紫草素诱导非小细胞肺癌衰老及分子机制

发布时间：2018-05-19 02:31

本文选题：紫草素 + 肺癌　；参考：《广州中医药大学》2017年硕士论文

【摘要】：目的:非小细胞肺癌(Non-small-cell lung cancer,NSCLC),是所有肺癌病人中占比最高的类型,在全世界范围内,其发病率和死亡率都较高,是最常见的恶性肿瘤。目前关于癌症的治疗,主要通过药物诱导肿瘤细胞凋亡来发挥作用。然而,细胞衰老,一种能够抑制细胞增殖的应激反应,它在抗肿瘤的研究中备受关注。衰老在癌前病变的组织中普遍存在,肿瘤向恶性发展必然要摆脱衰老的束缚。然而,即使是恶性肿瘤,如果能激活抑癌基因发挥功能或是触发衰老信号,恶性肿瘤仍有可能走向衰老,衰老在肿瘤发展过程中起到了关键的屏障作用。在体内,衰老的肿瘤细胞可通过免疫细胞或其他途径清除,因而有效的抑制了肿瘤的恶化。目前的常规化疗也具有诱导细胞衰老的潜能,这可能是其具有抗肿瘤作用的部分原因。尽管衰老疗法在动物模型中取得了令人满意的效果,但是要将其转化为临床抗肿瘤手段仍然是一个挑战。中药紫草已经在中国被沿用了上千年。紫草素(Shikonin,SHK),从紫草中分离提取分离的活性成分,在临床应用中具有解毒,抗炎,抗肿瘤和抗病毒的作用。研究表明,紫草素具有广泛的药理活性,能够抑制多种肿瘤细胞的生长,包括结肠癌,乳腺癌,胃癌,肺癌等。据报道,紫草素可以诱导A549癌细胞凋亡,坏死和早衰。然而,紫草素诱导肺癌细胞衰老的具体机制仍尚不明确。基于上述研究基础,本论文旨在研究紫草素对非小细胞肺癌的衰老影响及其作用机制,为紫草素新药开发及临床使用提供参考依据。方法:1.紫草素对非小细胞肺癌细胞生长与增殖的抑制作用采用MTT的实验方法检测紫草素作用于A549和H1299细胞24,48,72h后对细胞活力的影响,并计算得出每种细胞在不同时间点对应的IC50值。采用EdU掺入法检测紫草素作用72h后细胞的增殖情况。用流式细胞术检测给药后细胞周期阻滞于哪一周期,并结合western blot实验方法考察紫草素对细胞周期蛋白的作用。2.紫草素对非小细胞肺癌细胞衰老的影响采用β-半乳糖苷酶染色法检测紫草素作用细胞72 h后细胞的衰老情况,细胞染色后于荧光倒置显微镜下拍照,并观察衰老细胞和正常细胞的形态。细胞给药结束后,DAPI染色处理,采用激光共聚焦扫描显微镜观察衰老相关异染色质聚集(SAHF)情况。3.紫草素诱导非小细胞肺癌细胞衰老的作用机制采用流式细胞术检测给药后细胞内ROS的变化,以及给药后western blot检测DNA损伤相关蛋白p-H2A.X、Kdm2b/H3K36me2和p53/p21waf的表达情况。在加入抗氧化剂NAC预处理细胞后检测ROS的表达水平,在ROS耗竭后考察细胞活力、细胞衰老、p53变化以及DNA损伤情况。4.紫草素诱导非小细胞肺癌衰老与自噬、凋亡的相关性采用流式细胞术结合western blot的方法考察A549和H1299给药处理后细胞自噬的变化。采用AnnexinV-FITC/PI双染色法检测紫草素处理后,细胞的凋亡情况,同时采用western blot的方法检测与细胞凋亡相关的蛋白表达情况。5.考察紫草素在体内的抗肿瘤作用建立裸鼠移植瘤模型,将A549细胞接入裸鼠腋下,待肿瘤体积达到一定大小后灌胃给紫草素溶液。持续给药4周,每隔三天称量体重,测量肿瘤体积,实验结束后,称量肿瘤重量,β-半乳糖苷酶染色和免疫组化检测相关指标。结果:1.紫草素使细胞生长周期阻滞于G0/G1期而抑制肺癌细胞的生长与增殖细胞活力检测显示紫草素在24、48、72h的时间点都能够阻止肺癌细胞生长与增殖,并成浓度依赖性。A549和H1299细胞经紫草素处理72h后的IC50值分别为2.28 ±0.19 μ和0.64± 0.07 μM,EdU实验结果显示紫草素可以明显抑制A549和H1299细胞的增殖,而且细胞周期检测显示两种细胞在药物作用72 h后周期都阻滞于G0/G1期,细胞周期蛋白CyclinD1均受到抑制。2.紫草素能够诱导非小细胞肺癌细胞的衰老紫草素作用肺癌细胞72 h后,SA-β-gal阳性染色细胞数增多,表明其能够诱导肺癌细胞发生衰老样变化,并且细胞的衰老程度与给药浓度成正比。与此同时,也可以检测到与衰老相关的另外一些标志物:衰老相关异染色质聚集(SAHF)出现,细胞形态体积变大,变扁平。3.紫草素诱导细胞产生ROS,导致DNA损伤并通过下游信号通路促进细胞衰老紫草素作用于A549和H1299细胞后能够引起胞内ROS的水平升高,进而上调p-H2A.X、p53、p21waf蛋白的表达。同时,Kdm2b受到抑制,H3K36me2表达升高。抗氧化剂NAC能够抑制ROS的产生,逆转紫草素对细胞活力、衰老、DNA损伤和p53蛋白的影响。4.紫草素诱导的衰老细胞中能够检测到细胞凋亡流式细胞术结合western blot的实验结果均表明,紫草素处理三天后可以抑制细胞的自噬。Annexin V-FITC/PI双染色法检测给药后衰老组细胞的阳性染色比例升高,这表明衰老的细胞最终可能发生了凋亡。Western blot检测了与凋亡相关的蛋白,结果显示促凋亡蛋白Bax和非线粒体途径凋亡蛋白Cleaved Caspase-3的表达升高,同时,与Bax作用相反的凋亡抑制蛋白Bcl-2表达下调。5.紫草素诱导肿瘤细胞衰老而在体内发挥抗肿瘤活性在建立裸鼠移植瘤的模型中,紫草素能够抑制体内肿瘤的生长。在持续4周的灌胃给药过程中,各给药组小鼠体重无明显波动,但与空白溶剂处理组相比,给药组肿瘤的生长明显受到抑制。紫草素5 mg/kg和10 mg/kg给药组的肿瘤抑制率分别达到28.57%、55.84%。随后冰冻切片检测β-半乳糖苷酶活性结果显示,给药组衰老细胞明显增多,并成浓度依耐性。免疫组化结果显示,给药后DNA损伤蛋白p-H2A.X表达增多,同时p53也增多,相反的是自噬相关蛋白LC3-Ⅱ表达减少。结论:在体外实验中,紫草素能够诱导非小细胞肺癌细胞产生ROS,导致DNA损伤,通过p53/p21waf信号通路促进细胞衰老。在A549裸鼠移植瘤模型中,紫草素同样能够诱导肿瘤细胞衰老而达到与体外相同的抗肿瘤效果,这是以前所有关于紫草素抗肿瘤研究中所没有报道的。紫草素,一种新型的ROS依赖性衰老激动剂,可以作为肺癌治疗一种潜在和有前景的候选药物。
[Abstract]:Objective: non small cell lung cancer (Non-small-cell lung cancer, NSCLC) is the highest proportion of all lung cancer patients. In the world, the incidence and mortality are high and the most common malignant tumor. At present, the treatment of cancer is mainly used to induce tumor cell apoptosis to play a role. However, cell aging, cell aging, The stress response that can inhibit cell proliferation has attracted much attention in the antitumor research. Senescence is common in precancerous tissue, and the malignant development of tumor is bound to get rid of the shackles of aging. However, even if it is malignant, it may still be possible to activate the tumor suppressor gene to play the work or trigger the aging signal. Aging, aging plays a key role in the development of cancer. In the body, aging tumor cells can be removed by immune cells or other pathways, thus effectively inhibiting the deterioration of the tumor. The current