三氧化二砷联合粉防己碱对乳腺癌血管新生及肺转移的影响

发布时间：2018-02-25 23:37

本文关键词： 粉防己碱三氧化二砷乳腺癌肺转移 HCC1937 MDA-MB-435S　出处：《北京中医药大学》2016年博士论文　论文类型：学位论文

【摘要】：研究目的：本课题在既往研究基础上,分析粉防己碱、三氧化二砷及两药联合对MDA-MB-435S细胞和HCC1937细胞体外的抑制增殖作用及促凋亡机制。体内实验则利用MDA-MB-435S细胞接种的裸鼠乳腺癌肺转移模型来观察粉防己碱、三氧化二砷及两药联合与化疗药顺铂的抑瘤作用及其与肿瘤血管生成相关指标的表达,同时检测各组裸鼠肝肾功能以判断毒性中药安全性。以期为中医“以毒攻毒”指导下的中药配伍组合治疗乳腺癌及乳腺癌肺转移提供相关实验依据。研究方法：体外实验利用MTT方法检测经不同浓度粉防己碱、三氧化二砷及粉防己碱联合三氧化二砷对MDA-MB-435S及HCC1937乳腺癌细胞在不同时间点(24、48、72、96h)的抑制作用及药物浓度与时间关系。利用中效原理(Chou-Talalay合用指数法)判断联合用药效果最好的药物配比以指导后续实验。采用倒置相差荧光显微镜观察不同浓度粉防己碱、三氧化二砷及两药联合干预48小时后MDA-MB-435S及HCC1937乳腺癌细胞的形态学变化,探究药物的作用效果。采用AnnexinV-FITC、PI双染流式细胞术测定不同浓度粉防己碱、三氧化二砷及两药联合干预48小时后MDA-MB-435S及HCC1937乳腺癌细胞早期凋亡、晚期凋亡的比例变化,探究两药对肿瘤细胞凋亡影响。采用肿瘤侵袭实验检测不同浓度粉防己碱、三氧化二砷及两药联合干预48小时后MDA-MB-435S及HCC1937乳腺癌细胞的侵袭能力是否受到药物抑制及其抑制程度。采用RT-PCR及Western blot方法分析粉防己碱联合三氧化二砷干预48小时后MDA-MB-435S及HCC1937乳腺癌细胞凋亡相关基因蛋白Caspase-3、PARP、Bid、 Bax、Bcl-2、survivin、AKT的表达及相关作用机制。体内试验采用高肺转移率人乳腺癌细胞MDA-MB-435S种植于BALB/c裸鼠乳房垫下以形成乳腺癌肺转移模型,造模成功后采用汉防己甲素注射液、亚砷酸注射液、顺铂注射液及汉防己甲素注射液联合亚砷酸注射液观察裸鼠一般生存情况、抑瘤率、肺转移率、肝肾功能等指标,对瘤体及肺组织做HE染色切片,免疫组化、RT-PCR及Westernblot观察VEGF、MMPs等血管生成相关基因、蛋白表达情况,说明其作用通路。研究结果：体外实验中发现粉防己碱、三氧化二砷及粉防己碱联合三氧化二砷对MDA-MB-435S及HCC1937乳腺癌细胞的体外生长均有较为显著的抑制效果,与药物剂量、作用时间呈明显关系,抑制率随作用时间的延长及药物浓度增加上升,且时间与药物剂量间存在交互效应。根据中效原理(Chou-Talalay合用指数法)及calcusyn2.0软件得出两药联合作用于MDA-MB-435S及HCC1937乳腺癌细胞,在粉防己碱(1μg/mL)和三氧化二砷(1.5μg/mL)时协同作用最好,遂选取该组合作为后期实验选取的合药浓度。细胞形态学研究显示,粉防己碱、三氧化二砷及粉防己碱联合三氧化二砷均可使细胞形态发生改变且与药物剂量大小有关,且合药效果优于单药。细胞大量死亡、崩解,贴壁数量显著减少。流式细胞术双染检测凋亡可发现粉防己碱、三氧化二砷及两药联合干预MDA-MB-435S及HCC1937乳腺癌细胞48h小时早期凋亡率和晚期凋亡率均有上升,且合药较之单药细胞凋亡率增加。肿瘤侵袭实验发现在粉防己碱、三氧化二砷干预MDA-MB-435S及HCC1937乳腺癌细胞48小时后,穿膜进入下室的细胞数具有差异,且药物浓度越大穿膜细胞数越少。两药联合作用效果较之同浓度单药并无明显差异。通过RT-PCR对凋亡相关基因表达情况进行分析发现：经粉防己碱联合三氧化二砷干预48小时后,MDA-MB-435S及HCC1937细胞中Caspase-3、bax、bid相关基因表达随药物浓度变化升高,Bcl-2、suivivin调控基因的表达降低。且粉防己碱与三氧化二砷联合用药效果,优于等浓度的单一用药效果。Western bolt实验发现,MDA-MB-435S及HCC1937细胞经药物作用后Caspase 3前体表达量减少,反之切割体表达量增加。下游PRAP的酶切条带提示细胞凋亡的发生。Bcl-2表达降低,Bax、Bid表达增加,且合药作用优于等浓度单药。Survivin的表达在两药单独作用时与空白组无明显差异,合药则在该蛋白的表达上降低明显。体内试验表明汉防己甲素注射液、亚砷酸注射液及两药联合均可减慢肿瘤生长,抑制肺转移发生,但较之顺铂则效果略差。两药单用及合用均对裸鼠肝肾功能无明显损害。对裸鼠瘤体及肺组织进行采用RT-PCR及Western blot检测发现,汉防己甲素注射液、亚砷酸注射液及两药联合均可降低VEGF表达,且合药组比单药组表达更低。研究结论：粉防己碱、三氧化二砷及两药联合在体外MDA-MB-435S及HCC1937细胞增殖有明显抑制作用,且与药物剂量及作用时间相关性强。在粉防己碱(1μg/mL)和三氧化二砷(1.5μg/mL)时协同作用最好。不同浓度粉防己碱、三氧化二砷及两药联合干预48h后,MDA-MB-435S及HCC1937细胞在早期凋亡率和晚期凋亡率均有所提高,侵袭功能减弱。通过降低Bcl-2,升高Bax、 Bid表达,影响Caspase-3来诱导乳腺癌细胞的体外凋亡体内实验表明MDA-MB-435S细胞接种的裸鼠乳腺癌模型造模成功,肺转移率高,汉防己甲素注射液、亚砷酸注射液及两药联合均可抑制瘤体生长及肺转移发生。其作用机制与降低VEGF表达,影响肿瘤血管生成有关。
[Abstract]:Objective: in this study, based on previous study, analysis of tetrandrine, arsenic trioxide and the combination of the two drugs in vitro MDA-MB-435S cells and HCC1937 cell proliferation inhibition and apoptosis mechanism. In vivo experiments using MDA-MB-435S cells in nude mice inoculated with breast cancer lung metastasis model to observe the effect of tetrandrine on the expression of tumor related indicators, the effect of arsenic trioxide and the two drugs combined with cisplatin and tumor angiogenesis, and to detect the nude mice liver and kidney function to determine the toxicity of traditional Chinese medicine safety. In order to "fight" under the guidance of the traditional Chinese medicine combination compatibility of traditional Chinese medicine treatment of breast cancer and pulmonary metastasis of breast cancer to provide experimental basis. Methods: in vitro were detected by MTT. With