甘露糖化壳聚糖唑来膦酸纳米粒体外抗肿瘤效应的初步研究

发布时间：2018-04-03 19:44

本文选题：唑来膦酸纳米粒　切入点：M2型巨噬细胞　出处：《西南医科大学》2017年硕士论文

【摘要】：目的:将U937细胞诱导为M0巨噬细胞、M1型巨噬细胞、M2型巨噬细胞,了解不同类型巨噬细胞对唑来膦酸纳米粒的摄取情况;了解唑来膦酸纳米粒对共培养情况下肿瘤细胞的迁移、侵袭的影响;唑来膦酸纳米粒对共培养情况下肿瘤细胞的增殖抑制情况。方法:1、巨噬细胞诱导:将U937细胞平铺至六孔板中,加入佛波酯(PMA)诱导6个小时;2、M1型巨噬细胞诱导:将U937细胞平铺至六孔板中,加入佛波酯(MPA)(200ng/ml)诱导6个小时后再加入脂多糖(LPS)(100ng/ml)和IFN-γ(20ng/ml)继续诱导66小时(共72小时)。3、M2型巨噬细胞诱导:将U937细胞平铺至六孔板中,加入佛波酯(MPA)(200ng/ml)诱导6个小时后再加入IL-4(20ng/ml)和IL-13(20ng/ml)继续诱导66小时(共72小时)。并通过检测Arg-1酶及iNOS的表达情况,检测是否诱导成功。4、摄取实验:选取人体成纤维细胞、M1型巨噬细胞、M2巨噬细胞、SW620结肠癌细胞,加入荧光标记后的唑来膦酸纳米粒,反应3小时后,采用Dil对细胞膜进行染色,在荧光显微镜下观察四种细胞对唑来膦酸纳米粒的摄取情况。5、CCK-8增殖抑制实验:通过唑来膦酸纳米粒与不同种类细胞的相互作用,了解其对细胞的增殖能力的影响;6、细胞划痕实验及transwell小室迁移实验,设置sw620结肠癌组、sw620+m1型巨噬细胞组、sw620+m2型巨噬细胞组,每组分为加和不加唑来膦酸纳米粒组(20ng/ul),共6组实验组,观察每组各因素对肿瘤细胞迁移能力的影响。7、侵袭实验:将sw620结肠癌细胞放入transwell小室的上室中,下室依次为空白组(单独培养基)、唑来膦酸纳米粒组、m1型巨噬细胞组、m1型巨噬细胞+唑来膦酸纳米粒组、m2型巨噬细胞组、m2型巨噬细胞+唑来膦酸纳米粒组;并采用吉姆萨染色法计数,了解各因素对肿瘤细胞侵袭能力的影响;结果:1、arg-1及inos检测结果显示m2型巨噬细胞免疫组化呈阳性反应,而其余两组呈阴性反应,m1型巨噬细胞inos表达(74.4%)远高于其余两组(14.0%及14.7%),证实m1及m2型巨噬细胞诱导成功。2、m2型巨噬细胞摄取纳米颗粒的荧光强度远高于其他细胞;3、m1型巨噬细胞抑制肿瘤细胞迁移;m2型巨噬细胞促进肿瘤细胞的迁移,与唑来膦酸纳米粒相互作用可抑制m2巨噬细胞的这种促进作用(p0.01);4、m1型巨噬细胞抑制肿瘤细胞侵袭力;m2型巨噬细胞促进肿瘤细胞的侵袭,与唑来膦酸纳米粒相互作用可抑制m2巨噬细胞的这种促进作用(p0.01)5、纳米颗粒通过抑制m2型巨噬细胞,影响肿瘤细胞增殖(p0.01)。结论:唑来膦酸纳米粒能特异性被m2型巨噬细胞摄取并抑制m2巨噬细胞的增殖。m2巨噬细胞促进肿瘤的迁移、侵袭和增殖。唑来膦酸纳米粒通过抑制m2型巨噬细胞间接抑制肿瘤细胞的增殖、迁移及侵袭能力。
[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- 纬 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.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R73-36

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