生育三烯酚诱导结肠癌细胞死亡的方式及机制研究

发布时间：2018-04-17 01:39

本文选题：生育三烯酚 + 人结肠癌细胞SW620　；参考：《天津医科大学》2013年硕士论文

【摘要】：目的本研究旨在通过体外培养人结肠癌SW620细胞、HCT-8细胞,利用分子生物学实验技术,通过多个角度证实生育三烯酚诱导结肠癌细胞死亡的死亡方式为paraptosis样死亡,并以paraptosis样死亡方式为中心,探讨生育三烯酚诱导结肠癌细胞发生的机制。方法 1.生育三烯酚诱导人结肠癌细胞死亡的方式研究体外培养人结肠癌细胞,加入不同剂量的6-生育三烯酚(1、2.5、5、10、15、20、25、30和40μmol/L)和乙醇(溶剂对照)作用24h后,以MTT实验观察δ-生育三烯酚对SW620细胞存活率的影响；按照5、10、15、20μmol/L δ-生育三烯酚作用于细胞,以荧光染色(AO/EB染色,DAPI染色)观察细胞死亡情况。按照20μmol/L剂量加入δ-生育三烯酚,通过加入paraptosis抑制剂放线菌酮观察其对生育三烯酚作用的影响情况,来正面证实生育三烯酚诱导人结肠癌细胞死亡的方式中存在paraptosis样死亡。以不同剂量的δ-生育三烯酚作用于人结肠癌细胞SW620中,检测细胞中caspase-3的活性,并用抗癌阳性药物紫杉醇作为肿瘤细胞凋亡诱导剂作用于SW620细胞中,利用DNA Ladder方法检测细胞中DNA的降解情况,从而证实生育三烯酚诱导人结肠癌细胞死亡并非典型凋亡方式。利用免疫细胞化学检测细胞中细胞自噬标记蛋白LC3的表达情况来观察生育三烯酚诱导人结肠癌细胞产生的这种空泡化死亡现象与细胞自噬的关系,从而排除细胞自噬的可能,从反面证实生育三烯酚诱导人结肠癌细胞死亡的方式为paraptosis样死亡。为了排除细胞系的特异性,本实验中增加了另一种人结肠癌细胞系,HCT-8细胞。将γ-生育三烯酚作用于HCT-8细胞,观察细胞死亡情况,同时检测细胞中caspase-3的活性,作为细胞系平行实验。 2.δ-生育三烯酚诱导人结肠癌细胞SW620死亡的机制研究体外培养SW620细胞,加入的20μmol/Lδ-生育三烯酚作用24h后,通过免疫细胞化学和western bloting的方法检测细胞内质网膜蛋白标志物钙连蛋白和内质网应激标记蛋白Bip的表达情况,检测6-生育三烯酚诱导人结肠癌细胞出现空泡化死亡的现象与内质网应激的关系；在SW620细胞中加入wnt通路抑制剂FH535抑制细胞中wnt通路的激活,观察细胞的形态学变化,并利用western bloting方法检测wnt通路关键因子β-catenin的表达量变化,检测6-生育三烯酚诱导人结肠癌细胞死亡与Wnt通路的关系。结果 1.与乙醇对照组相比,6-生育三烯酚作用于结肠癌细胞SW62024h后,各剂量组均可以使SW620细胞存活率降低(p0.05),且细胞存活率随着6-生育三烯酚剂量的增加呈现降低的趋势,IC50值为15.18μmol/L。γ-生育三烯酚作用于结肠癌细胞HCT-824h后,各剂量组均可以使HCT-8细胞存活率降低(p0.05),且细胞存活率随着γ-生育三烯酚剂量的增加呈现降低的趋势,IC50值为32.69mol/L 2.生育三烯酚作用于结肠癌细胞SW620和HCT-8后,细胞内均未见细胞核不规整,核染色体DNA浓缩、着边呈新月状,凋亡小体生成等典型凋亡现象,取而代之的是细胞内出现大量空泡。6-生育三烯酚所致的SW620细胞死亡可被paraptosis抑制剂放线菌酮所抑制。紫杉醇可使SW620细胞核内DNA产生梯度断裂,而6-生育三烯酚并不能使SW620细胞核内DNA发生梯度降解现象。细胞自噬标记物LC3表达为阴性。证明6-生育三烯酚诱导结肠癌细胞SW620死亡的方式为paraptosis样死亡而非典型凋亡或细胞自噬。 3. FH535可导致SW620细胞死亡,形态学显示细胞胞浆内出现大量空泡,蛋白定量结果显示wnt通路关键因子β-catenin的表达量下降。证明生育三烯酚诱导结肠癌细胞paraptosis样死亡与抑制细胞中Wnt通路的激活有关。 4.免疫细胞化学结果显示,δ-生育三烯酚作用于结肠癌细胞SW62024h后,内质网膜钙连蛋白表达阳性,随着生育三烯酚剂量的增大,细胞内质网逐渐形成空泡。内质网应激标记物Bip蛋白表达量增高。证明生育三烯酚诱导结肠癌细胞paraptosis样死亡与细胞内质网应激有关结论 1.证实生育三烯酚诱导结肠癌死亡的死亡方式中存在paraptosis样死亡。 2.发现6-生育三烯酚对结肠癌细胞中wnt信号通路的影响以及引起细胞内质网应激可能是其诱导SW620细胞paraptosis样死亡的机制。
[Abstract]:Purpose

