兰州大学机构库 >生命科学学院
NaSeVO 和 SeMoV 的抗肿瘤作用及机理探讨
Alternative TitleThe Antitumor Effects and Mechanism of Selenium Compound NaSeVO and SeMoV
杨军英
Thesis Advisor王子仁
2006-01-20
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name博士
KeywordNaSeVO SeMoV 抗肿瘤活性 细胞凋亡 活性氧 细胞毒性 荷瘤小鼠 MTT 比色法 激光共聚焦显微镜检查法 流式细胞术 bcl-2 bax 细胞色素C IκBα NF-κB
Abstract

硒是自然界存在的一种珍稀微量元素,具有多种生物学功能,和多种疾病特别是和肿瘤的发生密切相关。近年来国内外关于硒化合物的研究很多,包括新的硒化合物的合成、研究已有化合物的抗炎、抗病毒、抗肿瘤作用以及探讨硒化合物的抗肿瘤作用机理等。众多的研究证实硒化合物具有明确的抗肿瘤作用,可以作为化学保护剂对抗化疗药物的副反应,增加药物耐受性,同时又不影响其抗癌活性,引起了人们的广泛兴趣。由于目前广泛用于预防克山病、癌症等的硒酸钠和亚硒酸钠,以及新合成的其它含硒药物,普遍具有安全范围小、毒性大的缺点,影响了它们的应用。所以发现高效低毒的含硒化合物,是我们努力的方向。本课题对新合成的 2 种含硒化合物 NaSeVO 和 SeMoV 的抗肿瘤作用进行研究,并从对肿瘤细胞周期及细胞凋亡的诱导、对细胞内离子浓度的影响等方面揭示其作用机理,为开发新的含硒化合物提供实验依据。实验结果如下:
1. NaSeVO 和 SeMoV 对 k562 细胞增殖的抑制作用。体外采用人红白血病 K562 细胞株,用 MTT 比色法观察药物对肿瘤细胞增殖的影响,结果显示 NaSeVO(0.625~20 mg/L)和 SeMoV(0.313~10 mg/L) 能时间、浓度依赖性地抑制 K562 细胞增殖(p<0.05)。用药 48 和 72 h 时的 IC50 值分别为: NaSeVO: 14.41 (4.45~46.60) mg/L 和 3.45 (2.29~5.22)mg/L; SeMoV: 14.41 (4.45~ 46.60) 和 3.45 (2.29~5.22) mg/L。2. NaSeVO 和 SeMoV 对 K562 细胞内[Ca2+]、 [Mg2+]、 ROS、 pH 值和线粒体膜电位(MMP)的荧光强度的影响用激光共聚焦显微技术测定细胞内[Ca2+]、 [Mg2+]、 ROS、 pH值和MMP荧光强度的变化。结果表明, NaSeVO和SeMoV 1.25~10 mg/L能够明显增加k562细胞内Ca2+、 Mg2+和ROS的荧光强度(p<0.01),但SeMoV 10 mg/L则使ROS显减少(p<0.001)。与此相反, pH和MMP显著降低(p<0.01)。3. NaSeVO 和 SeMoV 对 K562 细胞凋亡百分率和细胞周期的影响用流式细胞仪测定细胞凋亡百分率和 Multicycle 软件分析细胞周期。结果显示 NaSeVO 和 SeMoV 对 K562 细胞的周期分布有明显影响,且在大剂量时出现了明显的亚二倍体峰。用药24、48h时,小剂量NaSeVO诱导细胞聚集在G1期,而G2/M细胞数量减少;与此相反,大剂量NaSeVO使G0/G1细胞数量减少,而G2/M细胞数量增加。S期细胞数量明显增加。用药 24h 时,小剂量 SeMoV 诱导细胞聚集在 G1 期,S 期细胞减少;而大剂量 SeMoV 诱导细胞聚集在 S 期, G0/G1 期细胞减少。 48h 时, SeMoV 使细胞聚集在 S 期, G0/G1 期细胞减少。4. NaSeVO 和 SeMoV 对 K562 细胞的凋亡形态学特征影响用倒置显微镜和电子显微镜观察 NaSeVO 和 SeMoV 对 k562 细胞形态的影响。结果表明 NaSeVO(0.625~20 mg/L )和 SeMoV(0.313~10 mg/L) 24 h, 使 k562细胞出现典型的凋亡特征,在倒置显微镜下可观察到细胞体积缩小,细胞膜完整,核浓染、碎裂,伴有出泡现象和凋亡小体出现。 NaSeVO 10 mg/L 和 SeMoV 5mg/L 24 h 在电子显微镜下观察到典型的凋亡特征,染色质高度凝集,边集,出现典型的凋亡细胞和凋亡小体。5. NaSeVO 和 SeMoV 对体内移植瘤的生长抑制作用采用小鼠 S180 肉瘤和小鼠 H22 肝癌实验,结果显示 NaSeVO 和 SeMoV 5 、10 mg/kg 腹腔注射能够明显抑制 S180 和 H22 的生长(p<0.01),对 S180 的抑制率分别为 26.8 %、 58.4 %(NaSeVO),和 33.08%、 42.74(SeMoV);对 H22 的抑制率分别为 31.3 %、 47.4%(NaSeVO)和 38.19%、 39.17%(SeMoV)。6. NaSeVO和SeMoV对K562细胞Bcl-2/Bax表达的影响用免疫组化实验检测K562细胞内Bax 和Bcl-2基因的表达。 NaSeVO 10 mg/L、 SeMoV 5 mg/L能够明显抑制bcl-2 的表达,而bax 的表达增强。表明经NaSeVO 和SeMoV 处理后K562细胞内Bcl-2/Bax比率下降。7. NaSeVO 和 SeMoV 对 K562 细胞内蛋白 cytochrome c、 NF-κB 和 IκBα 的影响(western-bloting)用 western-blotting 法检测 K562 细胞中细胞色素 C、 IκBα 和 NF-κB 的含量。研究结果表明,与对照组比较,NaSeVO 和 SeMoV 处理 K562 细胞24h后,胞浆细胞色素 C 含量明显增多,胞浆 IκBα 含量明显增加。核内 NF-κB 含量随剂量增加而呈下降趋势。
从以上结果可得出以下结论:
1.NaSeVO 和 SeMoV 5、10 mg/kg 腹腔注射均能明显抑制小鼠S180 肉瘤和 H22肝癌的生长,说明 NaSeVO 和 SeMoV 具有体内抗肿瘤作用。2.NaSeVO 和 SeMoV 能够抑制 K562 的增殖,影响细胞周期的分布,表明体外有较强的抗肿瘤作用。3.亚二倍体峰的出现(流式细胞实验)和细胞形态学变化提示 NaSeVO 和SeMoV 的抗肿瘤作用可能与Ca2+、Mg2+、ROS、pH 值和线粒体膜电位(MMP)诱导的细胞凋亡有关。4.NaSeVO 和 SeMoV 抑制 K562 细胞 bcl-2 的表达,使 bax 的表达增强;胞浆细胞色素 C 含量明显增多,胞浆IκBα含量明显增加。NF-κB 含量随剂量增加而呈下降趋势。提示NaSeVO 和 SeMoV 具有抗肿瘤作用,其作用机理与细胞凋亡有关。

