腫瘤相關(guān)巨噬細(xì)胞對(duì)肺腺癌的發(fā)展及耐藥作用的研究
發(fā)布時(shí)間：2018-09-13 06:12
【摘要】：第一部分腫瘤相關(guān)巨噬細(xì)胞的誘導(dǎo)及鑒定目的:通過(guò)體外誘導(dǎo)確立人腫瘤相關(guān)巨噬細(xì)胞(TAM),并分析其表型特征。方法:一般觀點(diǎn)認(rèn)為,普通巨噬細(xì)胞為M1型巨噬細(xì)胞,TAM為M2型巨噬細(xì)胞。本部分首先運(yùn)用佛波酯(PMA)誘導(dǎo)人單核細(xì)胞株THP-1細(xì)胞貼壁分化成巨噬細(xì)胞(PMA only型),運(yùn)用流式細(xì)胞分析儀檢測(cè)巨噬細(xì)胞(PMA only型)表面分子CD14的表達(dá)水平,然后運(yùn)用人白細(xì)胞介素4(IL-4)或脂多糖(LPS)分別刺激巨噬細(xì)胞極化成為M2或M1型巨噬細(xì)胞;半定量RT-PCR法檢測(cè)不同組巨噬細(xì)胞IL-6m RNA的表達(dá)水平,ELISA實(shí)驗(yàn)分析不同組巨噬細(xì)胞TNF-α蛋白的表達(dá)情況。結(jié)果:THP-1細(xì)胞被PMA誘導(dǎo)后,其細(xì)胞表面CD14的表達(dá)水平明顯增高。和LPS誘導(dǎo)極化的M1型巨噬細(xì)胞相比,單獨(dú)運(yùn)用PMA誘導(dǎo)的巨噬細(xì)胞(PMA only型)和IL-4誘導(dǎo)的M2型巨噬細(xì)胞其IL-6m RNA的表達(dá)和TNF-α蛋白的表達(dá)水平明顯降低。結(jié)論:成功誘導(dǎo)了巨噬細(xì)胞,其CD14高表達(dá)且偏向于M2型巨噬細(xì)胞;成功誘導(dǎo)建立了TAM,其特征為M2型巨噬細(xì)胞,表現(xiàn)為IL-6及TNF-α低表達(dá)。第二部分腫瘤相關(guān)巨噬細(xì)胞對(duì)肺腺癌細(xì)胞遷移、侵襲的影響及初步機(jī)制研究目的:探討腫瘤相關(guān)巨噬細(xì)胞(TAM)對(duì)人肺腺癌A549細(xì)胞遷移和侵襲的影響,并初步分析其潛在的分子機(jī)制。方法:將肺腺癌A549細(xì)胞與腫瘤相關(guān)巨噬細(xì)胞(TAM)通過(guò)transwell小室進(jìn)行間接共培養(yǎng),然后收集其共培養(yǎng)液用作后續(xù)實(shí)驗(yàn)。用劃痕愈合實(shí)驗(yàn)檢測(cè)腫瘤相關(guān)巨噬細(xì)胞(TAM)對(duì)A549細(xì)胞遷移的影響;用transwell侵襲實(shí)驗(yàn)檢測(cè)腫瘤相關(guān)巨噬細(xì)胞(TAM)對(duì)A549細(xì)胞侵襲的影響;用半定量RT-PCR,q RT-PCR和western blot法檢測(cè)腫瘤相關(guān)巨噬細(xì)胞(TAM)對(duì)A549細(xì)胞VEGF-C,VEGF-D和VEGFR3表達(dá)的影響;用免疫組化法(IHC)檢測(cè)VEGFR3在肺腺癌組織中的表達(dá)分布。在腫瘤相關(guān)巨噬細(xì)胞(TAM)微環(huán)境中,VEGFR3的抑制劑(MAZ51)抑制VEGFR3后,用劃痕愈合實(shí)驗(yàn)和transwell侵襲實(shí)驗(yàn)分別檢測(cè)A549細(xì)胞的遷移、侵襲能力。結(jié)果:腫瘤相關(guān)巨噬細(xì)胞(TAM)促進(jìn)肺腺癌A549細(xì)胞的遷移和侵襲。腫瘤相關(guān)巨噬細(xì)胞(TAM)提高肺腺癌A549細(xì)胞VEGF-C和VEGFR3蛋白的表達(dá)。免疫組化(IHC)實(shí)驗(yàn)證實(shí)VEGFR3主要在人肺腺癌細(xì)胞中高表達(dá)。抑制VEGFR3后明顯降低肺腺癌A549細(xì)胞的遷移和侵襲。結(jié)論:腫瘤相關(guān)巨噬細(xì)胞(TAM)通過(guò)提高A549細(xì)胞VEGFR3的表達(dá)從而促進(jìn)腫瘤細(xì)胞的遷移和侵襲。第三部分腫瘤相關(guān)巨噬細(xì)胞介導(dǎo)的VEGFR3對(duì)腫瘤增殖的影響目的:探討腫瘤相關(guān)巨噬細(xì)胞(TAM)介導(dǎo)的人肺腺癌細(xì)胞VEGFR3的表達(dá)對(duì)肺腺癌細(xì)胞增殖的影響,并探究其潛在的分子機(jī)制。方法:用MTT實(shí)驗(yàn)分析在不同濃度梯度的VEGFR3抑制劑(MAZ51)作用下,其共培養(yǎng)液中A549細(xì)胞的活力變化;流式細(xì)胞分析儀檢測(cè)在MAZ51作用下,共培養(yǎng)A549細(xì)胞的凋亡情況;細(xì)胞集落形成實(shí)驗(yàn)分析MAZ51對(duì)共培養(yǎng)A549細(xì)胞增值的抑制作用。Western blot分析法檢測(cè)在MAZ51作用下共培養(yǎng)A549細(xì)胞中MAPK信號(hào)通路以及P53,PTEN,BCL-2和MMP-2蛋白表達(dá)變化;半定量RT-PCR法檢測(cè)ERK信號(hào)通路抑制劑U0126抑制ERK信號(hào)通路及si RNA干擾P53和PTEN的表達(dá)后,共培養(yǎng)A549細(xì)胞中P53和PTEN m RNA的表達(dá)變化;MTT實(shí)驗(yàn)分析si RNA干擾P53和PTEN的表達(dá)后,在MAZ51作用下,共培養(yǎng)A549細(xì)胞的活力變化。結(jié)果:MAZ51降低共培養(yǎng)A549細(xì)胞的增殖活力,促進(jìn)其凋亡,降低其細(xì)胞集落形成數(shù)。Western blot實(shí)驗(yàn)證實(shí)MAZ51能夠抑制p-ERK的活性,提高抑癌蛋白P53,PTEN的表達(dá),降低抗凋亡蛋白BCL-2和促侵襲蛋白MMP-2的表達(dá)。抑制ERK信號(hào)通路后,P53和PTEN m RNA的表達(dá)明顯提高。干擾P53和PTEN的表達(dá)后,提高了共培養(yǎng)A549細(xì)胞的增殖活力。結(jié)論:抑制VEGFR3后,可通過(guò)降低p-ERK的活性,提高抑癌蛋白P53和PTEN的表達(dá),進(jìn)而抑制腫瘤細(xì)胞的增殖活力。第四部分腫瘤相關(guān)巨噬細(xì)胞介導(dǎo)的VEGFR3對(duì)腫瘤藥物敏感性的影響目的:探討腫瘤相關(guān)巨噬細(xì)胞(TAM)介導(dǎo)的人肺腺癌細(xì)胞VEGFR3的表達(dá)對(duì)肺腺癌A549細(xì)胞化療藥物敏感性的影響,為臨床肺腺癌的治療提供新的策略。