本文選題：宮頸癌 + SAHA��； 參考：《深圳大學》2017年碩士論文

【摘要】：宮頸癌(cervical cancer)是女性宮頸上皮細胞癌變而形成的腫瘤,是全球女性最常見的惡性腫瘤之一,發(fā)病率位居惡性腫瘤第二位。同時,宮頸癌的發(fā)病率在中國居于第一的位置,死亡率位居第二。有研究資料顯示,宮頸癌的發(fā)病率正在以每年2%-3%的速度迅速增長。發(fā)達國家宮頸癌的防治經(jīng)驗表明,通過對易發(fā)人群的密切篩查和早期預防,宮頸癌的發(fā)病率可以降低70%-90%。因此,研究宮頸癌的發(fā)病的分子機制,篩查和防治宮頸癌的發(fā)生,是世界宮頸癌研究和防治中最為艱巨和緊迫的課題。目前宮頸癌的臨床治療方法主要以手術(shù)治療輔以化學治療療和放射治療及分子靶向治療,雖然近幾十年來宮頸癌的臨床治療取得了長足進步,早期患者采用手術(shù)或放療,能夠取得較好的療效,但對于中晚期宮頸癌患者,手術(shù)操作難度較大。并且,傳統(tǒng)的化療藥物在殺傷腫瘤細胞的同時,也殺傷大量的正常細胞,使患者產(chǎn)生較嚴重的不良反應。同時,隨著化療的繼續(xù),化療藥物的耐藥性問題也常使化療效果難以達到預期。因此在宮頸癌的傳統(tǒng)治療模式中,發(fā)展更有效的治療藥物與方法成為新的研究課題。本課題以人宮頸癌HeLa細胞為研究對象,研究組蛋白去乙�；敢种苿㏒AHA聯(lián)合紫杉醇對宮頸癌He La細胞的體外殺傷效果。MTT法檢測宮頸癌HeLa細胞增殖情況,分別用SAHA、紫杉醇、SAHA+紫衫醇處理HeLa細胞24h、48h后,發(fā)現(xiàn)相較于SAHA(53.62±3.66%、34.74±3.03%)和紫杉醇(74.07±5.32%、70.88±3.09%)單獨使用組,SAHA與紫衫醇聯(lián)合使用(45.73±4.02%、22.98±3.86%)組的HeLa細胞存活率更低,能夠顯著抑制He La細胞的增殖。采用SPSS16.0計算SAHA增敏前后紫杉醇對He La細胞IC50的影響,結(jié)果顯示SAHA增敏后,紫杉醇對He La細胞24h的半數(shù)致死劑量由(18.023±1.256)nM降低到(8.887±0.949)nM,48h的半數(shù)致死劑量由(12.119±1.083)n M降低到(3.994±0.601)nM,SAHA能夠顯著降低紫杉醇對HeLa細胞的IC50(P0.01);利用流式細胞術(shù)檢測He La細胞的凋亡率,結(jié)果顯示SAHA組、紫杉醇組及SAHA與紫杉醇聯(lián)合用藥組的HeLa細胞的凋亡率分別為(6.44±0.86)%,(7.23±1.49)%,(16.22±3.38)%,聯(lián)合用藥組HeLa細胞的凋亡率分別高于紫杉醇組、SAHA組(P0.05,P0.05),SAHA與紫杉醇聯(lián)合可以顯著增加HeLa細胞的凋亡率,提高誘導HeLa細胞凋亡的能力;利用流式細胞術(shù)檢測HeLa細胞周期,發(fā)現(xiàn)單獨SAHA處理24h的HeLa細胞主要處于G0/G1期(90.07±1.39)%,S期(3.88±1.47)%,單獨紫杉醇的HeLa細胞主要處于G0/G1期(33.48±6.64)%,S期(40.77±4.43)%,SAHA聯(lián)合紫杉醇的HeLa細胞主要處于G0/G1期(84.22±2.07)%,S期(11.67±1.28)%,提示SAHA與紫杉醇聯(lián)合使用能夠抑制HeLa細胞有絲分裂過程中的DNA合成和復制;利用流式細胞儀檢測HeLa細胞中活性氧(ROS)水平,對照組、SAHA、紫杉醇和SAHA+紫杉醇組處理24h后的He La細胞的ROS水平分別為(1.65±0.84)%、(35.19±4.23)%、(27.26±3.74)%和(44.04±10.77)%,SAHA+紫杉醇組中ROS水平明顯升高,且相較于紫杉醇組具有統(tǒng)計學意義(P0.05);免疫熒光法標記HeLa細胞微管和DNA,發(fā)現(xiàn)SAHA和紫杉醇聯(lián)合處理24h后的HeLa細胞的微管呈短小纖細,并向細胞核聚集;RT-PCR檢測P27抑癌基因mRNA的表達情況,發(fā)現(xiàn)紫杉醇組、SAHA組以及SAHA和紫杉醇聯(lián)合用藥組的HeLa細胞中抑癌基因p27基因的mRNA相對表達量分別為0.978±0.117,5.845±0.548和10.601±0.673,聯(lián)合用藥組相對表達量均高于紫杉醇組、SAHA組,具有統(tǒng)計學意義(P0.001,P0.001);Western blot檢測凋亡相關蛋白(caspase-3和caspase-9)和乙�；M蛋白(Ac-H4)的表達情況,發(fā)現(xiàn)SAHA+紫杉醇能夠激活caspase-3和caspase-9蛋白,增強組蛋白H4的乙酰化水平。綜上所述,在體外培養(yǎng)條件下,SAHA與紫杉醇聯(lián)合作用于宮頸癌HeLa細胞時,通過激活caspase蛋白途徑、提高細胞內(nèi)活性氧ROS水平、上調(diào)p27抑癌基因的表達、增強組蛋白H4的乙�；胶陀绊懳⒔z微管的結(jié)構(gòu)和功能,從而誘導細胞凋亡,抑制細胞增殖,阻滯細胞周期,最終在增強抗腫瘤的能力的同時減輕化療藥物的副作用。
[Abstract]:Cervical cancer (cervical cancer) is a tumor formed by canceration of cervical epithelial cells in women. It is one of the most common malignant tumors in women all over the world. The incidence of cancer is the second most malignant tumor. At the same time, the incidence of cervical cancer is the first in China and the mortality rate ranks second. The rate of 2%-3% is increasing rapidly in the year. The experience of prevention and control of cervical cancer in developed countries shows that the incidence of cervical cancer can be reduced by close screening and early prevention. The molecular mechanism of the study of cervical cancer, screening and preventing the occurrence of cervical cancer are the most arduous in the research and prevention of cervical cancer in the world. At present, the clinical treatment of cervical cancer is mainly based on surgical treatment, chemotherapy, radiotherapy and molecular targeting therapy. Although the clinical treatment of cervical cancer has made great progress in recent decades, early patients with surgery or radiotherapy can achieve better curative effect, but for patients with middle and late cervical cancer, hand It is difficult to operate, and the traditional chemotherapeutic drugs kill the tumor cells and kill a large number of normal cells, so that the patients produce more serious adverse reactions. At the same time, with the continuation of chemotherapy, the drug resistance of chemotherapeutic drugs often makes the effect of chemotherapy difficult to achieve expectations. More effective therapeutic drugs and methods have become a new research