different concentrations of tetrandrine, arsenic trioxide and tetrandrine combined with arsenic trioxide on MDA-MB-435S and HCC1937 in breast cancer cells At different time points (24,48,72,96h) and the inhibitory effect of the drug concentration and time. The median effect principle (Chou-Talalay share index) to determine the effect of drug combination the best drug ratio in order to guide the subsequent experiment. Using the inverted fluorescence microscope observation of different concentrations of tetrandrine, morphological changes of MDA-MB-435S and HCC1937 in breast cancer cells after 48 hours the joint intervention of arsenic trioxide and explore the effect of two drugs drugs. Using AnnexinV-FITC, PI double staining were measured at different concentrations of tetrandrine in flow cytometry, arsenic trioxide and two drug combination intervention after 48 hours of MDA-MB-435S and HCC1937 in breast cancer cells early apoptosis, late apoptosis ratio changes, explore the impact of the two drugs on the apoptosis of tumor cells. The tumor invasion assay with different concentrations of tetrandrine, HCC1937 and MDA-MB-435S after 48 hours and two drug intervention combined with arsenic trioxide Breast cancer cell invasion and the degree of inhibition by whether the drug inhibition by RT-PCR and Western. Blot analysis of tetrandrine combined with arsenic trioxide and apoptosis of MDA-MB-435S breast cancer cell HCC1937 gene protein Caspase-3, after 48 hours of treatment PARP, Bid, Bax, Bcl-2, survivin, AKT expression and mechanism in vivo by high. The rate of lung metastasis of human breast cancer cells grown in MDA-MB-435S BALB/c nude mice breast pad to form a model of pulmonary metastasis of breast cancer, after the success of modeling by Tetrandrine Hydrochloride Injection, Cisplatin Injection and Tetrandrine Hydrochloride Injection arsenious acid injection combined with Arsenious Acid Injection were observed general survival, tumor inhibition rate, the rate of lung metastasis, liver and kidney function and other indicators, do HE staining the tumor and lung tissue, immunohistochemistry, RT-PCR and Westernblot were VEGF, MMPs and other angiogenesis related genes, eggs White expression, indicating its pathway. Results: it was found that tetrandrine in vitro growth inhibitory effect was more significant in vitro arsenic trioxide and tetrandrine combined with arsenic trioxide on MDA-MB-435S and HCC1937 in breast cancer cells, and the drug dose, as time was, the inhibition rate with the increase of the incubation time and drug the concentration increased, and the time and dose effect. According to the interaction between the efficiency principle (Chou-Talalay combination index method) and calcusyn2.0 software to get the combined effects of two drugs on MDA-MB-435S and HCC1937 in breast cancer cells, tetrandrine (1 g/mL) and arsenic trioxide (1.5 g/mL) when the best synergism, and concentration then select the combination as a late experiment. Cell morphology showed that tetrandrine, arsenic trioxide and tetrandrine combined with three oxidation two arsenic can cause cell Changing the shape and size of the dose related and drug effect is better than the single drug. A large number of cell death, disintegration, the adherent population decreased significantly. Double staining of apoptosis can be found in tetrandrine