论文标题:硝酸甘油代谢对肿瘤生长繁殖调控研究
The Studies on Regulating Effects of Glyceryl Trinitrate Metabolism on Tumor Growth and Proliferation
论文作者 张阳东
论文导师 田亚平,论文学位 硕士,论文专业 临床检验诊断学
论文单位 中国人民解放军军医进修学院,点击次数 129,论文页数 63页File Size2106k
2004-05-01论文网 http://www.lw23.com/lunwen_2179317/ 临床医学;专业学位;临床能力;考评体系;应用
clinical medicine; medical graduate degrees (clinical);clinical competence; evaluation system; apply
一氧化氮(nitric oxide，NO)是一种无色、微溶于水、脂溶性较强的气体分子，在生物体内可以自由地通过生物膜，作为一种重要的生物活性分子，NO参与体内一系列生理和病理过程，相关研究显示NO具有抗肿瘤功效。硝酸甘油(glyceryl trinitrate，GTN)作为有机硝酸酯类扩血管药物已有百余年的应用史，近年的研究发现，GTN作为NO供体在体内可转化为NO，进而合成S-亚硝基硫醇(S-nitrosothiols，RSNO)或氧化为亚硝酸盐。利用GTN在体内代谢特性，本研究以细胞培养和动物实验模型，探索了GTN对肿瘤细胞生长繁殖的影响及其机制，结果显示其具有潜在的抗肿瘤功效。 1．GTN在细胞培养体系及动物体内经消化道吸收后代谢产生亚硝酸盐。将不同浓度的GTN加入1640培养液，放置于37℃ 5％CO_2孵箱中48h后加Griess试剂，于540 nm测亚硝酸盐。发现GTN浓度依赖性代谢产生亚硝酸盐。以实验兔为动物模型，应用电生理监测仪动态检测血压，血气分析仪检测高铁血红蛋白(Methemoglobin，MetHb)，全自动生化分析仪检测丙氨酸氨基转移酶(Alanine transferase，ALT)，比色法检测硝酸盐、亚硝酸盐、还原型谷胱甘肽(reduced glutathione，GSH)。结果发现GTN经消化道吸收后代谢产生亚硝酸盐，并导致红细胞GSH降低，此代谢过程具有潜硕士学位论文在抑制肿瘤细胞生长效能。Me tHb、ALT未见明显变化，说明GTN在代谢过程中未导致明显肝脏功能损伤.2.GTN可有效抑制肿瘤细胞的生长繁殖，并导致肿瘤细胞生长周期改变.取对数分裂期的人宫颈癌细胞株(human cervicalearcinoma ce一line，Hela)细胞，用0.125%的胰蛋白酶消化处理进行细胞计数、加入%孔板，调节细胞数为3 xl少/孔.分别加入不同浓度的GTN.将%孔板放置于37℃，5%C02孵箱中培养48h后，取上清液测亚硝酸盐，以四甲基偶氮吐蓝(3一(4，5一d imethylthiazol一2一yl)一2，5一dyphenyltetrazoliumbromide，MTT)方法测细胞的生长活性。发现高浓度GTN可导致细胞不贴壁，并引起细胞死亡。MTT检测结果显示，高浓度GTN可明显抑制肿瘤细胞的生长。同时利用对数分裂期的He la细胞，在培养体系中加入1.75X10一恤GTN，48h后消化细胞，70%冷乙醇4℃固定，澳化乙吮(Ethidium bromide，EB)染色，用流式细胞仪检测细胞周期。结果显示GTN可改变肿瘤细胞周期各时相的分布，表现为GZ~M期细胞所占比例增加，S期细胞所占比例下降。3.GTN代谢可导致肿瘤细胞内GSH被氧化，抑制肿瘤细胞Bcl一2墓因蛋白表达和诱导肿瘤细胞凋亡.GSH的检测参照文献报道的二硫双硝基苯甲酸(2一nitr。benzoic，DTNB)法并加以改进。在培养体系中加入1.75X10一‘M GTN，48h后消化细胞，调整细胞浓度为3X10‘，离心弃上清，加1.67%偏磷酸沉淀剂150川，混匀，超速离心，取上清60林l加入96孔板，每孔加0.3M Na:Hpo;液200林1，0.04%DTNB液25川，混匀，用酶标仪405nm波长比色.GTN可引起肿瘤细胞的硕士学位论文GSH明显下降(P<0.05)。 在培养体系中加入1.75X10一俪GTN，48h后消化细胞，调整细胞浓度为10‘，按试剂盒操作说明加细胞破膜剂，稳定剂，FITc标记的抗人Bcl一2抗体，用流式细胞仪检测荧光指数。结果显示，GTN作用后的肿瘤细胞荧光指数下降，GTN可抑制B。1一2基因蛋白的表达。在培养体系中加入1.7sxlo一‘M eTN，48h后消化细胞，用^nnexinV联合碘化丙咤(Propidium Iodide，Pl)染色细胞，用流式细胞仪检测细胞凋亡指数(APoPt。5 1 5 index，.AI)。肿瘤细胞在GTN的作用下，细胞的凋亡指数明显增高(杯0.05)，GTN可诱导细胞凋亡.4.动物实验证明GTN能够抑制小鼠移植性肿瘤的生长.将GTN灌胃处理后的5180肉瘤细胞接种到昆明小鼠腋窝皮下，用高低浓度的GTN灌胃后观察抑瘤率并检测小鼠血液的生化指标。结果显示GTN灌胃能明显抑制小鼠5 1 80实体瘤的生长，同时消耗GSH，使血糖浓度升高，尿素浓度下降。GTN对小鼠移植性肿瘤具有抑制作用，并提高荷瘤小鼠的生存质量.结论: 通过本系列研究表明，GTN作为N0供体，在生物体内代谢氧化还原性琉基，同时生成亚硝酸盐.此过程可通过抑制B。1一2基因的表达、诱导细胞凋亡、改变细胞周期等机制而抑制肿瘤细胞的生长繁殖，且动物实验证明其具有抑制5180实体瘤移植后生长的功效，具有潜在抗肿瘤功效.
