沙利度胺对人骨肉瘤细胞MG-63的体外作用及其机制研究

发布时间：2018-07-06 09:32

本文选题：骨肉瘤 + 沙利度胺　；参考：《广州医科大学》2017年硕士论文

【摘要】：研究背景:骨肉瘤是最常见的原发性恶性骨肿瘤,好发于青少年,其恶性程度高,预后差,死亡率高[1,2]。在近30多年的研究中发现,骨肉瘤患者经过新辅助化疗联合保肢手术治疗,5年生存率约为70%,并无显著变化,使骨肉瘤的治疗进入了瓶颈期[7]。因此,探索治疗骨肉瘤的新药物和新方法显得尤为必要。沙利度胺最早期主要用于治疗妊娠呕吐反应,后因其严重胎儿致畸作用而几乎遭全球禁用。而近些年随着对其药理机制的深入研究发现,沙利度胺具有较明显的抗肿瘤作用。在肿瘤治疗领域,沙利度胺已经成为复发和难治多发性骨髓瘤标准治疗的一部分,对其他肿瘤如前列腺癌、直肠癌、非小细胞肺癌、乳腺癌及肾细胞癌等多种实体肿瘤也被证实有一定疗效[18-24]。但沙利度胺对骨肉瘤细胞增殖、侵袭和转移有何影响,目前尚少见报道。本实验用不同浓度沙利度胺处理骨肉瘤细胞,通过体外实验探讨其抗肿瘤作用,并探究可能的凋亡通路,为沙利度胺可能应用于骨肉瘤的临床治疗提供实验基础与理论依据。研究目的通过体外实验观察沙利度胺对人骨肉瘤细胞MG-63增殖和凋亡作用的影响,并探讨沙利度胺对人骨肉瘤的抗肿瘤作用和可能的相关分子机制。研究方法1.CCK-8法检测不同浓度沙利度胺对人骨肉瘤U2OS、MG-63细胞株增殖抑制的影响。2.Hoechst-33258荧光染色法观察不同浓度沙利度胺对MG-63细胞胞核的形态学影响。3.流式细胞术检测不同浓度沙利度胺作用于MG-63细胞后细胞凋亡情况。4.流式细胞术检测不同浓度沙利度胺作用于MG-63细胞后细胞周期变化。5.JC-1荧光染色检测不同浓度的沙利度胺处理MG-63细胞后线粒体膜电位的变化。6.DCFH-DA荧光探针检测不同浓度的沙利度胺对MG-63细胞内活性氧水平的影响。7.蛋白免疫印迹法测定不同浓度沙利的度胺处理MG-63细胞后凋亡相关蛋白BCL2、BAX、Nf-κb和Caspase3表达变化。实验结果1.体外细胞毒性实验结果显示:沙利度胺能明显的抑制人骨肉瘤细胞U2OS和MG-63的细胞活性,并呈现剂量-时间依赖效应;对U2OS细胞的24h、48h和72h的半数抑制浓度(IC50)分别为:500μg/ml、476.13±93.3μg/ml和156.61±40.65μg/ml;沙利度胺对MG-63细胞的24h、48h和72h的IC50分别为;500μg/ml、151.05±8.09μg/ml和94.76±10.52μg/ml。2.经Hoechst-33258染色后荧光显微镜下观察细胞核显示:与实验组对比,空白对照组人骨肉瘤MG-63细胞胞核完整,着色均匀,荧光弥散且暗淡;而不同浓度的沙利度胺处理细胞后,染色质凝聚皱缩,着色不规则,细胞核可呈致密浓染,或呈碎块状,颜色较明亮,呈现细胞凋亡皱缩核碎裂等典型变化,而且与沙利度胺浓度具有正相关性。3.Annexin V-FITC/PI双染法检测细胞凋亡显示:沙利度胺能够诱导骨肉瘤细胞MG-63凋亡坏死,并且与剂量呈相关性。经不同浓度沙利度胺(0、50,100和200μg/ml)处理MG-63细胞后,各组凋亡率分别是(7.98±1.26)%,(10.58±1.18)%,(28.74±6.08)%和(38.00±6.40)%。4.流式细胞术检测沙利度胺处理MG-63细胞后周期变化显示:不同浓度的沙利度胺(0、50μg/ml、100μg/ml、200μg/ml)处理骨肉瘤细胞MG-63细胞48h后,流式细胞仪检测周期变化显示沙利度胺能起G0/G1期阻滞,并随剂量增加而G0/G1期阻滞效应对应升高,0、50μg/ml、100μg/ml、200μg/ml分别为(63.68±1.76)%、(71.9±0.83)%,(73.87±1.72)%和(76.37±1.12)%;而S期细胞比例则随浓度增高而逐渐减少,0、50μg/ml、100μg/ml、200μg/ml分别为(19.95±3.11)%、(15.08±3.35)%、(13.53±2.96)%和(12.38±2)%,G2/M期也呈下降趋势。5.JC-1探针法检测沙利度胺对线粒体膜电位变化结果显示:沙利度胺能明显降低MG-63细胞线粒体膜电位,不同浓度药物(0、50μg/ml、100μg/ml、200μg/ml)作用MG-63细胞24h后,流式细胞仪检测相应沙利度胺浓度对应线粒体膜电位降低细胞比例分别为:(4.84±0.31)%、(7.63±0.94)%,(9.57±7.63)%和(13.62±5.92)%;未受损线粒体膜电位细胞比例为:(95.17±0.31)%,(92.37±0.97)%,(90.4±2.62)%,(85.53±4.70)%。6.DCFH-DA荧光探针法检测沙利度胺对细胞内活性氧水平结果显示:沙利度胺在100ug/ml至400ug/ml浓度范围作用于MG63细胞可以增加其胞内的活性氧水平,并呈现浓度依赖效应(P0.05),沙利度胺组(100μg/ml、200μg/ml、400μg/ml)的DCF荧光强度与正常对照组的比值分别是(1.26±0.16),(1.40±1.40)和(1.88±0.32),而阳性对照组(ROSUP)与正常对照组的比值是(1.34±0.07)。沙利度胺高浓度组(100μg/ml至400μg/ml)与低浓度组(12.5ug/ml至-50μg/ml)活性氧水平也具有显著的统计学意义(P0.05)。7.蛋白免疫印迹法检测不同浓度沙利度胺作用于骨肉瘤细胞后凋亡相关蛋白BCL2、BAX、Caspase3和Nf-κb蛋白表达情况,结果显示:与阴性对照相比,不同浓度的沙利度胺(0、50μg/ml、100μg/ml、200μg/ml)处理骨肉瘤细胞MG-63细胞48h后,骨肉瘤细胞内抗凋亡蛋白Bcl2及Nf-κb蛋白表达水平下降,而促凋亡蛋白Caspase3和Bax表达水平逐渐升高。结论:沙利度胺能体外抑制骨肉瘤细胞MG-63增殖和促进凋亡,可能机制是通过提升细胞内活性氧水平,降低线粒体膜电位,促进促凋亡蛋白Bax和Caspase3表达,抑制抗凋亡蛋白Bcl2及Nf-κb蛋白表达,提示其可能机制是通过内源性线粒体凋亡途径诱导细胞凋亡。
[Abstract]:Background: osteosarcoma is the most common primary malignant bone tumor. It is found in adolescents with high malignancy, poor prognosis and high mortality rate [1,2].. In the recent 30 years of study, osteosarcoma patients were treated with neoadjuvant chemotherapy combined with limb salvage surgery, and the 5 year survival rate was about 70%, which made the treatment of osteosarcoma into the bottle. It is necessary to explore new drugs and new methods for the treatment of osteosarcoma in cervical [7].. Thalidomide is mainly used in the treatment of pregnancy vomiting, which is almost globally banned due to its severe fetal teratogenicity. In recent years, thalidomide has a more obvious antitumor effect