人胚肺成纤维细胞与肺腺癌细胞共培养时的相互影响和转化现象

发布时间：2018-11-18 14:10

【摘要】：目的:本实验通过人胚肺成纤维和肺腺癌细胞在体外共培养的方法,以明确肿瘤微环境中正常成纤维细胞与肿瘤细胞之间的相互作用和影响,免疫荧光染色初步揭示细胞外基质中a-SMA明显上调,说明正常成纤维细胞是被“激活”,具有肿瘤相关成纤维细胞的特征表达和性质,并且“活化”的成纤维细胞可以使经诱导的肺腺癌细胞在其细胞增殖能力、侵袭能力、迁移能力和对顺铂等常规化疗药物敏感性等生物学行为方面的改变。方法:通过研究正常成纤维细胞与肿瘤细胞的相互影响和转化现象,将体外模型用于肿瘤与基质环境相互作用的多因素过程中。1.通过人胚肺成纤维细胞和肺腺癌细胞共培养,利用免疫荧光技术明确“活化”的成纤维细胞特征性地高表达α-SMA的情况,以充分证明正常成纤维细胞向肿瘤相关成纤维细胞转变的可能。2.通过MTS细胞增殖实验,比较经诱导后的肺腺癌细胞的增殖能力的变化。3.通过流式细胞周期检测实验,检测细胞周期的分布和凋亡情况。4.通过划痕实验和Transwell法检测细胞迁移能力实验,比较经诱导后的肺腺癌细胞的迁移能力的变化。5.通过Transwell法检测细胞侵袭能力实验,比较经诱导后的肺腺癌细胞的侵袭能力的变化。6.通过药物抑制实验,利用MTS法检测和比较经诱导后的肺腺癌细胞对顺铂等化疗药物敏感性的变化。结果:正常成纤维细胞可以产生促进细胞增殖的间质基质和特异性地持续表达Vimentin蛋白,而α-SMA是几乎不表达的。肿瘤相关成纤维细胞(CAF)可以合成、分泌大量层粘连蛋白-1、α-SMA等,且随着处理时间的增加,α-SMA表达上调。1.人胚肺成纤维细胞与肺腺癌细胞体外共培养后可将正常的成纤维细胞“激活”,转变为肿瘤相关成纤维细胞(CAFs),免疫荧光染色显示“活化”的成纤维细胞高表达a-SMA,a-SMA为肿瘤相关成纤维细胞的特异性标志物。2.“活化”的成纤维细胞可以使经诱导的肺腺癌细胞在其细胞增殖能力、侵袭能力、迁移能力方面明显增强,而对顺铂等常规化疗药物敏感性明显降低。结论:1.正常的成纤维细胞在肿瘤细胞诱导下可转化为肿瘤相关成纤维细胞(CAFs),具有肿瘤相关成纤维细胞的性质,对肿瘤的发生、发展起着至关重要的作用。2.“活化”的成纤维细胞可以使经诱导的肿瘤细胞在其细胞增殖能力、侵袭能力、迁移能力方面明显增强,而对顺铂等常规化疗药物耐药。
[Abstract]:Objective: to investigate the interaction and effect of normal fibroblasts and tumor cells in tumor microenvironment by co-culture of human embryonic lung fibroblasts and lung adenocarcinoma cells in vitro. Immunofluorescence staining revealed that a-SMA was significantly up-regulated in extracellular matrix, indicating that normal fibroblasts were "activated" and had the characteristic expression and properties of tumor-associated fibroblasts. Moreover, activated fibroblasts can induce changes in cell proliferation, invasion, migration and sensitivity to conventional chemotherapeutic agents such as cisplatin. Methods: by studying the interaction and transformation between normal fibroblasts and tumor cells, the in vitro model was applied to the multi-factor process of the interaction between tumor and matrix environment. 1. By co-culture of human embryonic lung fibroblasts and lung adenocarcinoma cells, the expression of 伪-SMA in activated fibroblasts was identified by immunofluorescence technique. In order to fully demonstrate the normal fibroblasts to tumor-associated fibroblasts. 2. 2. The proliferative ability of lung adenocarcinoma cells after induction was compared by MTS cell proliferation assay. 3. 3. The distribution and apoptosis of cell cycle were detected by flow cytometry. 4. The migration ability of lung adenocarcinoma cells was measured by scratch test and Transwell assay. The invasive ability of lung adenocarcinoma cells was measured by Transwell assay. 6. 6%. MTS assay was used to detect and compare the sensitivity of induced lung adenocarcinoma cells to cisplatin and other chemotherapeutic agents. Results: normal fibroblasts could produce stromal matrix to promote cell proliferation and express Vimentin protein continuously, but 伪-SMA was almost unexpressed. (CAF) can be synthesized and secreted a lot of laminin 1, 伪-SMA, etc. The expression of 伪-SMA is up-regulated with the increase of treatment time. After co-culture of human embryonic lung fibroblasts and lung adenocarcinoma cells in vitro, normal fibroblasts were "activated" and transformed into tumor-associated fibroblasts. (CAFs), immunofluorescence staining showed that "activated" fibroblasts overexpressed a-SMA. A-SMA is a specific marker of tumor-associated fibroblasts. 2. "activated" fibroblasts could significantly enhance the ability of proliferation, invasion and migration of induced lung adenocarcinoma cells, while the sensitivity to conventional chemotherapy drugs such as cisplatin was significantly decreased. Conclusion: 1. Normal fibroblasts can be transformed into tumor-associated fibroblasts (CAFs),) under the induction of tumor cells, which have the properties of tumor-associated fibroblasts and play an important role in the development and development of tumor. 2. "activated" fibroblasts can significantly enhance the ability of tumor cells to proliferate, invade and migrate, but are resistant to conventional chemotherapeutic drugs such as cisplatin.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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