conventional chemotherapy also has the potential to induce cell senescence, which may be part of the reason for its anti-tumor effect. Although aging therapy has made a satisfactory effect in animal models, it is still a challenge to convert it into a clinical antitumor method. The Chinese herb has been used in China for thousands of years. Shikonin (SHK) has been used to separate and separate the active components from the herb, and it has detoxification, anti-inflammatory and anti swelling in clinical application. Tumor and antiviral action. Studies have shown that Zac has extensive pharmacological activity and can inhibit the growth of various tumor cells, including colon cancer, breast cancer, gastric cancer, lung cancer and so on. It is reported that A549 can induce apoptosis, necrosis and premature senility. However, the specific mechanism of induced senescence of lung cancer cells is still unclear. On the basis of these studies, the aim of this thesis is to study the effect and mechanism of shikonin on non small cell lung cancer and to provide reference for the development and clinical use of new medicine. Methods: 1. the inhibitory effect of 1. herb on the growth and proliferation of non small cell lung cancer cells was tested by MTT method to detect A549 and H129 The effects of 9 cell 24,48,72h on cell viability and the IC50 value of each cell at different time points were calculated. The proliferation of cells after 72h was detected by EdU incorporation. The cell cycle was blocked by flow cytometry and the cell cycle was blocked by the flow cytometry, and the Western blot experimental method was used to examine the cells of the cells. The effect of cyclin on the senescence of non small cell lung cancer cells by.2., using beta galactosidase staining to detect the aging of cells after 72 h of purple grass cells, the cells were photographed under fluorescence inverted microscope after staining, and observed the morphology of senescent cells and normal cells. After the end of the drug, the cells were stained with DAPI, Using laser confocal scanning microscope to observe the aging related heterochromatin aggregation (SAHF), the mechanism of.3. induced senescence of non small cell lung cancer cells was induced by flow cytometry, and the changes in intracellular ROS were detected by flow cytometry, and Western blot was used to detect DNA damage related proteins p-H2A.X, Kdm2b/H3K36me2 and p53/p21waf after Administration of Western blot. The expression level of ROS was detected after the addition of antioxidant NAC to the cells. After ROS depletion, cell viability, cell senescence, p53 changes and DNA damage were used to induce senescence and autophagy induced by.4. in non small cell lung cancer, and the correlation of apoptosis was investigated by flow cytometry combined with Western blot to investigate A549 and H1299 administration The changes in autophagy after treatment. The AnnexinV-FITC/PI double staining was used to detect the apoptosis of the cells and the expression of apoptosis related proteins by Western blot..5. was used to investigate the anti tumor effect of purple grass in the body, and the A549 cells were connected to the armpit of nude mice. After the volume of the tumor reached a certain size, it was administered to the herb solution for 4 weeks. The weight of the tumor was weighed every three days and the volume of the tumor was measured. After the experiment, the weight of the tumor, beta galactosidase staining and immunohistochemical staining were measured. Results: 1. the cell growth cycle was blocked at the stage of G0/G1 and the growth of lung