【作者】 张昭军；

【导师】 锡林宝勒日；

【作者基本信息】 新疆医科大学 ， 外科学， 2011， 硕士

【摘要】 目的：研究左旋棉酚联合低浓度阿霉素对软组织肉瘤人纤维肉瘤HT1080细胞和滑膜肉瘤SW982细胞的凋亡及其作用机制,为提高肿瘤治疗效果提供理论依据。方法：采用MTT法检测药物对肿瘤细胞的抗增殖作用,应用Wright-Giemsa染色、Hoechst 33258染色、透射电镜观察药物作用后肿瘤细胞的形态学变化,琼脂糖凝胶电泳分析药物作用后肿瘤细胞DNA有无出现梯状带型,用流式细胞仪检测药物作用后肿瘤细胞的凋亡率和细胞周期的变化。Western-Blot法检测药物作用后肿瘤细胞凋亡相关基因Bcl-2、Bax蛋白的表达,比色分析法测定药物作用后肿瘤细胞内Caspase-3和Caspase-9的活性变化。结果：左旋棉酚与低浓度阿霉素对软组织肉瘤人纤维肉瘤HT1080细胞和滑膜肉瘤SW982细胞具有抑制增殖作用,联合应用抑制增殖作用更强,具有浓度与时间依赖性。左旋棉酚与低浓度阿霉素能诱导软组织肉瘤人纤维肉瘤HT1080细胞和滑膜肉瘤SW982细胞凋亡,两药作用后肿瘤细胞出现典型的凋亡细胞所具有的特征性改变,DNA琼脂糖凝胶电泳观察到明显的梯状带型,流式细胞仪检测发现左旋棉酚与低浓度阿霉素能诱导肿瘤细胞凋亡,联合应用作用更强。左旋棉酚与低浓度阿霉素对软组织肉瘤人纤维肉瘤HT1080细胞和滑膜肉瘤SW982细胞具有细胞周期调节作用,左旋棉酚使细胞阻滞在G1期,阿霉素使细胞阻滞在S期,两药联合应用使S期明显增加。左旋棉酚与低浓度阿霉素均导致肿瘤细胞Bcl-2蛋白表达逐渐减少,Bax蛋白的表达逐渐增多,两药联合应用变化更加明显。在诱导肿瘤细胞凋亡的过程中,肿瘤细胞内出现Caspase-9、Caspase-3的活化,两药联合应用变化更加明显。结论：左旋棉酚与低浓度阿霉素对软组织肉瘤人纤维肉瘤HT1080和滑膜肉瘤SW982细胞有明显抑制增殖及诱导凋亡作用,联合应用作用更强,其机制可能与下调Bcl-2蛋白表达,上调Bax蛋白表达及影响细胞周期有关。本研究通过左旋棉酚与低浓度阿霉素联合应用的体外实验,探讨了其治疗软组织肉瘤的可行性及其相互作用机理,为筛选与阿霉素有效配伍的抗肿瘤药物提供了实验依据。更多还原

【Abstract】 Objective:To investigate Apoptosis Inducted by (-)-Gossypo with combination of low concentration of adriamycin in soft tissue sarcoma cells and Its Mechanisms, offer theoretical basis for the improvement of cancer treatment. Methods:The antiproliferation of drugs on cells was deteceted by MTT assay. The changes of cells morphology and quantity were observed by Wright-Giemsa staining、Hoechst 33258 staining and transmission electron microscopy. DNA ladder formation was determined in Agarose gel electrophoresis.The apoptosis rate and cell cycle of soft tissue sarcoma cells treated by drugs was detected by flow cytometry. And the protein expression of Bcl-2、Bax in soft tissue sarcoma cells treated by drugs were detected by Western-Blot. The changes of caspase-3 and caspase-9 was deteceted by colorimetric assay. Results:(-)-Gossypol and low concentration of adriamycin resulted in a dose and time dependent ihnibition of HT1080 and SW982 cells,(-)-Gossypo with combination of low concentration of adriamycin had more obviouse effet of antiporliefration.(-)-Gossypol and low concentration of adriamycin was found to be bale to induce apoptosis of HT1080 and SW982 cells, as evidenced by typical morphological changes. Agarose gel electrophoresis determined typical DNA ladder formation.Flow cytometry deteceted that (-)-gossypol and low concentration of adriamycin induce apoptosis of HT1080 and SW982 cells (-)-Gossypo with combination of low concentration of adriamycin had more obvious apoptosis-inducing effet. Cell cycle analysis by flow eytometry indicated that (-)-Gossypol and low concentration of adriamycin could change cell cycle.(-)-Gossypol can induce G1 arrest and low concentration of adriamycin can induce S arrest.(-)-Gossypo with combination of low concentration of adriamycin had more obvious increace of S arrest,which evidence that (-)-Gossypol increased the chemotherapy sensitivity of HT1080 and SW982 cells.(-)-Gossypol and low concentration of adriamycin can result in decrease of the protein expression level of Bcl-2 and decrease of the protein expression level of Bax.The combination had more obvious changes. Colorimertie assay showed an activation of caspase-9 and caspase-3. The combination had higher activation changes.Conclusion: In this sutdy,we showed that (-)-Gossypol and low concentration of adriamycin had the effet of antiprolierfation and inducetion of apoptosis on HT1080 and SW982 cells for the first time.(-)-Gossypo with combination of low concentration of adriamycin had more obvious effect. The mechanisms mihgt involve down-relulation of Bcl-2 and up-relulation of bax and affeting cell cycle. Experimental Study on (-)-Gossypo with combination of low concentration of adriamycin in vitro discuss feasibility and interactional mechanism on the effect of (-)-Gossypo with combination of low concentration of adriamycin in soft tissue sarcoma and provide Experimental evidence for screen effective antitumor drugs combining with adriamycin.更多还原