Nitric oxide (NO) is a simple and small free radical gas which is soluble both in water and lipid, and it is freely diffusible in the environment of cells. NO react with superoxide anion radical to produce molecules with strong oxidizing activity, such as nitrogen dioxide and peroxy nitrites. These oxidants are more toxic than NO itself. In recent years many studies showed that NO could modulate proliferation of tumor cells and had potential anti-tumor effect. Glyceryl trinitrate (GTN) had been used in clinic therapy as a vasodilator for more than a century. Many studies had been carried out on the metabolic mechanism of GTN and the results showed that as an NO donor, GTN could be metabolized to NO and then yielded S-nitrosothiols (RSNO). According to the metabolic mechanism of GTN in vivo, the present study was designed to evaluate the inhibitory effects and mechanism of GTN on tumor cells proliferation. For this purpose, experiment animals and cultured tumor cells had been used in these serial studies and the results indicated that GTN had potential anti-tumor efficacy. 1. GTN administered through alimentary canal or added in cellculture media yield nitrite. Different concentration of GTN was added to RMPI 1640 medium in 96-well plate incubated at 37 C for 48 hours and the nitrite was measured by Griess regent. The results of OD values was read by Bio-Rad Model 3550 - UV Microplate Reader both in measurement wavelength 545nm and reference wavelength 595nm. As results showed that different concentration of nitrite were released from different concentration of GTN. The higher concentration of GTN was administered, the higher concentration of nitrite was detected. In the study of rabbits, blood pressures were measured by dynamic analyzer. Methemoglobin (MetHb) was measured by blood gas analyzer and Alanine transferase (ALT) was measured by HITACHI 7600 automatic analyzer. Nitrate, nitrite and GSH were measured with colorimetry method. As results blood pressure and GSH in red blood cells showed significant decreases, nitrites showed significant increases. MetHb and ALT had no significant difference before and after GTN administration. According to these results we can conclude that metabolic GTN had no injury effects on hemoglobin and liver. 2 GTN could inhibit the growth of tumor cells and modulate cell cycle of tumor cell. Hela cells in early logarithmic growth phase (3 x 104/well) were incubated with different concentrations of GTN and incubated at 37 C for 48 hours. Then Griess reagent was added to measure nitrite or 3 -(4,5-dimethylthiazole-2-yI) -2,5-diphenyl tetrazolium-bromide ( MTT) was added to measure proliferationactivity. The results indicated that high concentration of GTN could inhibit the ability of Hela cells to adhere to culture bottle. Results of MTT assay showed high concentration of GTN could inhibit the growth of Hela cells. At the same time Hela cells were treated with 1.75X10~4M GTN and incubated at 37掳C for 48 hours. After treatment, cells were fixed by cool alcohol and Cell cycle distribution was analyzed by flow cytometer analysis of Pi-stained cells. Cell cycles could be modulated by GTN which could prompte S stage into G2/M compared to the control.3 GTN deplete GSH to initiate down- expression of Bcl-2 and induce apoptosis in tumor cells. After cells were treated with 1.75X10-4M GTN and incubated at 37 C for 48 hours, 150 1 1.67% metaphosphonic acid was added to 3X106 Hela cells, centrifuged (10,000 g for 15 minutes) and collected 60 1 supernatants to 96-well plate, 200 1 0.3M Na2HPO4 and 25 1 0.04% 2-nitrobenzoic (DTNB) were added to each well. OD was read by Bio-Rad Model 3550 - UV Microplate Reader in measurement wavelength 405nm. The result of OD value showed that concentration of GSH in Hela cells decreased significantly compared to the control. GTN could deplete GSH in tumor cells (p<0.05).Cells were treated with 1.75X10-4M GTN and incubated at 37掳C for 48 hours. After treatment, cells were digested and washed 2 times with phosphate buffered saline (PBS), then dealt
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