發(fā)布時(shí)間：2018-05-19 02:31

  本文選題：紫草素 + 肺癌�。� 參考：《廣州中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：目的:非小細(xì)胞肺癌(Non-small-cell lung cancer,NSCLC),是所有肺癌病人中占比最高的類型,在全世界范圍內(nèi),其發(fā)病率和死亡率都較高,是最常見(jiàn)的惡性腫瘤。目前關(guān)于癌癥的治療,主要通過(guò)藥物誘導(dǎo)腫瘤細(xì)胞凋亡來(lái)發(fā)揮作用。然而,細(xì)胞衰老,一種能夠抑制細(xì)胞增殖的應(yīng)激反應(yīng),它在抗腫瘤的研究中備受關(guān)注。衰老在癌前病變的組織中普遍存在,腫瘤向惡性發(fā)展必然要擺脫衰老的束縛。然而,即使是惡性腫瘤,如果能激活抑癌基因發(fā)揮功能或是觸發(fā)衰老信號(hào),惡性腫瘤仍有可能走向衰老,衰老在腫瘤發(fā)展過(guò)程中起到了關(guān)鍵的屏障作用。在體內(nèi),衰老的腫瘤細(xì)胞可通過(guò)免疫細(xì)胞或其他途徑清除,因而有效的抑制了腫瘤的惡化。目前的常規(guī)化療也具有誘導(dǎo)細(xì)胞衰老的潛能,這可能是其具有抗腫瘤作用的部分原因。盡管衰老療法在動(dòng)物模型中取得了令人滿意的效果,但是要將其轉(zhuǎn)化為臨床抗腫瘤手段仍然是一個(gè)挑戰(zhàn)。中藥紫草已經(jīng)在中國(guó)被沿用了上千年。紫草素(Shikonin,SHK),從紫草中分離提取分離的活性成分,在臨床應(yīng)用中具有解毒,抗炎,抗腫瘤和抗病毒的作用。研究表明,紫草素具有廣泛的藥理活性,能夠抑制多種腫瘤細(xì)胞的生長(zhǎng),包括結(jié)腸癌,乳腺癌,胃癌,肺癌等。據(jù)報(bào)道,紫草素可以誘導(dǎo)A549癌細(xì)胞凋亡,壞死和早衰。然而,紫草素誘導(dǎo)肺癌細(xì)胞衰老的具體機(jī)制仍尚不明確�；谏鲜鲅芯炕A(chǔ),本論文旨在研究紫草素對(duì)非小細(xì)胞肺癌的衰老影響及其作用機(jī)制,為紫草素新藥開發(fā)及臨床使用提供參考依據(jù)。方法:1.紫草素對(duì)非小細(xì)胞肺癌細(xì)胞生長(zhǎng)與增殖的抑制作用采用MTT的實(shí)驗(yàn)方法檢測(cè)紫草素作用于A549和H1299細(xì)胞24,48,72h后對(duì)細(xì)胞活力的影響,并計(jì)算得出每種細(xì)胞在不同時(shí)間點(diǎn)對(duì)應(yīng)的IC50值。采用EdU摻入法檢測(cè)紫草素作用72h后細(xì)胞的增殖情況。用流式細(xì)胞術(shù)檢測(cè)給藥后細(xì)胞周期阻滯于哪一周期,并結(jié)合western blot實(shí)驗(yàn)方法考察紫草素對(duì)細(xì)胞周期蛋白的作用。2.紫草素對(duì)非小細(xì)胞肺癌細(xì)胞衰老的影響采用β-半乳糖苷酶染色法檢測(cè)紫草素作用細(xì)胞72 h后細(xì)胞的衰老情況,細(xì)胞染色后于熒光倒置顯微鏡下拍照,并觀察衰老細(xì)胞和正常細(xì)胞的形態(tài)。細(xì)胞給藥結(jié)束后,DAPI染色處理,采用激光共聚焦掃描顯微鏡觀察衰老相關(guān)異染色質(zhì)聚集(SAHF)情況。3.紫草素誘導(dǎo)非小細(xì)胞肺癌細(xì)胞衰老的作用機(jī)制采用流式細(xì)胞術(shù)檢測(cè)給藥后細(xì)胞內(nèi)ROS的變化,以及給藥后western blot檢測(cè)DNA損傷相關(guān)蛋白p-H2A.X、Kdm2b/H3K36me2和p53/p21waf的表達(dá)情況。在加入抗氧化劑NAC預(yù)處理細(xì)胞后檢測(cè)ROS的表達(dá)水平,在ROS耗竭后考察細(xì)胞活力、細(xì)胞衰老、p53變化以及DNA損傷情況。4.紫草素誘導(dǎo)非小細(xì)胞肺癌衰老與自噬、凋亡的相關(guān)性采用流式細(xì)胞術(shù)結(jié)合western blot的方法考察A549和H1299給藥處理后細(xì)胞自噬的變化。采用AnnexinV-FITC/PI雙染色法檢測(cè)紫草素處理后,細(xì)胞的凋亡情況,同時(shí)采用western blot的方法檢測(cè)與細(xì)胞凋亡相關(guān)的蛋白表達(dá)情況。5.考察紫草素在體內(nèi)的抗腫瘤作用建立裸鼠移植瘤模型,將A549細(xì)胞接入裸鼠腋下,待腫瘤體積達(dá)到一定大小后灌胃給紫草素溶液。