topic. In this study, human cervical cancer HeLa cells were used to study the killing effect of histone deacetylase inhibitor SAHA combined with taxol on cervical cancer He La cells in vitro.MTT assay for the proliferation of cervical cancer HeLa cells, respectively, SAHA, paclitaxel, and SAHA+ After 24h and 48h, HeLa cells were found to be compared to SAHA (53.62 + 3.66%, 34.74 + 3.03%) and paclitaxel (74.07 + 5.32%, 70.88 + 3.09%) alone. The survival rate of HeLa cells in the group of SAHA and the combined use of (45.73 + 4.02%, 22.98 + 3.86%) group was lower, and the proliferation of He La cells could be suppressed significantly. SPSS16.0 calculation of paclitaxel before and after SAHA sensitization to SAHA was used. The effect of IC50 in He La cells showed that after SAHA sensitization, the median lethal dose of paclitaxel to He La cell 24h decreased from (18.023 + 1.256) nM to (8.887 + 0.949) nM, and the median lethal dose of 48h decreased from (12.119 + 1.083) n M to (3.994 + 0.601). The apoptosis rate of He La cells was measured. The results showed that the apoptosis rate of HeLa cells in group SAHA, paclitaxel group and SAHA and paclitaxel group was (6.44 + 0.86)%, (7.23 + 1.49)%, (16.22 + 3.38)%. The apoptosis rate of HeLa cells in combination group was higher than that of paclitaxel group, SAHA group (P0.05, P0.05), SAHA and paclitaxel could significantly increase HeLa. The apoptotic rate of cells increased the ability to induce apoptosis of HeLa cells, and HeLa cell cycle was detected by flow cytometry. The HeLa cells treated with single SAHA for 24h were mainly in G0/G1 phase (90.07 + 1.39)%, S phase (3.88 + 1.47)%, and HeLa cells of taxol alone in G0/G1 (33.48 + 6.64)%, S (40.77. 4.43)%, SAHA with paclitaxel HeL A cells were mainly in phase G0/G1 (84.22 + 2.07)% and S phase (11.67 + 1.28)%. The combination of SAHA and taxol could inhibit the synthesis and replication of DNA during the mitosis of HeLa cells, and the level of reactive oxygen (ROS) in HeLa cells was detected by flow cytometry. The control group, SAHA, paclitaxel and SAHA+ paclitaxel group treated 24h He cells The level of (1.65 + 0.84)%, (35.19 + 4.23)%, (27.26 + 3.74)% and (44.04 + 10.77)%, ROS level in SAHA+ paclitaxel group increased significantly, and compared with paclitaxel group (P0.05). The immunofluorescence method labeled HeLa cell microtubules and DNA, and the microtubules of HeLa cells after SAHA and paclitaxel combined with 24h were small and thin. The expression of P27 suppressor gene mRNA was detected by RT-PCR. The relative expression of the mRNA relative expression of the tumor suppressor gene p27 gene in the paclitaxel group, the SAHA group and the SAHA and paclitaxel group HeLa cells was 0.978 + 0.117,5.845 + 0.548 and 10.601 + 0.673 respectively. The relative expression of the combined drug group was higher than that of the paclitaxel group, the SAHA group, It was statistically significant (P0.001, P0.001); Western blot detected the expression of apoptosis related proteins (caspase-3 and caspase-9) and acetylation histone (Ac-H4). It was found that SAHA+ paclitaxel could activate caspase-3 and caspase-9 proteins and enhance the level of acetylation of histone H4. Under the conditions of culture, SAHA and taxol were combined in vitro. When used for cervical cancer HeLa cells, by activating the caspase protein pathway, increasing the intracellular reactive oxygen ROS level, up regulation of the expression of p27 suppressor gene, enhancing the level of acetylation of histone H4 and influencing the structure and function of microtubule microtubule, inducing cell apoptosis, inhibiting cell proliferation, blocking cell cycle, and finally enhancing the ability of anti-tumor. It also alleviated the side effects of chemotherapeutic drugs.
【學位授予單位】：深圳大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.33

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