with its pharmacological mechanism. In the field of cancer treatment, thalidomide has become a part of the standard treatment of recurrent and refractory multiple myeloma. Many solid tumors, such as prostate cancer, rectal cancer, non small cell lung cancer, breast cancer and renal cell carcinoma, have also been proved to have a certain effect [18-24]. but thalidomide proliferation, invasion, and invasion of osteosarcoma cells, and invasion and treatment of osteosarcoma cells. This experiment uses different concentrations of thalidomide to treat osteosarcoma cells and explore the antitumor effect in vitro, and explore the possible apoptosis pathway, which provides experimental basis and theoretical basis for the clinical treatment of thalidomide for osteosarcoma. The objective of this study is to observe the experimental observation in vitro. The effect of thalidomide on the proliferation and apoptosis of human osteosarcoma cell MG-63 and the antitumor effect and possible molecular mechanism of thalidomide on human osteosarcoma. The effect of different concentrations of thalidomide on the proliferation and inhibition of human osteosarcoma U2OS and MG-63 cell lines by 1.CCK-8 method was observed by.2.Hoechst-33258 fluorescence staining The effect of different concentrations of thalidomide on the cell nucleus of MG-63 cells.3. flow cytometry was used to detect the apoptosis of MG-63 cells with different concentrations of thalidomide,.4. flow cytometry was used to detect different concentrations of thalidomide in MG-63 cells and cell cycle changes after.5.JC-1 fluorescence staining to detect different concentrations of thalidomide treatment M Changes of mitochondrial membrane potential after G-63 cells.6.DCFH-DA fluorescence probe detected the effect of different concentrations of thalidomide on reactive oxygen levels in MG-63 cells.7. protein immunoblotting was used to determine the expression changes of apoptosis related protein BCL2, BAX, Nf- kappa B and Caspase3 after MG-63 cells with different concentrations of Sally in MG-63 cells. Experimental results 1. cytotoxic in vitro The results showed that thalidomide significantly inhibited the cell activity of U2OS and MG-63 cells in human osteosarcoma cells, and showed a dose time dependence effect. The median inhibitory concentration (IC50) for 24h, 48h and 72h (IC50) of U2OS cells was 500 mu g/ml, 476.13 + 93.3 micron and 156.61 + 40.65 micron g/ml, and thalidomide for MG-63 cells The nuclei of 500 mu g/ml, 151.05 + 8.09 mu g/ml and 94.76 + 10.52 mu g/ml.2. were stained with Hoechst-33258 after Hoechst-33258 staining. Compared with the experimental group, the nucleus of MG-63 cells in the blank control group was complete, coloured evenly, and the fluorescence was dispersed and dim; and the chromatin condensed and crinkled after the different concentration of thalidomide cells. The coloring is irregular, the nucleus can be dense and dense, or the nucleus is fragmented, the color is bright, and the cell apoptosis and crumbling nucleus fragmentation, and the positive correlation of thalidomide concentration with.3.Annexin V-FITC/PI double staining method to detect cell apoptosis: Thalidomide can induce osteosarcoma cell MG-63 apoptosis and necrosis, and the dose of thalidomide. After the treatment of MG-63 cells with different concentrations of thalidomide (0,50100 and 