利用一種簡易的3D共培養(yǎng)體系探究乳腺癌相關成纖維細胞在MDA-MB-231細胞上皮—間充質轉換中的作用
發(fā)布時間：2019-02-26 10:21
【摘要】：研究背景:乳腺癌是威脅女性健康最常見的一種上皮腫瘤。研究表明乳腺癌相關成纖維細胞(CAF)可以刺激乳腺癌細胞的侵蝕和轉移。然而,由于缺乏合適的模型,CAF對于腫瘤的發(fā)展和轉移所起到的作用仍然是不確切的。在這里我們建立了一種易得的共培養(yǎng)系統,它可以在體外提供一種與體內乳腺癌周邊細胞更相似的細胞微環(huán)境。這種3D冷凍凝膠共培養(yǎng)系統可以作為觀察乳腺癌細胞和基質細胞相互作用的理想平臺,并且它還可以直接評估CAF在3D共培養(yǎng)條件下對于乳腺癌轉移的影響�；谶@種系統我們發(fā)現CAF可以促進MDA-MB-231細胞在體外的EMT和在體內的惡化轉移。這種簡易的3D共培養(yǎng)體系也許能為研究EMT機制提供一種簡便方法并為抗腫瘤藥物研究提供一個實用的平臺。目的:1.利用甲基化的明膠制作彈性冷凍凝膠,并與人乳腺癌細胞MDA-MB-231及MCF-7細胞共培養(yǎng),檢測其表征,以建立適宜乳腺癌細胞生長的三維支架,并提供更優(yōu)越的平臺來進一步研究三維條件下EMT機制對腫瘤發(fā)生發(fā)展的作用。2.利用細胞與材料構建以CAFs為基質細胞層的三維人源性乳腺癌組織,探討體外乳腺癌三維構建對乳腺癌細胞遷移的影響。3.選用裸鼠為實驗對象,比較傳統二維培養(yǎng)條件,以冷凍凝膠為平臺的三維腫瘤模型及復合CAF細胞共培養(yǎng)的以冷凍凝膠為平臺的三維腫瘤模型下乳腺癌細胞在裸鼠體內的遷移情況,初步探討這種簡易的3D共培養(yǎng)體系相比于傳統培養(yǎng)模型的優(yōu)勢,為研究EMT機制提供一種簡便方法并為抗腫瘤藥物研究提供一個實用的平臺。方法:1.利用臨床乳腺癌腫瘤新鮮標本分離成纖維細胞(CAF)2.細胞培養(yǎng)。購買人乳腺癌細胞株MDA-MB-231,一組用于傳統二維培養(yǎng),一組與cryogel共培養(yǎng),一組與復合CAF細胞基底層的cryogel共培養(yǎng),構建3D腫瘤組織模型。3.體外驗證簡易的3D共培養(yǎng)體系對EMT影響。4.利用BALB/c-nu裸鼠進行體內實驗進一步驗證。5.統計學分析。統計分析采用SPSS13.0統計軟件完成,p0.05認為差異有統計學意義。結果:1.成功制備了與細胞相容性良好的彈性cryogel。2.成功分離并培養(yǎng)了人源性乳腺癌成纖維細胞(CAF)。3.構建了復合CAF細胞作為基質層的cryogel三維乳腺癌模型。4.該模型可促進MDA-MB-231細胞的EMT。5.體內實驗表明該模型可促進乳腺癌腫瘤的EMT和惡性度增強。結論:對于3D共培養(yǎng)條件下乳腺癌細胞和基質細胞間相互作用的研究,這種甲基化的明膠冷凍凝膠是一種理想的材料。根據我們發(fā)明的3D共培養(yǎng)體系,我們發(fā)現CAF細胞可以促進MDA-MB-231細胞在體外的EMT以及在體內的惡化和轉移。這種簡易的3D共培養(yǎng)體系可以成為一種方便的方法來研究EMT機制,并為體內抗乳腺癌藥物的研究提供一個有價值的平臺。
[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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.9
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