發(fā)布時(shí)間：2018-05-24 05:24

  本文選題：曲古抑素A + 順鉑　； 參考：《山東大學(xué)》2015年博士論文

【摘要】：表觀遺傳學(xué)是遺傳學(xué)分支學(xué)科,指在不改變DNA序列的前提下,基因或者蛋白表達(dá)發(fā)生變化,并可以在細(xì)胞的生長(zhǎng)和傳代過程中穩(wěn)定傳遞,主要包括組蛋白的共價(jià)修飾,DNA甲基化,基因沉默,染色質(zhì)重塑,以及非編碼RNA編輯等機(jī)制。表觀遺傳學(xué)與腫瘤的發(fā)生和轉(zhuǎn)移有密切聯(lián)系,腫瘤中存在表觀遺傳方式的異常。表觀遺傳學(xué)通過影響腫瘤抑制基因及其表達(dá),促進(jìn)腫瘤發(fā)生。組蛋白組成了核小體,它可以被多種化合物所修飾,如磷酸化、乙�；图谆�,稱為共價(jià)修飾,組蛋白的這些共價(jià)修飾作用主要發(fā)生在其N2末端,其中最為重要的是組蛋白乙酰化,組蛋白乙�；瘯�(huì)對(duì)腫瘤細(xì)胞的染色體結(jié)構(gòu)以及基因的表達(dá)產(chǎn)生影響,從而使腫瘤的治療更加樂觀。組蛋白乙�；腿ヒ阴；诩�(xì)胞信號(hào)轉(zhuǎn)導(dǎo)、分裂、凋亡、細(xì)胞內(nèi)的DNA修復(fù)、以及及染色體組裝等方面起著重要作用。各種抗腫瘤藥物有著不同的作用特點(diǎn),不同類型的腫瘤使用不同的抗腫瘤藥物,其作用機(jī)制各不相同,但多種針對(duì)實(shí)體瘤或血液系統(tǒng)腫瘤的抗腫瘤藥物均可與組蛋白去乙酰化酶抑制劑聯(lián)合,起到協(xié)同作用,研究發(fā)現(xiàn),抗腫瘤藥物與組蛋白去乙�；敢种苿┞�(lián)合后,比單藥使用抗腫瘤藥物組生存率高,對(duì)腫瘤細(xì)胞殺傷作用顯著,并且其療效有統(tǒng)計(jì)學(xué)差異。組蛋白去乙�；敢种苿┰趷盒阅[瘤的治療作用已受到廣泛認(rèn)可。組蛋白去乙�；敢种苿┠壳俺３Ｓ脕砼cDNA甲基轉(zhuǎn)移酶抑制劑、拓?fù)洚悩?gòu)酶抑制劑、激素、化療藥物如紫杉醇及鉑類等、酪氨酸激酶受體阻滯劑等藥物聯(lián)合使用。目前在肺癌的研究中,組蛋白去乙酰化酶抑制劑聯(lián)合鉑類藥物的報(bào)道較少,為研究組蛋白去乙�；敢种苿┡c順鉑聯(lián)合用藥,對(duì)肺癌細(xì)胞的影響,并對(duì)其作用機(jī)制進(jìn)一步探索,設(shè)計(jì)了本次的實(shí)驗(yàn)。目的：研究曲古抑菌素A (trichostatin A,TSA)聯(lián)合順鉑(cisplatin)對(duì)肺腺癌細(xì)胞株A549凋亡的影響,以及藥物處理后A549細(xì)胞中cFLIP、caspase-8蛋白的表達(dá)情況。方法：1、使用PRMI1640培養(yǎng)液培養(yǎng)肺腺癌A549細(xì)胞株,將細(xì)胞分為四組：1)、對(duì)照組2)、TSA250nmol/L處理組3)、Cisplatin 10μg/mL處理組4)、TSA250nmol/L+ Cisplatin 10μg/m L聯(lián)合處理組,藥物處理24h后,使用光學(xué)顯微鏡觀察各個(gè)組細(xì)胞形態(tài)學(xué)的變化。2、使用四甲基偶氮唑藍(lán)(MTT)比色法測(cè)定細(xì)胞的抑制率,將細(xì)胞分為10組：1)、空白對(duì)照組2)、TSA 250nmol/L處理組3) Cisplatin 5.3μg/mL處理組4)TSA250nmol/L+ Cisplatin 5.3μg/mL處理組5) Cisplatin 9μg/m處理組6)TSA250nmol/L+Cisplatin 9μg/mL處理組7) Cisplatin 13μg/mL處理組8)TSA250nmol/L+ Cisplatin 13μg/mL處理組9) Cisplatin 20μg/mL處理組10)TSA250nmol/L+ Cisplatin 20μg/mL處理組。測(cè)定不同濃度順鉑處理細(xì)胞后,對(duì)細(xì)胞抑制率,以及不同濃度順鉑聯(lián)合TSA處理細(xì)胞后,對(duì)細(xì)胞的抑制率。3、按照1中的分組方法將細(xì)胞分為4組,使用Hoechst33258染色法顯示不同處理組細(xì)胞凋亡的特點(diǎn)。4、按照1中的分組方法將細(xì)胞分為四組,使用Western blot法檢測(cè)cFLIP、 Pro-caspase-8及Caspase-8蛋白表達(dá)的變化。實(shí)驗(yàn)數(shù)據(jù)使用SPSS 15軟件進(jìn)行χ2檢驗(yàn)、t檢驗(yàn),p0.05表示差異有統(tǒng)計(jì)學(xué)意義。結(jié)果：1、光學(xué)顯微鏡下觀察四個(gè)組藥物處理后細(xì)胞形態(tài)的變化①對(duì)照組：細(xì)胞排列規(guī)則,緊密貼壁,細(xì)胞邊界清晰,未看到懸浮死細(xì)胞。②TSA250nmol/L處理組：細(xì)胞變大,有些細(xì)胞呈并指狀,細(xì)胞間隙增大,可見少量懸浮死細(xì)胞。③Cisplatin 10μg/mL處理組：細(xì)胞比對(duì)照組變小、變圓,發(fā)生皺縮,可見較多死細(xì)胞。④TSA250nmol/L+ cisplatin 10μg/mL聯(lián)合處理組：與前三組相比,細(xì)胞密度明顯下降,細(xì)胞發(fā)生皺縮,可見大量死細(xì)胞懸浮。2、四甲基偶氮唑鹽(MTT法)檢測(cè)不同濃度順鉑對(duì)細(xì)胞的抑制作用,以及不同濃度順鉑聯(lián)合TSA對(duì)細(xì)胞的抑制作用使用不同濃度的順鉑處理細(xì)胞,發(fā)現(xiàn)順鉑對(duì)細(xì)胞的抑制作用與順鉑的濃度呈正相關(guān)。隨著順鉑濃度的增加,細(xì)胞凋亡率增加。當(dāng)聯(lián)合TSA后,各組對(duì)細(xì)胞的抑制率均升高。Cisplatin 5.3μg/mL、TSA250nmol/L+ Cisplatin 5.3μg/mL細(xì)胞抑制率分別為13.68%、26.42%。Cisplatin 9μg/mL、TSA250nmol/L+ Cisplatin 9μg/mL細(xì)胞抑制率分別為28.73%、46.72%。Cisplatin 13μg/mL、TSA250nmol/L+ Cisplatin 13μg/mL細(xì)胞抑制率分別為44.58%、49.65%。Cisplatin 20μg/mL、TSA250nmol/L+ Cisplatin 20μg/mL細(xì)胞抑制率分別為51.12%、61.92%。順鉑與TSA聯(lián)合對(duì)細(xì)胞的抑制明顯高于單藥組(p0.05)。