方法:在體外,用流式細(xì)胞分析儀檢測(cè)VEGFR3抑制劑(MAZ51)與化療藥物多柔比星在不同的藥物組合方案中,其共培養(yǎng)液中肺腺癌A549細(xì)胞凋亡的情況。在體內(nèi),構(gòu)建裸鼠皮下成瘤模型,檢測(cè)VEGFR3抑制劑(MAZ51)對(duì)肺腺癌化療藥物敏感性的影響。結(jié)果:體外實(shí)驗(yàn)證實(shí),與單獨(dú)運(yùn)用MAZ51、多柔比星或者兩者同時(shí)運(yùn)用相比,先用MAZ51處理腫瘤細(xì)胞,而后用多柔比星處理腫瘤細(xì)胞后,其細(xì)胞的凋亡比例比單用MAZ51、多柔比星或者同時(shí)運(yùn)用兩種藥物更加明顯。體內(nèi)實(shí)驗(yàn)證實(shí),與單獨(dú)運(yùn)用多柔比星相比,MAZ51與多柔比星聯(lián)合運(yùn)用后,其裸鼠腫瘤的生長(zhǎng)體積明顯降低。結(jié)論:抑制VEGFR3后可以增強(qiáng)藥物多柔比星對(duì)肺腺癌的化療敏感性。
[Abstract]:Part I: Induction and identification of tumor-associated macrophages Objective: To establish human tumor-associated macrophages (TAM) by induction in vitro and analyze their phenotypic characteristics. Methods: It is generally believed that normal macrophages are M1 macrophages and TAM is M2 macrophages. The expression of CD14 on the surface of macrophages (PMA only type) was detected by flow cytometry, and then the polarization of macrophages was stimulated by human interleukin 4 (IL-4) or lipopolysaccharide (LPS) to M2 or M1 type macrophages respectively. IL-6mR was detected by semi-quantitative RT-PCR. Results: The expression of CD14 on the surface of THP-1 cells was significantly increased after PMA induction. Compared with LPS-induced polarized M 1 macrophages, the expression of IL-6 in PMA-induced macrophages and IL-4-induced M2 macrophages was significantly increased. CONCLUSION: Macrophages were successfully induced to express CD14 over-expression and tend to M2 type macrophages, and TAM was successfully induced, characterized by low expression of IL-6 and TNF-alpha in M2 type macrophages. Objective: To investigate the effect of tumor-associated macrophages (TAM) on the migration and invasion of human lung adenocarcinoma A549 cells, and to analyze its potential molecular mechanism. Methods: TAM and A549 cells were co-cultured indirectly in Transwell chamber, and then the co-culture medium was collected for follow-up. Scratch healing test was used to detect the effect of tumor-associated macrophages (TAM) on the migration of A549 cells; Transwell invasion test was used to detect the effect of tumor-associated macrophages (TAM) on the invasion of A549 cells; semi-quantitative RT-PCR, Q RT-PCR and Western blot were used to detect the expression of VEGF-C, VEGF-D and VEGFR3 in A549 cells. Immunohistochemistry (IHC) was used to detect the expression and distribution of VEGF R3 in lung adenocarcinoma. In tumor-associated macrophage (TAM) microenvironment, the inhibitor of VEGF R3 (MAZ51) inhibited the expression of VEGF R3. Scratch healing test and Transwell invasion test were used to detect the migration and invasion ability of A549 cells. Tumor-associated macrophages (TAM) increased the expression of VEGF-C and VEGFR3 protein in lung adenocarcinoma A549 cells. Immunohistochemistry (IHC) showed that the expression of VEGF-R3 was mainly high in human lung adenocarcinoma cells. Inhibition of VEGF-R3 significantly decreased the migration