种易得的共培养系统,它可以在体外提供一种与体内乳腺癌周边细胞更相似的细胞微环境。这种3D冷冻凝胶共培养系统可以作为观察乳腺癌细胞和基质细胞相互作用的理想平台,并且它还可以直接评估CAF在3D共培养条件下对于乳腺癌转移的影响。基于这种系统我们发现CAF可以促进MDA-MB-231细胞在体外的EMT和在体内的恶化转移。这种简易的3D共培养体系也许能为研究EMT机制提供一种简便方法并为抗肿瘤药物研究提供一个实用的平台。目的:1.利用甲基化的明胶制作弹性冷冻凝胶,并与人乳腺癌细胞MDA-MB-231及MCF-7细胞共培养,检测其表征,以建立适宜乳腺癌细胞生长的三维支架,并提供更优越的平台来进一步研究三维条件下EMT机制对肿瘤发生发展的作用。2.利用细胞与材料构建以CAFs为基质细胞层的三维人源性乳腺癌组织,探讨体外乳腺癌三维构建对乳腺癌细胞迁移的影响。3.选用裸鼠为实验对象,比较传统二维培养条件,以冷冻凝胶为平台的三维肿瘤模型及复合CAF细胞共培养的以冷冻凝胶为平台的三维肿瘤模型下乳腺癌细胞在裸鼠体内的迁移情况,初步探讨这种简易的3D共培养体系相比于传统培养模型的优势,为研究EMT机制提供一种简便方法并为抗肿瘤药物研究提供一个实用的平台。方法:1.利用临床乳腺癌肿瘤新鲜标本分离成纤维细胞(CAF)2.细胞培养。购买人乳腺癌细胞株MDA-MB-231,一组用于传统二维培养,一组与cryogel共培养,一组与复合CAF细胞基底层的cryogel共培养,构建3D肿瘤组织模型。3.体外验证简易的3D共培养体系对EMT影响。4.利用BALB/c-nu裸鼠进行体内实验进一步验证。5.统计学分析。统计分析采用SPSS13.0统计软件完成,p0.05认为差异有统计学意义。结果:1.成功制备了与细胞相容性良好的弹性cryogel。2.成功分离并培养了人源性乳腺癌成纤维细胞(CAF)。3.构建了复合CAF细胞作为基质层的cryogel三维乳腺癌模型。4.该模型可促进MDA-MB-231细胞的EMT。5.体内实验表明该模型可促进乳腺癌肿瘤的EMT和恶性度增强。结论:对于3D共培养条件下乳腺癌细胞和基质细胞间相互作用的研究,这种甲基化的明胶冷冻凝胶是一种理想的材料。根据我们发明的3D共培养体系,我们发现CAF细胞可以促进MDA-MB-231细胞在体外的EMT以及在体内的恶化和转移。这种简易的3D共培养体系可以成为一种方便的方法来研究EMT机制,并为体内抗乳腺癌药物的研究提供一个有价值的平台。
[Abstract]:Background: breast cancer is one of the most common epithelial tumors that threaten women's health. Studies have shown that breast cancer-associated fibroblast (CAF) can stimulate invasion and metastasis of breast cancer cells. However, due to the lack of appropriate models, the role of CAF in the development and metastasis of tumors is still uncertain. Here we establish an easy-to-obtain co-culture system that provides a cell microenvironment in vitro that is more similar to that of breast cancer peripheral cells in vivo. This 3-D frozen gel co-culture system can be used as an ideal platform for observing the interaction between breast cancer cells and stromal cells, and it can also directly evaluate the effect of CAF on breast cancer metastasis under 3-D co-culture conditions. Based on this system, we found that CAF can promote the deterioration and metastasis of MDA-MB-231 cells in vitro and in vivo. This simple 3D co-culture system may provide a simple method for studying the mechanism of EMT and a practical platform for the study of anticancer drugs. Objective: 1. Elastic frozen gel was made from methylated gelatin and co-cultured with human breast cancer cell line MDA-MB-231 and MCF-7 cells to detect their characterization in order to establish a three-dimensional scaffold suitable for the growth of breast cancer cells. And to provide a better platform for further study of the three-dimensional EMT mechanism in the carcinogenesis and development of the role. 2. Three-dimensional human breast cancer tissue with CAFs as stroma cell layer was constructed by using cells and materials, and the effect of three-dimensional construction of breast cancer in vitro on migration of breast cancer cells was investigated. 3. Nude mice were selected as the experimental object, and the traditional two-dimensional culture conditions were compared. The migration of breast cancer cells in nude mice under three-dimensional tumor model co-cultured with frozen gel and co-cultured with CAF cells in nude mice. The advantages of this simple 3D co-culture system compared with the traditional culture model are discussed. It provides a simple method for studying the mechanism of EMT and a practical platform for the research of anti-tumor drugs. Methods: 1. Isolation of fibroblasts from fresh specimens of clinical breast cancer (CAF) 2. Cell culture. Human breast cancer cell line MDA-MB-231, was purchased for traditional two-dimensional culture, one group was co-cultured with cryogel, one group was co-cultured with cryogel in basal layer of CAF cells, and the other group was co-cultured with cryogel in basal layer of CAF cells to construct 3D tumor tissue model. 3. The effect of simple 3D co-culture system on EMT was verified in vitro. 4. BALB/c-nu nude mice were used for further verification in vivo. 5. Statistical analysis. Statistical analysis using SPSS13.0 statistical software, p0.05 that the difference is statistically significant. Results: 1. Elastic cryogel.2. with good cell compatibility was successfully prepared. (CAF). 3. Human breast cancer fibroblasts were successfully isolated and cultured. A three-dimensional model of cryogel breast cancer with CAF cells as matrix layer was constructed. 4. This model can promote the EMT.5. of MDA-MB-231 cells. In vivo experiments show that the model can promote the enhancement of EMT and malignant degree of breast cancer. Conclusion: the methylated gelatin freeze gel is an ideal material for the study of the interaction between breast cancer cells and stromal cells in 3D co-culture. According to the 3D co-culture system we invented, we found that CAF cells can promote the degradation and metastasis of MDA-MB-231 cells in vitro and in vivo. This simple 3D co-culture system can be used as a convenient method to study the mechanism of EMT and provide a valuable platform for the study of anti-breast cancer drugs in vivo.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
