丹参素对黑色素瘤细胞粘附和侵袭生物学行为的影响及其机理研究

发布时间：2018-06-17 20:08

  本文选题：丹参素 + 黑色素瘤细胞　； 参考：《南京中医药大学》2010年硕士论文

【摘要】： [研究目的] 课题组前期实验研究表明丹参素(Danshensu, DLA)对B16F10黑色素瘤细胞转移具有抑制作用。运用分子对接技术进行丹参素分子靶标预测,推测丹参素可能对Neutrophil Collagenase、Ras-Ras Gap等蛋白具有一定作用,这些分子靶标则与肿瘤细胞粘附和侵袭环节相关。粘附和侵袭是黑色素瘤细胞血行转移过程中的关键步骤,本课题为了确证DLA对肿瘤转移中粘附和侵袭环节的作用,以黑色素瘤细胞粘附和侵袭生物学行为作为研究重点,并进行初步的机制探讨。 [研究内容] 实验研究部分共分为三部分,第一部分从肿瘤细胞粘附方面探讨DLA的作用,包括DLA对黑色素瘤细胞血行转移中粘附生物学行为的影响(与基质粘附的能力、细胞聚集能力、与血小板粘附的能力),第二部分进一步观察DLA对黑色素瘤细胞血行转移中相关粘附分子表达的影响,第三部分主要探讨DLA对黑色素瘤细胞血行转移中侵袭生物学行为的影响,包括降解基底膜和细胞运动两个方面。 具体实验方法如下：粘附：细胞染色检测黑色素瘤细胞与基质粘附能力,慢速聚集法观察黑色素瘤细胞聚集能力,流式细胞技术以及荧光标记血小板的方法检测血小板与黑色素瘤细胞粘附的能力,并用Real-time PCR测定对黑色素瘤细胞粘附过程中重要的粘附分子MCAM、β3 integrin, E-cadherin的mRNA水平,Western blot测定MCAM蛋白表达,ELISA法检测sICAM-1释放量。侵袭：趋化小室法观察黑色素瘤细胞基底膜侵袭能力；划痕法观察黑色素瘤细胞运动能力的变化；应用Real-time PCR对黑色素瘤细胞降解基底膜酶类MMP2、MMP9及其抑制因子TIMP2和RECK进行mRNA水平表达的测定,并用Westernblot蛋白测定法对黑色素瘤细胞MMP2和TIMP2进行测定。 [实验结果及结论] DLA对黑色素瘤细胞与基质粘附能力以及细胞聚集具有抑制作用,对血小板聚集和血小板与黑色素瘤细胞的粘附则无明显作用；同时DLA对A375黑色素瘤细胞粘附分子MCAM的mRNA和蛋白表达具有抑制作用,对A375细胞sICAM-1的释放亦有抑制作用。DLA对黑色素瘤细胞侵袭能力具有抑制作用,对侵袭过程中细胞运动能力和降解基底膜酶类表达均有一定的抑制作用。通过上述结果,可以初步看出DLA抑制黑色素瘤细胞血行转移,可能是通过影响黑色素瘤细胞粘附和侵袭行为而实现的。
[Abstract]:[objective] our previous study showed that Danshensu (DLA) could inhibit the metastasis of B16F10 melanoma cells. Using molecular docking technique to predict the molecular target of Danshensu, it is speculated that Danshensu may play a role in Neutrophil Collagenase- Ras-Ras Gap and other proteins. These molecular targets are related to the adhesion and invasion of tumor cells. Adhesion and invasion are the key steps in the process of blood metastasis of melanoma cells. In order to confirm the role of DLA in adhesion and invasion of melanoma cells, the biological behavior of adhesion and invasion of melanoma cells is the focus of this study. The preliminary mechanism was discussed. The experimental study is divided into three parts. The first part discusses the role of DLA in tumor cell adhesion. Including the effect of DLA on the adhesion biological behavior of melanoma cells during blood metastasis (the ability to adhere to matrix, the ability of cell aggregation, In the second part, the effect of DLA on the expression of adhesion molecules in melanoma cells was observed. In the third part, the effect of DLA on the invasive biological behavior of melanoma cells was studied. It includes degradation of basement membrane and cell motion. The specific experimental methods were as follows: adhesion: the adhesion ability of melanoma cells to matrix was detected by cell staining, and the aggregation ability of melanoma cells was observed by slow aggregation method. The ability of platelet adhesion to melanoma cells was detected by flow cytometry and fluorescent labeling of platelets. The mRNA levels of MCAM, 尾 3 integrin and E-cadherin in melanoma cell adhesion were determined by Real-time PCR. The expression of MCAM protein was detected by Elisa. The release of sICAM-1 was detected by Elisa. Invasion: the invasion ability of melanoma cell basement membrane was observed by chemotaxis chamber method, and the change of melanoma cell motility was observed by scratch method. Real-time PCR was used to detect the mRNA expression of MMP2MMP9 and its inhibitors TIMP2 and Reck in melanoma cells, and MMP2 and TIMP2 in melanoma cells were detected by Western blot protein assay. [results and conclusion] DLA can inhibit the adhesion of melanoma cells to matrix and cell aggregation, but has no effect on platelet aggregation and adhesion of platelets to melanoma cells. At the same time, DLA inhibited the expression of MCAM mRNA and protein in A375 melanoma cells, and inhibited the release of sICAM-1 from A375 cells. DLA also inhibited the invasion of A375 melanoma cells. It can inhibit the cell motility and the expression of basement membrane enzymes in the process of invasion. These results suggest that DLA inhibits the blood metastasis of melanoma cells, which may be due to the effect of DLA on the adhesion and invasion of melanoma cells.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R285;R739.5
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本文编号：2032295