【作者】 陈欣；

【导师】 张桂梅； 冯作化；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 目的本文拟研究LPS,肿瘤细胞坏死释放物质(NTC-Ms)对小鼠黑色素瘤B16细胞侵袭、转移能力的影响,并从分子水平探讨其影响的可能机制,从而为肿瘤治疗提供新的思路。方法(1)分别用LPS,NTC-Ms,以及Resveratrol联合NTC-Ms作用于B16细胞48h,粘附实验检测三组刺激因素分别对B16细胞粘附能力的影响。(2)分别用LPS,NTC-Ms,以及Resveratrol联合NTC-Ms作用于B16细胞48h,软琼脂迁移实验检测三组刺激因素分别对B16细胞迁移能力的影响。(3)分别用LPS,NTC-Ms,以及Resveratrol联合NTC-Ms作用于B16细胞48h,运动实验(Transwell小室法)检测三组刺激因素分别对B16细胞侵袭和趋化运动能力的影响。(4)分别用LPS,NTC-Ms,以及Resveratrol联合NTC-Ms作用于B16细胞48h,RT-PCR法检测B16细胞TLR4,MMP-9 mRNA的表达。(5)分别用LPS,NTC-Ms,以及Resveratrol联合NTC-Ms作用于B16细胞48h,,明胶酶谱实验检测B16细胞分泌的MMP-9的活性。结果(1)LPS,NTC-Ms刺激后,B16细胞粘附基底膜的能力均增强;Resveratrol联合NTC-Ms刺激后,B16细胞对基底膜的粘附能力较单用NTC-Ms减弱。(2)LPS,NTC-Ms刺激后,迁移至软琼脂底面的B16细胞数均增多;Resveratrol联合NTC-Ms刺激后,迁移至软琼脂底面的B16细胞数减少。(3)LPS,NTC-Ms刺激后,B16细胞侵袭和趋化运动能力均增强;Resveratrol联合NTC-Ms刺激后,B16细胞侵袭和趋化运动能力减弱。(4)LPS,NTC-Ms刺激后,B16细胞TLR4, MMP-9 mRNA表达均上调;Resveratrol联合NTC-Ms刺激后,B16细胞TLR4, MMP-9 mRNA表达下调;(5)LPS,NTC-Ms刺激后, B16细胞分泌的MMP-9活性均增强;Resveratrol联合NTC-Ms刺激后,B16细胞分泌的MMP-9活性较单用NTC-Ms组减弱。结论LPS,肿瘤细胞坏死释放物质可以通过激活TLR4信号通路,提高B16细胞的侵袭转移能力。更多还原

【Abstract】 Objective To investigate the effects of LPS and materials released from necrosis tumor cells on the invasion and metastasis of B16 malenoma cells, and analyze the possible molecular mechanism of TLR4 pathway on the invasion and metastasis of tumor cells.Methods (1) Stimulate B16 cells with LPS, NTC-Ms and Resveratrol + NTC-Ms for 48 hours separately , tumor cell endothelium adhesion assay was used to detect the adhesion ability of B16 cells. (2) Stimulate B16 cells with LPS, NTC-Ms and Resveratrol + NTC-Ms for 48 hours separately, soft agar migration assay was used to detect the migration ability of B16 cells. (3) Stimulate B16 cells with LPS, NTC-Ms and Resveratrol + NTC-Ms for 48 hours separately, Transwell assay was used to detect the invasion ability of B16 cells. (4) Stimulate B16 cells with LPS, NTC-Ms and Resveratrol + NTC-Ms for 48 hours separately, RT-PCR was used to detect the expression level of TLR4 and MMP-9 in B16 cells. (5) Stimulate B16 cells with LPS, NTC-Ms and Resveratrol + NTC-Ms for 48 hours separately, Gelatin zymography assay was used to detect the activity of MMP-9.Results (1) The adhesion ability of B16 cells was improved after the stimulation of LPS and NTC-Ms separately; but after the stimulation of Resveratrol + NTC-Ms, the adhesion ability of B16 cells was reduced compared to those stimulated by NTC-Ms only. (2) The number of B16 cells which migrated to the bottom of soft agar was increased after the stimulation of LPS and NTC-Ms separately; and was decreased after the stimulation of Resveratrol + NTC-Ms. (3) The invasion ability of B16 cells was improved after the stimulation of LPS and NTC-Ms separately; and was deteriorated after the stimulation of Resveratrol + NTC-Ms. (4) The expression levels of TLR4 and MMP-9 in B16 cells were increased after the stimulation of LPS and NTC-Ms separately; and was decreased after the stimulation of Resveratrol + NTC-Ms. (5) The activity of MMP-9 secreted from B16 cells was increased after the stimulation of LPS and NTC-Ms separately; and was decreased after the stimulation of Resveratrol + NTC-Ms.Conclusion LPS and materials released from necrosis tumor cells could activate toll-like receptor 4 signaling pathway, and improve the invasion and metastasis ability of tumor cells.更多还原