3、Hoechst33258染色法觀察各組使用藥物處理后細(xì)胞凋亡的特點(diǎn)各個(gè)組藥物處理24h后,使用Hoeschst33258染色法觀察細(xì)胞的凋亡特點(diǎn),相比于對(duì)照組,TSA及Cisplatin藥物單藥處理組凋亡細(xì)胞增多,出現(xiàn)染色質(zhì)凝集,核染色熒光增強(qiáng),聯(lián)合用藥上述現(xiàn)象更加明顯,熒光度增強(qiáng),并可以觀察到核裂解及凋亡小體。4、TSA聯(lián)合Cisplatin對(duì)cFLIP蛋白表達(dá)量的影響使用TSA 250nmol/L聯(lián)合Cisplatin 10μg/mL處理細(xì)胞24h后,單藥處理組cFLIP蛋白表達(dá)量減少,相比對(duì)照組及單藥處理組,聯(lián)合用藥組cFLIP蛋白表達(dá)明顯減少。測(cè)定各個(gè)處理組蛋白灰度值,結(jié)果顯示灰度值差異有統(tǒng)計(jì)學(xué)意義。5、TSA聯(lián)合Cisplatin對(duì)caspase-8及其前體表達(dá)的影響TSA 250nmol/L聯(lián)合Cisplatin 10μg/mL處理細(xì)胞24h后,單藥處理組Pro-caspase-8表達(dá)減少,caspase-8蛋白表達(dá)增多,與對(duì)照組與單藥處理組相比,聯(lián)合用藥組Pro-caspase-8明顯減少,caspase-8蛋白明顯增多,測(cè)定四個(gè)處理組蛋白灰度值,結(jié)果顯示其蛋白表達(dá)差異有統(tǒng)計(jì)學(xué)意義。結(jié)論：TSA聯(lián)合順鉑能夠通過影響Caspase-相關(guān)信號(hào)通路增加A549細(xì)胞株的凋亡,TSA和順鉑聯(lián)合具有協(xié)同作用。
[Abstract]:Epigenetics, a branch of genetics, refers to changes in gene or protein expression without changing the DNA sequence, and can be transferred steadily during cell growth and generation, mainly including covalent modification of histone, DNA methylation, gene silencing, chromatin remodeling, and non coded RNA editing. Epigenetic inheritance. It is closely related to the occurrence and metastasis of tumor. Epigenetics is abnormal in the tumor. Epigenetics promotes the occurrence of tumor by affecting the tumor suppressor gene and its expression. The histone is composed of nucleosomes, which can be modified by a variety of compounds, such as phosphorylation, acetylation and methylation, called covalent modification, histone These covalent modifications mainly occur at the end of the N2, the most important of which is histone acetylation. Histone acetylation will affect the chromosome structure and gene expression of tumor cells, thus making the cancer treatment more optimistic. Histone acetylation and deacetylation in cell signal transduction, division, apoptosis, cells. DNA repair, and chromosomal assembly play an important role. Various antitumor drugs have different characteristics, different types of tumor use different antitumor drugs, their mechanisms are different, but a variety of antitumor drugs against solid tumors or blood system tumors can be inhibited with histone deacetylase. As a synergistic effect, the study found that the combination of antitumor drugs and histone deacetylase inhibitors has a higher survival rate than the single drug use antitumor drug group and has a significant effect on tumor cells, and its efficacy is statistically different. Histone deacetylase inhibitors have been widely recognized in the treatment of malignant tumors. Histone deacetylase inhibitors are currently used in combination with DNA methyltransferase inhibitors, topoisomerase inhibitors, hormones, chemotherapeutic drugs such as Taxol and platinum, tyrosine kinase receptor blockers and other drugs. In the present study of lung cancer, histone deacetylase inhibitors combined with platinum drugs are reported