【作者】 李大鸣；

【导师】 王凤山；

【作者基本信息】 天津医科大学 ， 卫生毒理学， 2011， 硕士

【摘要】 目的本研究旨在通过体外培养人结肠癌SW620细胞,利用分子生物学实验技术,以Wnt信号途径为中心,探讨生育三烯酚诱导结肠癌细胞发生死亡的分子机制。方法1.生育三烯酚对SW620细胞增殖的影响体外培养SW620细胞,分别加入不同浓度生育三烯酚(δ-生育三烯酚终浓度分别为0、1、2.5、5、10、15、20、25、30和40μmol/L；Υ-生育三烯酚终浓度分别为0、5、10、15、20、25、30、40、50、60、70、80、90和100μmol/L)和乙醇(溶剂对照)作用24 h后,以MTT实验分别观察Υ-、δ-生育三烯酚对SW620细胞存活率的影响。分别以浓度为5、10、15、20μmo1/L的δ-生育三烯酚及15、30、45、60μmol/L的Υ-生育三烯酚和乙醇(溶剂对照)作用于细胞,利用荧光染色(AO/EB染色,DAPI染色)、电镜超微结构观察、流式细胞术、Caspase-3活性测定等方法检测细胞的死亡情况,并比较加入z.VAD.fmk与否时细胞的存活率2.生育三烯酚抑制SW620细胞增殖的机制研究体外培养SW620细胞,分别以浓度为5、10、15、20μmol/L的6-生育三烯酚及15、30、45、60-tmol/L的Υ-生育三烯酚和乙醇(溶剂对照)作用于细胞。24h后,提取细胞内总RNA进行Real-time RT-PCR检测wnt-1、β-catenin、cyclin D1、c-jun、MMP-7的mRNA表达。同时,提取总蛋白进行Westernblot检测上述蛋白的表达情况。结果1.Υ、δ-生育三烯酚分别作用24 h后,与乙醇对照组相比,各剂量组Υ-、6-生育三烯酚均可以使SW620细胞存活率降低(P<0.05),且细胞存活率分别随着Υ-、δ-生育三烯酚剂量的增加呈现降低的趋势。Υ-、6-生育三烯酚对SW620细胞的IC50值分别为31.35μmol/L,15.18μmol/L。2.通过细胞形态学观察发现,Υ-、δ-生育三烯酚分别作用SW620细胞6和24 h后,细胞内未见细胞核不规整,膜泡化,核染色体DNA浓缩、着边呈新月状,凋亡小体形成等典型的凋亡现象,取而代之的是细胞内出现大量空泡。电镜结果显示线粒体发生了肿胀及空泡变。3. Caspase抑制剂z.VAD.fmk加入后,SW620细胞生存率仍随着6-生育三烯酚的剂量增加而呈现下降的趋势(P<0.05),相同剂量组间细胞生存率的差异不具有统计学意义(P>0.05)；6-生育三烯酚分别作用SW620细胞6和24 h后,处理组细胞凋亡率与对照组比较,差异没有统计学意义(P>0.05)；6-生育三烯酚作用SW620细胞24 h后,随作用剂量的增加,Caspase-3活性并未增强(P>0.05)。Υ-生育三烯酚的结果与6-生育三烯酚的结果相类似。4.在不同剂量的δ-生育三烯酚作用24h后,SW620细胞内wnt-1、β-catenin的mRNA和蛋白的表达量下降；对Wnt信号途径下游癌基因cyclin D1、MMP-7的mRNA表达无影响,但是能够降低cyclin、D1、MMP-7蛋白量的水平。5.在不同剂量的Υ-生育三烯酚作用24h后,SW620细胞内(3-catenin、c-jun、cyclinD1的mRNA和蛋白表达量有不同程度的下降；对wnt-1、MMP-7的mRNA表达无影响,但可使其蛋白表达量有不同程度的下降。结论1.证实生育三烯酚抑制SW620细胞增殖。同时,发现δ-生育三烯酚对SW620细胞的抑制作用强于Υ-生育三烯酚。2.生育三烯酚可以诱导SW620细胞产生paraptosis样死亡3.生育三烯酚对Wnt信号途径的影响可能是其诱导SW620细胞paraptosis样死亡的机制之-。更多还原

【Abstract】 ObjectiveThe purpose of this study was to determine the mechanism of tocotrienols induced cell death on the Wnt signaling pathway in colon carcinoma cells (SW620) by the method of cell culture in vitro and molecular biological techniques.Methods1. The effects of tocotrienol on the proliferation of SW620 cellsAfter treated with different concentrations ofδ-tocotrienol or y-tocotrienol for 24h, cell viability was tested by MTT assay. The SW620 cells were treated with 0、5、10、15、20μmol/Lδ-tocotrienol and 15、30、45、60μmol/Lγ-tocotrienol respectively. Then, the cell death was determined via DAPI and AO/EB staining assay, transmission electron microscopy assay, flow cytometry assay, Caspase-3 activity assay, cell viability with/without z.VAD.fmk.2. The mechanism of tocotrienol induced cell death on SW620 cellsThe SW620 cells were treated with 0、5、10、15、20μmol/Lδ-tocotrienol and 15, 30,45,60μmol/Lγ-tocotrienol respectively. After treated for 24h, wnt-1、β-catenin、cyclin Dl、c-jun and MMP-7 mRNA and protein expression were observed by Real-time RT-PCR and Western blotting assay.Results1. Compared with control group, both y-tocotrienol andδ-tocotrienol dose-dependently decreased SW620 cell viability (P<0.05). The IC50 value of y-tocotrienol andδ-tocotrienol on SW620 cells was 31.35,15.2μmol/L respectively.2. The morphological assays showed that classic apoptotic changes were absent in tocotrienol-treated cells, such as cell shrinkage, membrane blebbing, chromatin condensation with margination at the nuclear periphery and formation of apoptotic body, but there were a plenty of large or small cytoplasmic vacuoles in treated cells. Swelling of mitochondria was observed from transmission electron microscopy and mitochondria had vacuolized. 3. The cell viability in both the 8-tocotrienol groups andδ-tocotrienol with z.VAD.fmk groups were decreased in a dose-dependent manner. There was no difference within the same concentration ofδ-tocotrienol with or without z.VAD.fmk (P>0.05). When compared to the negative control group, there was not a significant increase in cell apoptosis ratio an the levels of caspase-3 activity in the 8-tocotrienol-treated groups (P>0.05). The results of y-tocotrienol were the similar toδ-tocotrienor’s.4. After treated withδ-tocotrienol for 24h, the expression ofδ-catenin and wnt-1 mRNA and proteins were reduced.δ-Tocotrienol had no effects on the mRNA expression of cyclin Dl and MMP-7, but decreased the cyclin D1 and MMP-7 protein expression.5. In the treated groups, y-tocotrienol reduced the expression ofβ-catenin, c-jun and cyclin Dl mRNA and proteins in varying degrees. y-Tocotrienol decreased the wnt-1 and MMP-7 protein expression, but had no effects on wnt-1 and MMP-7 mRNA expression.Conclusions1. It is a study to prove the inhibitory effect of tocotrienols on SW620 cells. The inhibitory effect ofδ-tocotrienols is stronger than y-tocotrienol’s.2. Tocotrienols can induce paraptosis-like cell death on SW620 cells.3. Having an influence on the Wnt signaling pathways may be one of the mechanisms of paraptosis-like cell death induced by tocotrienols on SW620 cells.更多还原