and invasion of lung adenocarcinoma A549 cells. Cell (TAM) promotes the migration and invasion of tumor cells by increasing the expression of VEGFR3 in A549 cells. Part III Effect of tumor-associated macrophages-mediated VEGFR3 on tumor proliferation Objective: To investigate the effect of tumor-associated macrophages (TAM)-mediated expression of human lung adenocarcinoma cell line VEGFR3 on the proliferation of lung adenocarcinoma cells and explore its potential. Methods: MTT assay was used to analyze the changes of A549 cell viability in co-culture medium with different concentration gradient of inhibitor of VEGF R3 (MAZ51); flow cytometry was used to detect the apoptosis of A549 cells in co-culture with MAZ51; cell colony formation assay was used to analyze the inhibitory effect of MAZ51 on the proliferation of co-cultured A549 cells. Western blot assay was used to detect the expression of MAPK signaling pathway and P53, PTEN, BCL-2 and MMP-2 proteins in A549 cells co-cultured with MAZ51. Semi-quantitative RT-PCR was used to detect the inhibition of ERK signaling pathway by ERK signaling pathway inhibitor U0126 and the interference of SIRNA with P53 and PTEN. After interfering with the expression of P53 and PTEN by Si RNA, A549 cells were co-cultured with MAZ51. Results: MAZ51 decreased the proliferation, apoptosis and colony formation of A549 cells. Western blot showed that MAZ51 could inhibit the activity of p-ERK, increase the expression of tumor suppressor P53 and PTEN, and decrease the number of colony formation. The expression of anti-apoptotic protein BCL-2 and pro-invasive protein MMP-2 was significantly increased after inhibition of ERK signaling pathway. Interference with the expression of P53 and PTEN increased the proliferative activity of co-cultured A549 cells. Conclusion: Inhibition of VEGF R3 can inhibit the expression of tumor suppressor protein P53 and PTEN by decreasing the activity of p-ERK. Part IV Effect of tumor-associated macrophages-mediated VEGF R3 on tumor drug sensitivity Objective: To investigate the effect of tumor-associated macrophages (TAM)-mediated expression of human lung adenocarcinoma cell line VEGF R3 on chemosensitivity of lung adenocarcinoma A549 cells, and to provide a new strategy for clinical treatment of lung adenocarcinoma. In vitro, the apoptosis of lung adenocarcinoma A549 cells was detected by flow cytometry (FCM) in the co-culture medium of the inhibitor of VEGF R3 (MAZ51) and the chemotherapeutic drug doxorubicin in different regimens. In vivo, a nude mouse model of subcutaneous tumor formation was established to detect the effect of the inhibitor of VEGF R3 (MAZ51) on the chemosensitivity of lung adenocarcinoma. Compared with MAZ51, doxorubicin or both, the apoptotic rate of tumor cells treated with MAZ51 and then doxorubicin was more obvious than that treated with MAZ51, doxorubicin or both. Conclusion: Inhibition of VEGF R3 can enhance the chemosensitivity of doxorubicin to lung adenocarcinoma.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R734.2