cancer cells was inhibited. The activity of long and proliferating cells showed that the time point of 24,48,72h could prevent the growth and proliferation of lung cancer cells, and the IC50 values of concentration dependent.A549 and H1299 cells treated with 72h were 2.28 + 0.19 and 0.64 + 0.07 Mu respectively. The results of EdU test showed that the purple herb could obviously inhibit A549 and H1299 cells. Proliferation, and cell cycle detection showed that two cells were blocked in the period of G0/G1 after the drug action of 72 h, and cyclin CyclinD1 was inhibited to induce the senescence of non small cell lung cancer cells to induce the senescence of lung cancer cells of 72 h, and the number of SA- beta -gal positive chromating cells increased, indicating that it could induce lung cancer. At the same time, other markers related to senescence can be detected: aging related heterochromatin aggregation (SAHF), larger cell morphology, and flattened.3. lure inducing cells to produce ROS, resulting in DNA damage and through downstream signals. A549 and H1299 cells could increase the level of intracellular ROS and increase the expression of p-H2A.X, p53, p21WAF protein. Meanwhile, Kdm2b was inhibited and H3K36me2 expression increased. Antioxidant NAC could inhibit the production of ROS, reversing the effects of purple grass on cell vitality, senescence, DNA damage and p53 protein. The results of the apoptosis flow cytometry combined with Western blot in the senescent cells induced by Zac showed that the positive staining ratio of the cells in the senescence group increased after three days after the treatment of the autophagy and the autophagy was detected by the autophagy.Annexin V-FITC/PI double staining method, which indicated that the senescent cells might eventually hair. Apoptotic.Western blot was used to detect the apoptosis related proteins, and the results showed that the expression of apoptotic protein Bax and non mitochondrial pathway apoptotic protein Cleaved Caspase-3 increased, while the expression of apoptosis suppressor protein Bcl-2, which was opposite to Bax, downregulated the anti-tumor activity of.5. to induce tumor cells to induce tumor cells to be naked in the body. In the model of rat transplanted tumor, the growth of the tumor in the body was inhibited. The weight of the mice was not significantly fluctuated during the 4 weeks of gavage, but compared with the blank solvent treatment group, the growth of the tumor was significantly inhibited. The tumor inhibition rate of the 5 mg/kg and 10 mg /kg group was 28.57%, 55. respectively. 84%. then the results of the detection of beta galactosidase activity in the frozen section showed that the senescent cells in the administration group increased obviously and were concentration dependent. The results of immunohistochemistry showed that the expression of DNA damaged protein p-H2A.X increased after the administration, and the p53 increased, on the contrary, the expression of autophagy related protein LC3- II expression decreased. It is sufficient to induce ROS in non small cell lung cancer cells to cause DNA damage and promote cell senescence through the p53/p21waf signaling pathway. In the A549 nude mouse model, it can also induce tumor cell senescence to achieve the same antitumor effect as in vitro. This is not reported in previous studies on the Antitumor of purpura. ROS, a new type of aging dependent agonist, can be used as a potential and promising candidate for treatment of lung cancer.
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285

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