本文選題：唑來膦酸納米粒　切入點：M2型巨噬細胞　出處：《西南醫(yī)科大學》2017年碩士論文

【摘要】：目的:將U937細胞誘導為M0巨噬細胞、M1型巨噬細胞、M2型巨噬細胞,了解不同類型巨噬細胞對唑來膦酸納米粒的攝取情況;了解唑來膦酸納米粒對共培養(yǎng)情況下腫瘤細胞的遷移、侵襲的影響;唑來膦酸納米粒對共培養(yǎng)情況下腫瘤細胞的增殖抑制情況。方法:1、巨噬細胞誘導:將U937細胞平鋪至六孔板中,加入佛波酯(PMA)誘導6個小時;2、M1型巨噬細胞誘導:將U937細胞平鋪至六孔板中,加入佛波酯(MPA)(200ng/ml)誘導6個小時后再加入脂多糖(LPS)(100ng/ml)和IFN-γ(20ng/ml)繼續(xù)誘導66小時(共72小時)。3、M2型巨噬細胞誘導:將U937細胞平鋪至六孔板中,加入佛波酯(MPA)(200ng/ml)誘導6個小時后再加入IL-4(20ng/ml)和IL-13(20ng/ml)繼續(xù)誘導66小時(共72小時)。并通過檢測Arg-1酶及iNOS的表達情況,檢測是否誘導成功。4、攝取實驗:選取人體成纖維細胞、M1型巨噬細胞、M2巨噬細胞、SW620結(jié)腸癌細胞,加入熒光標記后的唑來膦酸納米粒,反應3小時后,采用Dil對細胞膜進行染色,在熒光顯微鏡下觀察四種細胞對唑來膦酸納米粒的攝取情況。5、CCK-8增殖抑制實驗:通過唑來膦酸納米粒與不同種類細胞的相互作用,了解其對細胞的增殖能力的影響;6、細胞劃痕實驗及transwell小室遷移實驗,設(shè)置sw620結(jié)腸癌組、sw620+m1型巨噬細胞組、sw620+m2型巨噬細胞組,每組分為加和不加唑來膦酸納米粒組(20ng/ul),共6組實驗組,觀察每組各因素對腫瘤細胞遷移能力的影響。7、侵襲實驗:將sw620結(jié)腸癌細胞放入transwell小室的上室中,下室依次為空白組(單獨培養(yǎng)基)、唑來膦酸納米粒組、m1型巨噬細胞組、m1型巨噬細胞+唑來膦酸納米粒組、m2型巨噬細胞組、m2型巨噬細胞+唑來膦酸納米粒組;并采用吉姆薩染色法計數(shù),了解各因素對腫瘤細胞侵襲能力的影響;結(jié)果:1、arg-1及inos檢測結(jié)果顯示m2型巨噬細胞免疫組化呈陽性反應,而其余兩組呈陰性反應,m1型巨噬細胞inos表達(74.4%)遠高于其余兩組(14.0%及14.7%),證實m1及m2型巨噬細胞誘導成功。2、m2型巨噬細胞攝取納米顆粒的熒光強度遠高于其他細胞;3、m1型巨噬細胞抑制腫瘤細胞遷移;m2型巨噬細胞促進腫瘤細胞的遷移,與唑來膦酸納米粒相互作用可抑制m2巨噬細胞的這種促進作用(p0.01);4、m1型巨噬細胞抑制腫瘤細胞侵襲力;m2型巨噬細胞促進腫瘤細胞的侵襲,與唑來膦酸納米粒相互作用可抑制m2巨噬細胞的這種促進作用(p0.01)5、納米顆粒通過抑制m2型巨噬細胞,影響腫瘤細胞增殖(p0.01)。結(jié)論:唑來膦酸納米粒能特異性被m2型巨噬細胞攝取并抑制m2巨噬細胞的增殖。m2巨噬細胞促進腫瘤的遷移、侵襲和增殖。唑來膦酸納米粒通過抑制m2型巨噬細胞間接抑制腫瘤細胞的增殖、遷移及侵襲能力。
[Abstract]:Objective: to study the uptake of zoledronate nanoparticles by different types of macrophages and the migration of tumor cells in co-culture of U937 cells induced as M 0 macrophages and M 1 type macrophages.The effect of zoledronic acid nanoparticles on the proliferation of tumor cells in co-culture.Methods: 1, macrophage induction: U937 cells were tiled into a six-well plate, and PMA was added to induce the induction of M 1 macrophages for 6 hours. U937 cells were tiled into a six-well plate.After 6 hours of induction, we added LPSN 100 ng / ml and IFN- 緯 20 ng / ml) and continued to induce 66 hours (72 hours in all, M 2 macrophage induction: tiling U937 cells into a six-well plate,After 6 hours of induction, IL-4 + 20 ng / ml and IL-13 + 20 ng / ml) were added for 66 hours (72 hours in total).By detecting the expression of Arg-1 enzyme and iNOS, we detected whether the induction was successful or not. The uptake experiment: human fibroblast M 1 macrophage M 2 macrophage was selected as the colon cancer cell line SW620, and the fluorescent labeled zoledronate nanoparticles were added.After 3 hours of reaction, the cell membrane was stained with Dil, and the uptake of zoledronate nanoparticles by four kinds of cells was observed under fluorescence microscope. The inhibition of proliferation of zoledronate nanoparticles was measured by the interaction between zoledronate nanoparticles and different kinds of cells.To understand its effect on cell proliferation, cell scratch test and transwell chamber migration test, we set up sw620 colon cancer group with sw620m1 macrophage group and SW620m2 macrophage group.Each group was divided into two groups with or without zoledronic acid nanoparticles (n = 6). The influence of each factor on the migration ability of tumor cells was observed. The invasion experiment: sw620 colon cancer cells were put into the upper chamber of transwell chamber.The lower chamber was divided into blank group (culture medium alone, zoledronic acid nanoparticles group, M 1 macrophage group, zoledronic acid nanoparticles group, zoledronic acid nanoparticles group, zoledronic acid nanoparticles group, M 2 macrophage group, M 2 macrophage group, zoledronic acid nanoparticles group;The effect of various factors on the invasiveness of tumor cells was investigated by means of Gimsa staining. Results the results of 1: 1 arg-1 and inos showed that the macrophages of type 2 were immunocytochemical positive.However, the other two groups showed negative reaction. The inos expression of M 1 type macrophages was much higher than that of the other two groups (14. 0% and 14. 7%, respectively). It was proved that the fluorescence intensity of M 1 and m 2 macrophages induced successfully by M 2 M 2 macrophages was much higher than that of other cells.Macrophages inhibit the migration of tumor cells and type M 2 macrophages promote the migration of tumor cells.The interaction with zoledronate nanoparticles can inhibit the promotion of m2 macrophage.The interaction with zoledronic acid nanoparticles can inhibit the promotion of m2 macrophages. The nanoparticles can inhibit the proliferation of tumor cells by inhibiting m2 macrophages.Conclusion: zoledronate nanoparticles can be specifically ingested by m2 macrophages and inhibit the proliferation of m2 macrophages. M2 macrophages promote tumor migration, invasion and proliferation.Zoledronate nanoparticles indirectly inhibit the proliferation, migration and invasion of tumor cells by inhibiting m 2 macrophages.
【學位授予單位】：西南醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R73-36

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