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土槿皮乙酸对人胰腺癌PANC-1细胞增殖和凋亡的影响
Effects of Pseudolaric Acid B on the Proliferation and Apoptosis of Human Pancreatic Carcionma PANC-1Cell Lines
【作者】 黄伟；
【导师】 曹明溶；
【作者基本信息】 暨南大学 ， 外科学， 2012， 硕士
【摘要】 目的：研究土槿皮乙酸(Pseudolaric Acid B，PAB)对人胰腺癌PANC-1细胞的增殖、凋亡作用，为进一步的临床研究及应用提供依据。方法：体外培养人胰腺癌PANC-1细胞，观察PAB对人胰腺癌PANC-1细胞的作用：通过CCK-8实验和细胞克隆形成实验，检测PAB对胰腺癌PANC-1细胞的生长抑制作用；通过Hoechst33258染色，观察PAB对PANC-1细胞的凋亡形态学改变；通过PI单染流式细胞术，检测PAB对PANC-1细胞周期的影响；通过Annexin V-PI双染，检测PAB对PANC-1细胞凋亡的影响；通过Western blot技术，检测凋亡相关蛋白caspase-3，caspase-9和JNK蛋白的表达量。结果：不同浓度的PAB对PANC-1细胞的生长抑制呈现出剂量和时间相关性：随着药物浓度的增加，对细胞的抑制作用逐渐增强，浓度为16μmol/l作用24h抑制率为52.58%；相同浓度的PAB对细胞的抑制作用随着时间的延长不断增强，浓度为2μmol/l作用72h抑制率为52.61%。对细胞形成克隆也具有浓度相关性，浓度越高，抑制作用越明显。Hoechst33258染色和PI单染流式检测加Annexin V-PI双染检测显示：PAB能够促进细胞的凋亡，将细胞周期阻滞于G2/M期。浓度为16μmol/l时，G2/M期细胞比例明显升高，凋亡率为37.47%±1.50%，与对照组相比具有统计学差异。Western blot结果显示：PAB能够提高caspase-3、caspase-9和JNK蛋白的表达量，且PAB浓度越高，与凋亡信号相关蛋白的表达量也越高。结论：1.土槿皮乙酸作用于人胰腺癌PANC-1细胞24、48、72h，细胞增殖明显受到抑制，且表现为剂量依赖性和时间依赖性；2.土槿皮乙酸作用于人胰腺癌PANC-1细胞7天后，能有效抑制PANC-1细胞形成的克隆数；3.土槿皮乙酸能够诱导人胰腺癌PANC-1细胞凋亡：土槿皮乙酸作用于PANC-1细胞48h后，Hoechst33258染色可见实验组细胞数量减少，部分细胞出现胞质减少，胞核固缩、边聚、裂解和凋亡小体等凋亡形态学变化；4.土槿皮乙酸作用于人胰腺癌PANC-1细胞48h后，通过PI单染流式细胞仪检测细胞周期发现：实验组G0/G1期细胞比例明显减少，G2/M期细胞比例明显增多，提示土槿皮乙酸可以将PANC-1细胞周期阻滞于G2/M期；5.土槿皮乙酸能够诱导细胞凋亡，作用机制与激活caspase-3，caspase-9和JNK信号通路相关。
【Abstract】 Objective：To detect the effects of PAB on the proliferation and apoptosis of PANC-1celllines. Investigate the probable mechanisms of suppresses and apoptosis and provide a basistheory for further clinical research and application.Methods: Test the effect of PAB on inhibiting proliferation of PANC-1cell lines by CCK-8test. The morphology of apoptosis of PANC-1cell lines was examined by fluorescencemicroscope after hoechst33258staining, while the clone formation of PANC-1cell lineswas detected by clone formation assay. The cell cycle and the percentage of hypodiploidcells of PANC-1cell lines treated with PAB for48h were determined with propidiumiodide (PI) staining by flow cytometry. Apoptosis was detected with Annexin v-pi dualparameter by flow cytometry. Western blot technique was used to detect apoptosis relatedproteins caspase-3, caspase-9and JNK expression levels.Results: PAB inhibited the growth of PANC-1cell lines in a time-and dose-dependentmanner. When the concentration is higher, the inhibitory rate is higher. Compared withcontrol group, the clone formation of PANC-1cell lines treated with PAB for7days wasalso significantly inhibited. After treated with PAB for48h, apoptosis morpholoficalalterations, such as cell number reduced obviously, some cells cytoplasmic reduce,chromation sondensation and apoptotic bodies were observed by staining with Hoechst33258under the fluorescence microscope. Compared with control group, After the cellswere treated with different concentration of PAB for48h,the ratio of G0/G1phasedecreased, ratio of G2/M phase increased obviously. The western blot results showed thatthe PAB can improve the caspase-3, caspase-9and JNK protein expression. And high PABconcentration could rise the protein expression levels. Conclusion:1. PAB could inhibite the proliferation of Human Pancreatic Carcionma PANC-1cell linesand have dose-dependent and time-dependent.2. The numbers of clone of PANC-1cells can be inhibited by PAB after7days effectively.3. PAB could induced apoptosis of Human Pancreatic Carcionma PANC-1cell lines. PABcould reduce cell number and cytoplasm, nucleus condensation and induce apoptoticmorphology change by Hoechest33258staining.4. PAB could inhibit cell proliferation at the G2/M phase.5. PAB can induced apoptosis, and the mechanism maybe related with activation ofcaspase-3, caspase-9and the JNK signaling pathway.