石蒜碱抑制肺癌NCI-H460细胞增殖及Lewis肺癌小鼠肿瘤生长作用研究

发布时间：2019-04-13 20:53

【摘要】：目的:通过研究石蒜碱在体外对人肺癌NCI-H460细胞增殖的抑制作用及对NCI-H460细胞凋亡的影响,和体内对Lewis肺癌小鼠的抑瘤作用,探讨石蒜碱抗肿瘤机制,旨在为临床非小细胞肺癌的治疗寻找新的高效、低毒的化疗药物。方法:1.石蒜碱对NCI-H460的细胞毒作用(1)MTT法检测石蒜碱抑制NCI-H460细胞增殖的作用,(2)用流式细胞仪检测NCI-H460细胞的凋亡率,(3)比色法检测凋亡相关因子Caspase 3的活性;2.石蒜碱对Lewis肺癌小鼠的抑瘤作用(1)Lewis肺癌细胞株的培养,(2)小鼠Lewis肺癌移植瘤模型的建立,(3)处死小鼠,计算抑瘤率,脾脏指数及胸腺指数,(4)HE染色后观察细胞形态。结果:1.石蒜碱作用NCI-H460细胞24 h后,显微镜下观察细胞形态,经石蒜碱作用后,NCI-H460细胞外形不规则,细胞皱缩,大小不一,细胞破碎程度随给药剂量增加而增大,细胞数量逐渐减少。2.用石蒜碱处理NCI-H460细胞24 h后,,NCI-H460细胞增殖受到明显的抑制,与对照组相比差异显著(p0.05,p0.01),并呈现一定的浓度相关性。IC50为5.79±0.11μmol/L,表明石蒜碱能够有效抑制肺癌NCI-H460细胞增殖。3.随着石蒜碱处理浓度的升高,处理组细胞凋亡率逐渐升高,与生理盐水组相比差异显著(p0.01)。最高浓度16μmol/L时,凋亡率达到46.2±3.46%。上述结果表明石蒜碱对NCI-H460细胞的凋亡具有诱导效应,并呈现一定的剂量依赖性。4.随着石蒜碱浓度的增加,Caspase 3的活性逐渐增高。石蒜碱处理组与生理盐水对照组比较,Caspase 3的活性显著升高(p0.01)。结果表明石蒜碱可增强肺癌NCI-H460细胞的Caspase 3的活性,从而诱导NCI-H460细胞凋亡。5.与生理盐水组比较,石蒜碱低、中低、中、中高、高剂量组对Lewis肺癌小鼠肿瘤生长均有抑制作用,其中以高剂量组(40 mg/kg)的作用最为显著,抑瘤率56.8%(p0.01)。6.荷瘤小鼠实体瘤组织的HE染色实验结果显示生理盐水组肿瘤细胞生长均匀紧密,核固缩较少,无坏死或点状坏死;石蒜碱各剂量组及环磷酰胺组肿瘤细胞可见大量核固缩,核碎裂,肿瘤细胞数量减少,排列疏松,尤其以高剂量组(40 mg/kg)的作用最为显著,肿瘤细胞明显散在分布,数量明显减少。结论:石蒜碱在体外能有效地抑制NCI-H460细胞增殖并促进其凋亡,其机制可能与激活了Caspase 3的表达从而诱导肿瘤细胞凋亡有关;石蒜碱在体内亦能有效抑制小鼠Lewis肺癌细胞生长。
[Abstract]:Objective: to study the inhibitory effect of Lycorine on the proliferation of human lung cancer NCI-H460 cells in vitro and the apoptosis of NCI-H460 cells, and the antitumor effect of Lycorine on Lewis lung cancer mice in vivo, and to explore the anti-tumor mechanism of garlicine. The aim of this study is to find new effective and low toxic chemotherapeutic drugs for the treatment of clinical non-small cell lung cancer (NSCLC). Methods: 1. Cytotoxicity of allicin to NCI-H460 (1) the inhibitory effect of allicin on the proliferation of NCI-H460 cells was detected by MTT assay; (2) the apoptosis rate of NCI-H460 cells was detected by flow cytometry; (3) the activity of apoptosis-related factor Caspase-3 was detected by colorimetry; 2. Antitumor effect of allicin on Lewis lung cancer mice (1) culture of Lewis lung cancer cell line, (2) establishment of transplanted tumor model of Lewis lung cancer in mice, (3) killing mice, calculation of tumor inhibition rate, spleen index and thymus index, (4) the morphology of the cells was observed after HE staining. Results: 1. After treated with allicin for 24 h, the morphology of NCI-H460 cells was observed under microscope. After treated with garlicine, the shape of NCI-H460 cells was irregular, the cells shrank, the size of the cells was different, and the degree of cell fragmentation increased with the increase of dosages. The number of cells gradually decreased. 2. When NCI-H460 cells were treated with garlicine for 24 h, the proliferation of NCI-H460 cells was significantly inhibited (p0.05, p0.01), and the IC50 was 5.79 卤0.11 渭 mol / L, compared with the control group (p0.05, p0.01), and the IC50 was 5.79 卤0.11 渭 mol / L, and the IC50 was 5.79 卤0.11 渭 mol / L. It is suggested that allicin can effectively inhibit the proliferation of lung cancer NCI-H460 cells. 3. With the increase of the concentration of allicin, the apoptosis rate of the treated group increased gradually, and the difference was significant compared with that of the saline group (p0.01). At the highest concentration of 16 渭 mol / L, the apoptosis rate reached 46.2 卤3.46%. These results suggest that Lycorine can induce apoptosis of NCI-H460 cells in a dose-dependent manner. 4. With the increase of the concentration of allicin, the activity of Caspase-3 increased gradually. Compared with the control group, the activity of Caspase-3 was significantly increased in the garlicine treatment group (p0.01). The results showed that allicin could enhance the activity of Caspase-3 in lung cancer NCI-H460 cells and induce the apoptosis of NCI-H460 cells. 5. Compared with normal saline group, Lycorine had inhibitory effect on tumor growth of Lewis lung cancer mice in low, middle and high dose groups, among which the high dose group (40 mg/kg) had the most significant effect. The tumor inhibition rate was 56.8% (p0.01). The results of HE staining in solid tumor tissue of tumor-bearing mice showed that the tumor cells in the saline group grew uniformly and closely with less nuclear condensation and no necrosis or punctate necrosis. A large number of nuclear pyknosis, nuclear fragmentation, decrease in the number of tumor cells and loose arrangement were observed in various dose groups and cyclophosphamide groups, especially in the high dose group (40 mg/kg), and the distribution of tumor cells was obvious. There was a marked decrease in the number. Conclusion: garlicine can effectively inhibit the proliferation and promote the apoptosis of NCI-H460 cells in vitro. The mechanism may be related to the activation of Caspase-3 expression and the induction of apoptosis of tumor cells. Allicin can also inhibit the growth of Lewis lung cancer cells in vivo.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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