Other Abstract

As a trace element, selenium is essential for nutrition and exhibits a wide range of biological functions. It is also shown to have anticarcinogenic or preventive chemicals from inducing tumors. Selenium compounds have been reported to possess a wide range of biological and pharmacological activities including anti-inflammatory, antiviral, anti tumor and used to a lot of disease such as Keshan disease. These diverse pharmacological activities of selenium compounds have demonstrated that they have anti-tumor activity. It is commonly accepted that the mechanism is attributed partially to its strong antioxidative properties which may relate to apoptosis and selenium compounds served as one of the most potent preventive chemicals from inducing tumors could against the side effect of chemotherapeutic agents and enhance the activity of chemotherapeutic agents and diminish normal cells’ toxicity. Nevertheless, its clinical effectiveness is restricted due to the dose-limiting toxicity of normal and healthy cells action, studies have focused on searching for new selenium compound which possess higher anti-tumor activity and lower toxicity. Therefore, the purpose of this study was to investigate the anti-tumor activities of selenium compound in vitro as well as in vivo, and to explore the mechanism. The main experimental results were as follows:
1. Anti-proliferation activity of NaSeVO and SeMoV in vitro NaSeVO 0.625~20 mg/L and SeMoV 0.313~10 mg/L could significantly inhibite proliferation of K562 cells in vitro in a time and dose-dependent manner. The IC50 values were 14.41 (4.45-46.60) and 3.45 (2.29-5.22) mg/L after treated with NaSeVO 0.625~20 mg/L for 48 h and 72 h, respectively. The IC50 values were 14.41 (4.45~ 46.60) and 3.45 (2.29~5.22) mg/L after treated with SeMoV 0.313~10 mg/L for 48 h and 72 h, respectively. 2. Anti- tumor activity in vivo The anti-tumor effect of NaSeVO and SeMoV in vivo was evaluated by the inhibition rate of tumor mass. Results showed that i.p. NaSeVO 5 and 10 mg/kg had a significant anti-tumor effect on the growth of S180 with inhibition rate 26.8 % and 58.4 %, and 31.3 % and 47.4% on H22, respectively. Inhibition rate of SeMoV was 31.3 %、47.4% and 38.19%、39.17%, respectively. 3. Effect of NaSeVO and SeMoV on cell cycle distribution of K562 Result showed that the proportion of G0/G1 phase was increased at low concentration, whereas the proportion of G2/M was decreased. the proportion of G0/G1 phase was decreased and the proportion of G2/M was increased at high concentration after treatment with NaSeVO 0.625~20 mg/L for 24, 48 h. It also caused cells gathered in S phase. There are significantly sub-G occurred at high concentration, sub-G% were 3.3 and 3.5, respectively. The proportion of G0/G1 phase was increased and S was decreased at low concentration after treatment with SeMoV 0.313~10 mg/L for 24 h. the proportion of; whereas the proportion of S increased and G0/G1 was decreased at high concentration.