本文編號(hào)：2240314
[Abstract]:Part I: Induction and identification of tumor-associated macrophages Objective: To establish human tumor-associated macrophages (TAM) by induction in vitro and analyze their phenotypic characteristics. Methods: It is generally believed that normal macrophages are M1 macrophages and TAM is M2 macrophages. The expression of CD14 on the surface of macrophages (PMA only type) was detected by flow cytometry, and then the polarization of macrophages was stimulated by human interleukin 4 (IL-4) or lipopolysaccharide (LPS) to M2 or M1 type macrophages respectively. IL-6mR was detected by semi-quantitative RT-PCR. Results: The expression of CD14 on the surface of THP-1 cells was significantly increased after PMA induction. Compared with LPS-induced polarized M 1 macrophages, the expression of IL-6 in PMA-induced macrophages and IL-4-induced M2 macrophages was significantly increased. CONCLUSION: Macrophages were successfully induced to express CD14 over-expression and tend to M2 type macrophages, and TAM was successfully induced, characterized by low expression of IL-6 and TNF-alpha in M2 type macrophages. Objective: To investigate the effect of tumor-associated macrophages (TAM) on the migration and invasion of human lung adenocarcinoma A549 cells, and to analyze its potential molecular mechanism. Methods: TAM and A549 cells were co-cultured indirectly in Transwell chamber, and then the co-culture medium was collected for follow-up. Scratch healing test was used to detect the effect of tumor-associated macrophages (TAM) on the migration of A549 cells; Transwell invasion test was used to detect the effect of tumor-associated macrophages (TAM) on the invasion of A549 cells; semi-quantitative RT-PCR, Q RT-PCR and Western blot were used to detect the expression of VEGF-C, VEGF-D and VEGFR3 in A549 cells. Immunohistochemistry (IHC) was used to detect the expression and distribution of VEGF R3 in lung adenocarcinoma. In tumor-associated macrophage (TAM) microenvironment, the inhibitor of VEGF R3 (MAZ51) inhibited the expression of VEGF R3. Scratch healing test and Transwell invasion test were used to detect the migration and invasion ability of A549 cells. Tumor-associated macrophages (TAM) increased the expression of VEGF-C and VEGFR3 protein in lung adenocarcinoma A549 cells. Immunohistochemistry (IHC) showed that the expression of VEGF-R3 was mainly high in human lung adenocarcinoma cells. Inhibition of VEGF-R3 significantly decreased the migration and invasion of lung adenocarcinoma A549 cells. Cell (TAM) promotes the migration and invasion of tumor cells by increasing the expression of VEGFR3 in A549 cells. Part III Effect of tumor-associated macrophages-mediated VEGFR3 on tumor proliferation Objective: To investigate the effect of