[Abstract]:Background: breast cancer is one of the most common epithelial tumors that threaten women's health. Studies have shown that breast cancer-associated fibroblast (CAF) can stimulate invasion and metastasis of breast cancer cells. However, due to the lack of appropriate models, the role of CAF in the development and metastasis of tumors is still uncertain. Here we establish an easy-to-obtain co-culture system that provides a cell microenvironment in vitro that is more similar to that of breast cancer peripheral cells in vivo. This 3-D frozen gel co-culture system can be used as an ideal platform for observing the interaction between breast cancer cells and stromal cells, and it can also directly evaluate the effect of CAF on breast cancer metastasis under 3-D co-culture conditions. Based on this system, we found that CAF can promote the deterioration and metastasis of MDA-MB-231 cells in vitro and in vivo. This simple 3D co-culture system may provide a simple method for studying the mechanism of EMT and a practical platform for the study of anticancer drugs. Objective: 1. Elastic frozen gel was made from methylated gelatin and co-cultured with human breast cancer cell line MDA-MB-231 and MCF-7 cells to detect their characterization in order to establish a three-dimensional scaffold suitable for the growth of breast cancer cells. And to provide a better platform for further study of the three-dimensional EMT mechanism in the carcinogenesis and development of the role. 2. Three-dimensional human breast cancer tissue with CAFs as stroma cell layer was constructed by using cells and materials, and the effect of three-dimensional construction of breast cancer in vitro on migration of breast cancer cells was investigated. 3. Nude mice were selected as the experimental object, and the traditional two-dimensional culture conditions were compared. The migration of breast cancer cells in nude mice under three-dimensional tumor model co-cultured with frozen gel and co-cultured with CAF cells in nude mice. The advantages of this simple 3D co-culture system compared with the traditional culture model are discussed. It provides a simple method for studying the mechanism of EMT and a practical platform for the research of anti-tumor drugs. Methods: 1. Isolation of fibroblasts from fresh specimens of clinical breast cancer (CAF) 2. Cell culture. Human breast cancer cell line MDA-MB-231, was purchased for traditional two-dimensional culture, one group was co-cultured with cryogel, one group was co-cultured with cryogel in basal layer of CAF cells, and the other group was co-cultured with cryogel in basal layer of CAF cells to construct 3D tumor tissue model. 3. The effect of simple 3D co-culture system on EMT was verified in vitro. 4. BALB/c-nu nude mice were used for further verification in vivo. 5. Statistical analysis. Statistical analysis using SPSS13.0 statistical software, p0.05 that the difference is statistically significant. Results: 1. Elastic cryogel.2. with good cell compatibility was successfully prepared. (CAF). 3. Human breast cancer fibroblasts were successfully isolated and cultured. A three-dimensional model of cryogel breast cancer with CAF cells as matrix layer was constructed. 4. This model can promote the EMT.5. of MDA-MB-231 cells. In vivo experiments show that the model can promote the enhancement of EMT and malignant degree of breast cancer. Conclusion: the methylated gelatin freeze gel is an ideal material for the study of the interaction between breast cancer cells and stromal cells in 3D co-culture. According to the 3D co-culture system we invented, we found that CAF cells can promote the degradation and metastasis of MDA-MB-231 cells in vitro and in vivo. This simple 3D co-culture system can be used as a convenient method to study the mechanism of EMT and provide a valuable platform for the study of anti-breast cancer drugs in vivo.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
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