本文選題：丹參素 + 黑色素瘤細(xì)胞��； 參考：《南京中醫(yī)藥大學(xué)》2010年碩士論文

【摘要】： [研究目的] 課題組前期實(shí)驗(yàn)研究表明丹參素(Danshensu, DLA)對B16F10黑色素瘤細(xì)胞轉(zhuǎn)移具有抑制作用。運(yùn)用分子對接技術(shù)進(jìn)行丹參素分子靶標(biāo)預(yù)測,推測丹參素可能對Neutrophil Collagenase、Ras-Ras Gap等蛋白具有一定作用,這些分子靶標(biāo)則與腫瘤細(xì)胞粘附和侵襲環(huán)節(jié)相關(guān)。粘附和侵襲是黑色素瘤細(xì)胞血行轉(zhuǎn)移過程中的關(guān)鍵步驟,本課題為了確證DLA對腫瘤轉(zhuǎn)移中粘附和侵襲環(huán)節(jié)的作用,以黑色素瘤細(xì)胞粘附和侵襲生物學(xué)行為作為研究重點(diǎn),并進(jìn)行初步的機(jī)制探討。 [研究內(nèi)容] 實(shí)驗(yàn)研究部分共分為三部分,第一部分從腫瘤細(xì)胞粘附方面探討DLA的作用,包括DLA對黑色素瘤細(xì)胞血行轉(zhuǎn)移中粘附生物學(xué)行為的影響(與基質(zhì)粘附的能力、細(xì)胞聚集能力、與血小板粘附的能力),第二部分進(jìn)一步觀察DLA對黑色素瘤細(xì)胞血行轉(zhuǎn)移中相關(guān)粘附分子表達(dá)的影響,第三部分主要探討DLA對黑色素瘤細(xì)胞血行轉(zhuǎn)移中侵襲生物學(xué)行為的影響,包括降解基底膜和細(xì)胞運(yùn)動兩個方面。 具體實(shí)驗(yàn)方法如下：粘附：細(xì)胞染色檢測黑色素瘤細(xì)胞與基質(zhì)粘附能力,慢速聚集法觀察黑色素瘤細(xì)胞聚集能力,流式細(xì)胞技術(shù)以及熒光標(biāo)記血小板的方法檢測血小板與黑色素瘤細(xì)胞粘附的能力,并用Real-time PCR測定對黑色素瘤細(xì)胞粘附過程中重要的粘附分子MCAM、β3 integrin, E-cadherin的mRNA水平,Western blot測定MCAM蛋白表達(dá),ELISA法檢測sICAM-1釋放量。侵襲：趨化小室法觀察黑色素瘤細(xì)胞基底膜侵襲能力；劃痕法觀察黑色素瘤細(xì)胞運(yùn)動能力的變化；應(yīng)用Real-time PCR對黑色素瘤細(xì)胞降解基底膜酶類MMP2、MMP9及其抑制因子TIMP2和RECK進(jìn)行mRNA水平表達(dá)的測定,并用Westernblot蛋白測定法對黑色素瘤細(xì)胞MMP2和TIMP2進(jìn)行測定。 [實(shí)驗(yàn)結(jié)果及結(jié)論] DLA對黑色素瘤細(xì)胞與基質(zhì)粘附能力以及細(xì)胞聚集具有抑制作用,對血小板聚集和血小板與黑色素瘤細(xì)胞的粘附則無明顯作用；同時(shí)DLA對A375黑色素瘤細(xì)胞粘附分子MCAM的mRNA和蛋白表達(dá)具有抑制作用,對A375細(xì)胞sICAM-1的釋放亦有抑制作用。DLA對黑色素瘤細(xì)胞侵襲能力具有抑制作用,對侵襲過程中細(xì)胞運(yùn)動能力和降解基底膜酶類表達(dá)均有一定的抑制作用。通過上述結(jié)果,可以初步看出DLA抑制黑色素瘤細(xì)胞血行轉(zhuǎn)移,可能是通過影響黑色素瘤細(xì)胞粘附和侵襲行為而實(shí)現(xiàn)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.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R285;R739.5
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本文編號：2032295