tumor-associated macrophages (TAM)-mediated expression of human lung adenocarcinoma cell line VEGFR3 on the proliferation of lung adenocarcinoma cells and explore its potential. Methods: MTT assay was used to analyze the changes of A549 cell viability in co-culture medium with different concentration gradient of inhibitor of VEGF R3 (MAZ51); flow cytometry was used to detect the apoptosis of A549 cells in co-culture with MAZ51; cell colony formation assay was used to analyze the inhibitory effect of MAZ51 on the proliferation of co-cultured A549 cells. Western blot assay was used to detect the expression of MAPK signaling pathway and P53, PTEN, BCL-2 and MMP-2 proteins in A549 cells co-cultured with MAZ51. Semi-quantitative RT-PCR was used to detect the inhibition of ERK signaling pathway by ERK signaling pathway inhibitor U0126 and the interference of SIRNA with P53 and PTEN. After interfering with the expression of P53 and PTEN by Si RNA, A549 cells were co-cultured with MAZ51. Results: MAZ51 decreased the proliferation, apoptosis and colony formation of A549 cells. Western blot showed that MAZ51 could inhibit the activity of p-ERK, increase the expression of tumor suppressor P53 and PTEN, and decrease the number of colony formation. The expression of anti-apoptotic protein BCL-2 and pro-invasive protein MMP-2 was significantly increased after inhibition of ERK signaling pathway. Interference with the expression of P53 and PTEN increased the proliferative activity of co-cultured A549 cells. Conclusion: Inhibition of VEGF R3 can inhibit the expression of tumor suppressor protein P53 and PTEN by decreasing the activity of p-ERK. Part IV Effect of tumor-associated macrophages-mediated VEGF R3 on tumor drug sensitivity Objective: To investigate the effect of tumor-associated macrophages (TAM)-mediated expression of human lung adenocarcinoma cell line VEGF R3 on chemosensitivity of lung adenocarcinoma A549 cells, and to provide a new strategy for clinical treatment of lung adenocarcinoma. In vitro, the apoptosis of lung adenocarcinoma A549 cells was detected by flow cytometry (FCM) in the co-culture medium of the inhibitor of VEGF R3 (MAZ51) and the chemotherapeutic drug doxorubicin in different regimens. In vivo, a nude mouse model of subcutaneous tumor formation was established to detect the effect of the inhibitor of VEGF R3 (MAZ51) on the chemosensitivity of lung adenocarcinoma. Compared with MAZ51, doxorubicin or both, the apoptotic rate of tumor cells treated with MAZ51 and then doxorubicin was more obvious than that treated with MAZ51, doxorubicin or both. Conclusion: Inhibition of VEGF R3 can enhance the chemosensitivity of doxorubicin to lung adenocarcinoma.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R734.2
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 許金華;朱文玉;;肺腺癌化療方案及輔助藥物使用分析[J];中國(guó)藥房;2013年22期
，本文編號(hào)：2240314
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