to be more effective. For the purpose of studying the effect of trichostatin A (TSA) combined with cisplatin (cisplatin) on the apoptosis of lung adenocarcinoma cell line A549, and the A549 fine after treatment, the effect of the combination of protein deacetylase inhibitor and cisplatin on lung cancer cells was further explored. The expression of cFLIP and caspase-8 protein in the cell. Methods: 1, the A549 cell lines of lung adenocarcinoma were cultured with PRMI1640 culture, and the cells were divided into four groups: 1), 2 in the control group, 3 in the TSA250nmol/L treatment group, 4 in the Cisplatin 10 g/mL treatment group, and TSA250nmol/L+ Cisplatin 10 micron g/m L combined treatment group. After the drug processing 24h, the optical microscope observation of each group was used. The cell morphology changes of the group.2, using the four methyl azazolium blue (MTT) colorimetric assay to determine the cell inhibition rate, the cells were divided into 10 groups: 1), the blank control group 2), the TSA 250nmol/L treatment group 3) Cisplatin 5.3 mu g/mL treatment group 4) TSA250nmol/L+ Cisplatin 5.3 micron rational group 5) Cisplatin 9 mu g/m processing group 6) TSA250nmol/L+Cisplatin 9 Mu L treatment group 7) Cisplatin 13 mu g/mL treatment group 8) TSA250nmol/L+ Cisplatin 13 g/mL treatment group 9) Cisplatin 20 g/mL treatment group 10) TSA250nmol/L+ Cisplatin 20 mu g/mL treatment group. After the determination of cisplatin treated cells with different concentrations, the inhibitory rate of cisplatin, and the inhibition rate of cisplatin with TSA treated cells, 1. In the group, the cells were divided into 4 groups. Hoechst33258 staining was used to show the characteristics of cell apoptosis in different treatment groups.4. The cells were divided into four groups according to the group method of 1. The changes of cFLIP, Pro-caspase-8 and Caspase-8 protein expression were detected by Western blot method. The experiment number was tested by SPSS 15 software for chi 2 test, t test, P0.05. The difference was statistically significant. Results: 1, the morphological changes of the four groups were observed under the optical microscope (1) the control group: the cells were arranged regularly, the cells were closely adhered, the cell boundaries were clear, and the dead cells were not seen. TSA250nmol/L treatment group: the cells became larger, some cells showed and finger like, the space gap was enlarged, and a small amount could be seen. Suspension of dead cells. (3) Cisplatin 10 mu g/mL treatment group: cells were smaller, round, crinkle and more dead cells than the control group. (4) TSA250nmol/L+ cisplatin 10 mu g/mL combined treatment group: compared with the first three groups, cell density decreased significantly, cell shrinkage, large number of dead cells suspended.2, four methyl azazolium salt (MTT method) detection The inhibitory effects of cisplatin on cells and the inhibition of cisplatin with different