【摘要】：目的:本實(shí)驗(yàn)通過人胚肺成纖維和肺腺癌細(xì)胞在體外共培養(yǎng)的方法,以明確腫瘤微環(huán)境中正常成纖維細(xì)胞與腫瘤細(xì)胞之間的相互作用和影響,免疫熒光染色初步揭示細(xì)胞外基質(zhì)中a-SMA明顯上調(diào),說明正常成纖維細(xì)胞是被“激活”,具有腫瘤相關(guān)成纖維細(xì)胞的特征表達(dá)和性質(zhì),并且“活化”的成纖維細(xì)胞可以使經(jīng)誘導(dǎo)的肺腺癌細(xì)胞在其細(xì)胞增殖能力、侵襲能力、遷移能力和對(duì)順鉑等常規(guī)化療藥物敏感性等生物學(xué)行為方面的改變。方法:通過研究正常成纖維細(xì)胞與腫瘤細(xì)胞的相互影響和轉(zhuǎn)化現(xiàn)象,將體外模型用于腫瘤與基質(zhì)環(huán)境相互作用的多因素過程中。1.通過人胚肺成纖維細(xì)胞和肺腺癌細(xì)胞共培養(yǎng),利用免疫熒光技術(shù)明確“活化”的成纖維細(xì)胞特征性地高表達(dá)α-SMA的情況,以充分證明正常成纖維細(xì)胞向腫瘤相關(guān)成纖維細(xì)胞轉(zhuǎn)變的可能。2.通過MTS細(xì)胞增殖實(shí)驗(yàn),比較經(jīng)誘導(dǎo)后的肺腺癌細(xì)胞的增殖能力的變化。3.通過流式細(xì)胞周期檢測實(shí)驗(yàn),檢測細(xì)胞周期的分布和凋亡情況。4.通過劃痕實(shí)驗(yàn)和Transwell法檢測細(xì)胞遷移能力實(shí)驗(yàn),比較經(jīng)誘導(dǎo)后的肺腺癌細(xì)胞的遷移能力的變化。5.通過Transwell法檢測細(xì)胞侵襲能力實(shí)驗(yàn),比較經(jīng)誘導(dǎo)后的肺腺癌細(xì)胞的侵襲能力的變化。6.通過藥物抑制實(shí)驗(yàn),利用MTS法檢測和比較經(jīng)誘導(dǎo)后的肺腺癌細(xì)胞對(duì)順鉑等化療藥物敏感性的變化。結(jié)果:正常成纖維細(xì)胞可以產(chǎn)生促進(jìn)細(xì)胞增殖的間質(zhì)基質(zhì)和特異性地持續(xù)表達(dá)Vimentin蛋白,而α-SMA是幾乎不表達(dá)的。腫瘤相關(guān)成纖維細(xì)胞(CAF)可以合成、分泌大量層粘連蛋白-1、α-SMA等,且隨著處理時(shí)間的增加,α-SMA表達(dá)上調(diào)。1.人胚肺成纖維細(xì)胞與肺腺癌細(xì)胞體外共培養(yǎng)后可將正常的成纖維細(xì)胞“激活”,轉(zhuǎn)變?yōu)槟[瘤相關(guān)成纖維細(xì)胞(CAFs),免疫熒光染色顯示“活化”的成纖維細(xì)胞高表達(dá)a-SMA,a-SMA為腫瘤相關(guān)成纖維細(xì)胞的特異性標(biāo)志物。2.“活化”的成纖維細(xì)胞可以使經(jīng)誘導(dǎo)的肺腺癌細(xì)胞在其細(xì)胞增殖能力、侵襲能力、遷移能力方面明顯增強(qiáng),而對(duì)順鉑等常規(guī)化療藥物敏感性明顯降低。結(jié)論:1.正常的成纖維細(xì)胞在腫瘤細(xì)胞誘導(dǎo)下可轉(zhuǎn)化為腫瘤相關(guān)成纖維細(xì)胞(CAFs),具有腫瘤相關(guān)成纖維細(xì)胞的性質(zhì),對(duì)腫瘤的發(fā)生、發(fā)展起著至關(guān)重要的作用。2.“活化”的成纖維細(xì)胞可以使經(jīng)誘導(dǎo)的腫瘤細(xì)胞在其細(xì)胞增殖能力、侵襲能力、遷移能力方面明顯增強(qiáng),而對(duì)順鉑等常規(guī)化療藥物耐藥。
[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 偽-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 偽-SMA was almost unexpressed. (CAF) can be synthesized and secreted a lot of laminin 1, 偽-SMA, etc. The expression of 偽-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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R734.2

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