持續(xù)給藥4周,每隔三天稱量體重,測(cè)量腫瘤體積,實(shí)驗(yàn)結(jié)束后,稱量腫瘤重量,β-半乳糖苷酶染色和免疫組化檢測(cè)相關(guān)指標(biāo)。結(jié)果:1.紫草素使細(xì)胞生長(zhǎng)周期阻滯于G0/G1期而抑制肺癌細(xì)胞的生長(zhǎng)與增殖細(xì)胞活力檢測(cè)顯示紫草素在24、48、72h的時(shí)間點(diǎn)都能夠阻止肺癌細(xì)胞生長(zhǎng)與增殖,并成濃度依賴性。A549和H1299細(xì)胞經(jīng)紫草素處理72h后的IC50值分別為2.28 ±0.19 μ和0.64± 0.07 μM,EdU實(shí)驗(yàn)結(jié)果顯示紫草素可以明顯抑制A549和H1299細(xì)胞的增殖,而且細(xì)胞周期檢測(cè)顯示兩種細(xì)胞在藥物作用72 h后周期都阻滯于G0/G1期,細(xì)胞周期蛋白CyclinD1均受到抑制。2.紫草素能夠誘導(dǎo)非小細(xì)胞肺癌細(xì)胞的衰老紫草素作用肺癌細(xì)胞72 h后,SA-β-gal陽(yáng)性染色細(xì)胞數(shù)增多,表明其能夠誘導(dǎo)肺癌細(xì)胞發(fā)生衰老樣變化,并且細(xì)胞的衰老程度與給藥濃度成正比。與此同時(shí),也可以檢測(cè)到與衰老相關(guān)的另外一些標(biāo)志物:衰老相關(guān)異染色質(zhì)聚集(SAHF)出現(xiàn),細(xì)胞形態(tài)體積變大,變扁平。3.紫草素誘導(dǎo)細(xì)胞產(chǎn)生ROS,導(dǎo)致DNA損傷并通過(guò)下游信號(hào)通路促進(jìn)細(xì)胞衰老紫草素作用于A549和H1299細(xì)胞后能夠引起胞內(nèi)ROS的水平升高,進(jìn)而上調(diào)p-H2A.X、p53、p21waf蛋白的表達(dá)。同時(shí),Kdm2b受到抑制,H3K36me2表達(dá)升高�？寡趸瘎㎞AC能夠抑制ROS的產(chǎn)生,逆轉(zhuǎn)紫草素對(duì)細(xì)胞活力、衰老、DNA損傷和p53蛋白的影響。4.紫草素誘導(dǎo)的衰老細(xì)胞中能夠檢測(cè)到細(xì)胞凋亡流式細(xì)胞術(shù)結(jié)合western blot的實(shí)驗(yàn)結(jié)果均表明,紫草素處理三天后可以抑制細(xì)胞的自噬。Annexin V-FITC/PI雙染色法檢測(cè)給藥后衰老組細(xì)胞的陽(yáng)性染色比例升高,這表明衰老的細(xì)胞最終可能發(fā)生了凋亡。Western blot檢測(cè)了與凋亡相關(guān)的蛋白,結(jié)果顯示促凋亡蛋白Bax和非線粒體途徑凋亡蛋白Cleaved Caspase-3的表達(dá)升高,同時(shí),與Bax作用相反的凋亡抑制蛋白Bcl-2表達(dá)下調(diào)。5.紫草素誘導(dǎo)腫瘤細(xì)胞衰老而在體內(nèi)發(fā)揮抗腫瘤活性在建立裸鼠移植瘤的模型中,紫草素能夠抑制體內(nèi)腫瘤的生長(zhǎng)。在持續(xù)4周的灌胃給藥過(guò)程中,各給藥組小鼠體重?zé)o明顯波動(dòng),但與空白溶劑處理組相比,給藥組腫瘤的生長(zhǎng)明顯受到抑制。紫草素5 mg/kg和10 mg/kg給藥組的腫瘤抑制率分別達(dá)到28.57%、55.84%。隨后冰凍切片檢測(cè)β-半乳糖苷酶活性結(jié)果顯示,給藥組衰老細(xì)胞明顯增多,并成濃度依耐性。免疫組化結(jié)果顯示,給藥后DNA損傷蛋白p-H2A.X表達(dá)增多,同時(shí)p53也增多,相反的是自噬相關(guān)蛋白LC3-Ⅱ表達(dá)減少。結(jié)論:在體外實(shí)驗(yàn)中,紫草素能夠誘導(dǎo)非小細(xì)胞肺癌細(xì)胞產(chǎn)生ROS,導(dǎo)致DNA損傷,通過(guò)p53/p21waf信號(hào)通路促進(jìn)細(xì)胞衰老。在A549裸鼠移植瘤模型中,紫草素同樣能夠誘導(dǎo)腫瘤細(xì)胞衰老而達(dá)到與體外相同的抗腫瘤效果,這是以前所有關(guān)于紫草素抗腫瘤研究中所沒(méi)有報(bào)道的。紫草素,一種新型的ROS依賴性衰老激動(dòng)劑,可以作為肺癌治療一種潛在和有前景的候選藥物。
[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.
【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285

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