flow cytometry combined intervention of arsenic trioxide and two drugs MDA-MB-435S and HCC1937 breast cancer cells 48h hours apoptotic rate the rate increased, compared with single drug and drug cell apoptosis rate increased. Tumor invasion was found in tetrandrine, arsenic trioxide intervention MDA-MB-435S and HCC1937 breast cancer cells 48 hours after penetrating the lower chamber of the number of cells with different drug concentration, and larger transmembrane cell number less. Significant difference between the two the medicine combined effect compared with the same concentration. There is no single drug case analysis found by RT-PCR on the expression of apoptosis related genes by tetrandrine combined with arsenic trioxide 48 hours after intervention, MDA-MB-435S and HCC Caspase-3, 1937 in Bax cells, bid gene expression changes with the concentration of drug increased Bcl-2, reduce the expression of suivivin gene. And the effects of tetrandrine combined with arsenic trioxide treatment effect, found that the effect of.Western bolt experiment is better than single drug concentration, MDA-MB-435S and HCC1937 cells after drug action Caspase expression before 3 to reduce the amount of instead of cutting body weight increases. The expression of downstream PRAP enzyme bands indicated that cell apoptosis decreased expression of.Bcl-2, Bax, Bid expression increased, expression and drug effect is better than the single drug concentration of.Survivin in the two drug alone and blank group had no significant difference, and medicine in the expression of the protein on the lower in vivo tests show obvious. Tetrandrine Hydrochloride Injection, combined with Arsenious Acid Injection and two drugs can slow tumor growth, inhibit the occurrence and metastasis to the lungs, but the effect is slightly worse than cisplatin two drugs used. And combined liver and kidney function of nude mice without obvious damage. The body and lung tissue of nude mice tumor were performed by RT-PCR and Western blot detection, Tetrandrine Hydrochloride Injection, combined with Arsenious Acid Injection and two drugs can reduce the expression of VEGF, and the combined drug group expressed lower than the single drug group. Conclusions: tetrandrine, arsenic trioxide and two drugs United has obvious inhibitory effect on the proliferation of the MDA-MB-435S cells and HCC1937 cells, and the drug dose and action time. The strong correlation between tetrandrine (1 g/mL) and arsenic trioxide (1.5 g/mL) at different concentration. The best synergistic effect of tetrandrine combined intervention of arsenic trioxide and two 48h after the administration of MDA-MB-435S and HCC1937 cells the increase in the early and late apoptosis rate were decreased. Invasion function by decreasing Bcl-2, increasing Bax, Bid expression in vitro apoptosis in vivo to induce breast cancer cells Caspase-3 Experiments show that the nude mice model of breast cancer MDA-MB-435S cell vaccination model was successful, lung metastasis rate is high, Tetrandrine Hydrochloride Injection, combined with Arsenious Acid Injection and two drugs can inhibit the growth and metastasis of lung tumor. The mechanism is related to the decreased expression of VEGF, tumor angiogenesis.

【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R273

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