After treatment for 48h, the proportion of S increased and G0/G1 was decreased at high concentration. There are significantly sub-G occurred at 5mg/L(24h) , 1.25, 2.5 mg/L(48h)4. Morphological features of apoptosis Typical apoptosis character was present in the K562 cells treated with NaSeVO and SeMoV for 24h. Nuclear condensation, chromosome fragmentation and apoptosis bodies were observed by inverted microscope. The electron microscopic observation also revealed typical apoptotic features, including shrinkage of cellular and nuclear membranes, condensed heterochromatin around the nuclear periphery, and cytoplasmic vacuolation in the K562 cells treated with NaSeVO and SeMoV for 24 h. 5. Eexpression of Bax and Bcl-2 Buffy precipitate appearanced in endochylema or cell membrane was regarded as positive expression. The study of immunocytochemistry shows that the expression of bcl-2 is significantly inhibited by NaSeVO 10 mg/L and SeMoV 5 mg/L and bax increased. 6. Effect of NaSeVO and SeMoV on intracellular Ca2+, Mg2+ and ROS concentration, pH value and MMP The fluorescence intensity of intracellular Ca2+ and Mg2+ was greatly increased after treatment with NaSeVO and SeMoV as compared with control group. Similar to the change of intracellular Ca2+ and Mg2+, fluorescence intensity of intracellular ROS also increased. However, treatment with NaSeVO and SeMoV markedly lowered the fluorescence intensity of intracellular pH value and mitochondrial membrane potential. 7. Effect of NaSeVO and SeMoV on content of cytochrome C, IκBαand NF-κB Results of western-blotting assay showed that the intracellular content of cytochrome C and IκBαmarkedly increased and NF-κB decreased when treatment with NaSeVO and SeMoV for 24h.
Taken together, the above results suggest:
1. NaSeVO and SeMoV 5, 10 mg/kg has anti-tumor activity in vivo. 2. NaSeVO and SeMoV could inhibit the proliferation of K562 in vitro and arrest cell cycle. 3. The occurrence of sub-G and change of K562 morphology indicated that the mechanism of NaSeVO and SeMoV is attributed partially to apoptosis induced through the elevation of intracellular Ca2+, and Mg2+ concentration, and the reduction of pH value and mitochondrial membrane potential 4. NaSeVO and SeMoV could inhance the expression of bax and inhibit the expression of bcl-2. They also increased intracellular content of cytochrome C and IκBαand decreased NF-κB . In summary, the above results suggest: NaSeVO and SeMoV have anti-tumor activity in vivo and in vitro, the mechanism of which may relat to apoptosis.

URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221929
Collection生命科学学院
Recommended Citation
GB/T 7714
杨军英. NaSeVO 和 SeMoV 的抗肿瘤作用及机理探讨[D]. 兰州. 兰州大学,2006.
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