concentrations of cisplatin combined with TSA on cells were treated with cisplatin at different concentrations. It was found that the inhibition of cisplatin to cisplatin was positively correlated with the concentration of cisplatin. With the increase of cisplatin concentration, the rate of cell apoptosis increased. When combined with TSA, the inhibition of cells was inhibited. The rate of.Cisplatin 5.3 mu g/mL, TSA250nmol/L+ Cisplatin 5.3 g/mL cell inhibition rate were 13.68%, 26.42%.Cisplatin 9 g/mL, TSA250nmol/L+ Cisplatin 9 micron g/mL cell inhibition rate was 28.73%, 46.72%.Cisplatin 13 micron g/mL, 13 micron cell inhibition rate was 44.58%, 20 micron, respectively. The inhibitory rate of TSA250nmol/L+ Cisplatin 20 mu g/mL cells was 51.12% respectively. The inhibition of 61.92%. cisplatin and TSA was significantly higher than that of single drug group (P0.05).3. Hoechst33258 staining method observed the characteristics of cell apoptosis after the use of drug treatment in each group. After the treatment of 24h, the apoptotic characteristics of the cells were observed by Hoeschst33258 staining. Compared with the control group, the number of apoptotic cells in the TSA and Cisplatin drug treatment group increased, the chromatin agglutination and the nuclear staining were enhanced. The above phenomenon was more obvious, the fluorophore was enhanced, and the nuclear fragmentation and the apoptotic body.4 could be observed. The effect of TSA combined with Cisplatin on the expression of cFLIP protein was combined with TSA 250nmol/L combined Cisplatin 1. After 0 mu g/mL treatment of 24h, the expression of cFLIP protein in the single drug treatment group decreased. Compared with the control group and the single drug treatment group, the expression of cFLIP protein in the combination group decreased obviously. The gray value of each processing group was measured, the results showed that the difference of gray value was statistically significant.5, TSA combined with Cisplatin on the expression of Caspase-8 and its precursors in TSA 250nmo. After l/L combined with Cisplatin 10 mu g/mL to treat 24h, the expression of Pro-caspase-8 in the single drug treatment group decreased and the expression of caspase-8 protein increased. Compared with the control group, the Pro-caspase-8 of the combined drug group decreased obviously, the caspase-8 protein increased obviously, and the gray value of the four treated group proteins was measured. The results showed that the protein expression was different. Conclusion: TSA combined with cisplatin can increase the apoptosis of A549 cell line by affecting Caspase- related signaling pathway. TSA and cisplatin have synergistic effect.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R734.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 伍俊;胡成平;;曲古抑素A下調(diào)Bcl-2水平經(jīng)線粒體途徑誘導(dǎo)耐順鉑人肺腺癌細(xì)胞凋亡[J];中國(guó)肺癌雜志;2009年11期

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本文編號(hào)：1927841