The aim of this study was to investigate the mechanism of tocotrienol - induced apoptosis in colon cancer cells by means of molecular biology experiments by culturing human colon cancer SW620 cells and HCT - 8 cells in vitro .

method

1 . Method for inducing human colon cancer cell death by tocotrienol

The effect of delta - tocotrienol on the survival rate of SW620 cells was observed by MTT assay after 24 h of incubation with 6 - tocotrienol ( 1 , 2.5 , 5 , 10 , 15 , 20 , 25 , 30 and 40 渭mol / L ) and ethanol ( solvent control ) in vitro .
The cell death was observed by fluorescence staining ( AO / EB staining , DAPI staining ) in the presence of 5 , 10 , 15 , 20 渭mol / L 未 - tocotrienol .

未 - tocotrienol ( 未 - tocotrienol ) was added at 20 渭mol / L dose , and the effect of tocotrienol on tocotrienol was observed by the addition of antimicrobial agents . The positive effect of tocotrienol on the death of human colon cancer cells was confirmed .

The activity of caspase - 3 in human colon cancer cells was detected with different doses of delta - tocotrienol , and the activity of caspase - 3 in human colon cancer cells was detected by using anti - cancer positive drug taxol as inducer of tumor cell apoptosis .

In order to exclude the specificity of cell lines , another human colon cancer cell line , HCT - 8 cells was added to the experiment .

2 . Mechanism of 未 - tocotrienol - induced death of human colon cancer cell SW620

SW620 cells were cultured in vitro . After 24 h of 20 渭mol / L 未 - tocotrienol , the expression of calcium connexin and endoplasmic reticulum stress marker protein Bip in human colon cancer cells were detected by immunohistochemistry and western bloting , and the relationship between apoptosis and endoplasmic reticulum stress was detected in 6 - tocotrienol - induced human colon cancer cells .
wnt pathway inhibitor was added to SW620 cells to inhibit the activation of wnt pathway in the cells , to observe the morphological changes of the cells , and to detect the change of the expression of 尾 - catenin in wnt pathway by western bloting method , and the relationship between the death of human colon cancer cells and Wnt pathway was detected by 6 - tocotrienol .

Results

1 . Compared with the ethanol control group , 6 - tocotrienol can decrease the survival rate of SW620 cells ( P < 0.05 ) , and the cell survival rate decreases with the increase of 6 - tocotrienol . The IC50 value is 15.18 渭mol / L . 纬 - tocotrienol acts on colon cancer cells HCT - 824h , and the cell survival rate decreases with increasing the dose of 纬 - tocotrienol , the IC50 value is 32.69mol / L .

2 . When tocotrienol acts on colon cancer cells SW620 and HCT - 8 , there is no abnormal cell nucleus in the cells . The DNA concentration of nuclear chromosomes , the formation of apoptotic bodies , and the like are inhibited . The death of SW620 cells induced by 6 - tocotrienol can be inhibited .

3 . The cell death of SW620 cells could result in the death of SW620 cells , and a large amount of vacuoles appeared in the cytoplasm of the cells . The results showed that the expression of 尾 - catenin was decreased in the wnt pathway .

4 . Immunocytochemistry showed that 未 - tocotrienol acts on colon cancer cells SW620 h after SW620 h . The expression of 尾 - tocotrienol increased with the increase of tocotrienol dose . The expression of 尾 - tocotrienol increased with the increase of tocotrienol dose . The expression of Bip protein in the endoplasmic reticulum stress marker was increased . It was shown that the death of the tocotrienol - induced colon cancer cells was related to the stress of the endoplasmic reticulum stress .

Conclusion

1 . It was confirmed that the death of a tocotrienol - induced death of colon cancer was fatal .

2 . It was found that the effects of 6 - tocotrienol on the wnt signal pathway in colon cancer cells and the possible mechanism of inducing the death of SW620 cells in SW620 cells .

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114;R735.35

【参考文献】