200 u g/ml), the apoptosis rate of each group was (7.98 + 1.26)%, (10.58 + 1.18)%, (28.74 + 6.08)% and (38 + 6.40)%.4. flow cytometry for the detection of thalidomide treatment MG-63 cell cycle changes showed that the different concentrations of thalidomide (0,50 mu g/ml, 100 u g/ml, 200 g/m) L) after treating osteosarcoma cell MG-63 cell 48h, the detection cycle of flow cytometry showed that thalidomide could block the G0/G1 phase, and the G0/G1 phase block effect increased with the dose increase, 0,50 mu g/ml, 100 u g/ml, (63.68 + 1.76)%, (71.9 + 0.83)%, (73.87 + 1.72)% and (76.37 + 1.12)%, respectively, while the ratio of S phase cells followed the concentration. Increasing and decreasing gradually, 0,50 mu g/ml, 100 mu g/ml, 200 mu g/ml (19.95 + 3.11)%, (15.08 + 3.35)%, (13.53 + 2.96)% and (12.38 + 2)%, and G2/M stage also showed a downward trend.5.JC-1 probe to detect the changes of thalidomide on mitochondrial membrane potential: salidomide can significantly reduce the mitochondrial membrane potential of MG-63 cells, different concentrations of drugs (0,50) The ratio of thalidomide to the mitochondrial membrane potential was (4.84 + 0.31)%, (7.63 + 0.94)%, (9.57 + 7.63)% and (13.62 + 5.92)% and (95.17 + 0.31)%, (95.17 + 0.31)%, (95.17 + 0.31)%, (92.37 + 4.84)%, and (7.63)%, and (7.63)%, (9.57 +)%, (9.57 +)%, and (7.63)%, respectively (4.84 + 0.31)%, (9.57 + 7.63)% and (13.62 + 5.92)%, respectively, after 24h. 3 + 4.70)%.6.DCFH-DA fluorescence probe was used to detect the intracellular reactive oxygen species of thalidomide. The effect of thalidomide on MG63 cells in the concentration range of 100ug/ml to 400ug/ml could increase the intracellular reactive oxygen level, and the concentration dependent effect (P0.05), the DCF fluorescence intensity of salidomide group (100 u g/ml, 200 Mu g/ml, 400 mu g/ml) The ratio of the normal control group was (1.26 + 0.16), (1.40 + 1.40) and (1.88 + 0.32), while the ratio of the positive control group (ROSUP) and the normal control group was (1.34 + 0.07). The active oxygen level of the thalidomide high concentration group (100 to 400 mu g/ml) and the low concentration group (12.5ug/ml to -50 Mu ml) also had significant statistical significance (P0.05).7. protein immunity The expression of apoptosis related protein BCL2, BAX, Caspase3 and Nf- kappa B protein in osteosarcoma cells was detected with different concentrations of thalidomide in osteosarcoma cells. The results showed that the anti apoptotic protein Bcl2 in osteosarcoma cells treated with different concentrations of thalidomide (0,50 mu g/ml, 100 u g/ml, 200 u g/ml) in the osteosarcoma cells. And the expression level of Nf- kappa B protein decreased, while the expression level of apoptotic protein Caspase3 and Bax increased gradually. Conclusion: Thalidomide can inhibit the proliferation and apoptosis of osteosarcoma cells MG-63 in vitro. The possible mechanism is to increase the intracellular reactive oxygen level, reduce the mitochondrial membrane potential, promote the expression of apoptotic protein Bax and Caspase3, and inhibit the resistance to withering. The expression of dead protein Bcl2 and Nf- kappa B protein suggests that the mechanism may be induced by endogenous mitochondrial apoptosis pathway.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R738

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