【摘要】：目的：上皮性卵巢癌（epithelial ovarian cancer, EOC）為最常見的卵巢惡性腫瘤，不易早期診斷，復(fù)發(fā)及死亡率高。卵巢癌目前標(biāo)準(zhǔn)治療方法主要為腫瘤細(xì)胞減滅術(shù)及以鉑類為基礎(chǔ)的化療，但約70-80%的患者最終會(huì)出現(xiàn)化療耐藥，引起腫瘤的復(fù)發(fā)及轉(zhuǎn)移，為卵巢癌治療失敗及患者死亡的重要原因，故其5年生存率一直徘徊在30%左右。因此，探討化療耐藥腫瘤的侵襲轉(zhuǎn)移機(jī)制，尋找有效的干預(yù)靶點(diǎn)，為EOC患者延長(zhǎng)生存期和改善預(yù)后的迫切需求。 近年來研究發(fā)現(xiàn)，上皮間質(zhì)轉(zhuǎn)化（epithelial-mesenchymal transition,EMT）不僅參與胚胎發(fā)育及創(chuàng)傷修復(fù)，在腫瘤的侵襲轉(zhuǎn)移過程中也發(fā)揮了重要作用。EMT是指特定的生理或病理情況下，上皮細(xì)胞丟失上皮表型的特性轉(zhuǎn)化為間質(zhì)表型細(xì)胞過程。發(fā)生EMT的細(xì)胞極性丟失，細(xì)胞間粘附力下降，運(yùn)動(dòng)能力增強(qiáng)，細(xì)胞形態(tài)延伸，抗凋亡能力增強(qiáng)，從而促進(jìn)腫瘤侵襲轉(zhuǎn)移。EMT與多種細(xì)胞因子、轉(zhuǎn)錄因子及信號(hào)通路相關(guān)。EMT過程伴隨著Notch、Wnt、Hedgehog等多種信號(hào)通路的活化；轉(zhuǎn)錄因子Snail和Slug等表達(dá)上調(diào)；上皮表型標(biāo)志物E-cadherin等表達(dá)下調(diào)，間質(zhì)表型標(biāo)志物vimentin等表達(dá)上調(diào)。此外，發(fā)生EMT的腫瘤細(xì)胞可獲得自我更新等干細(xì)胞樣特性，這可能與腫瘤細(xì)胞發(fā)生化療抵抗相關(guān)。 多年研究表明，Notch信號(hào)通路在多種實(shí)體腫瘤及血液系統(tǒng)腫瘤中均異常表達(dá)，且與腫瘤的發(fā)生發(fā)展密切相關(guān)。新近研究表明，Notch信號(hào)通路活化可誘導(dǎo)多種腫瘤細(xì)胞發(fā)生EMT，促進(jìn)腫瘤的轉(zhuǎn)移和復(fù)發(fā)。γ-分泌酶為激活Notch信號(hào)通路的核心環(huán)節(jié)，而成為腫瘤治療的研究熱點(diǎn)。本實(shí)驗(yàn)通過體外培養(yǎng)人卵巢漿液性囊腺癌細(xì)胞株SKOV3及其順鉑耐藥株SKOV3/DDP，檢測(cè)其Notch1mRNA表達(dá)量的差異，并進(jìn)一步研究γ分泌酶抑制劑DAPT作用于SKOV3/DDP細(xì)胞后，E-cadherin及vimentin的表達(dá)情況，以及其遷移及穿膜能力的改變。旨在從分子水平探討DAPT能否通過抑制Notch信號(hào)通路，從而抑制化療耐藥EOC的侵襲轉(zhuǎn)移能力，，為化療耐藥EOC的治療策略提供實(shí)驗(yàn)依據(jù)。 方法：用含10%胎牛血清的RPMI-1640培養(yǎng)基，在37℃，飽和濕度5%CO2的恒溫培養(yǎng)箱中常規(guī)培養(yǎng)人卵巢漿液性囊腺癌細(xì)胞株SKOV3、及其順鉑耐藥株SKOV3/DDP。取對(duì)數(shù)生長(zhǎng)期細(xì)胞進(jìn)行實(shí)驗(yàn)。 1倒置顯微鏡觀察SKOV3細(xì)胞及SKOV3/DDP細(xì)胞的形態(tài)并拍照。 2實(shí)時(shí)熒光定量PCR(PT-PC)檢測(cè)SKOV3細(xì)胞及SKOV3/DDP細(xì)胞Notch1mRNA表達(dá)水平。 3四甲基偶氮唑藍(lán)(MTT)法：對(duì)SKOV3/DDP細(xì)胞以不同濃度的DAPT(25，50，75μmol/L)作用不同時(shí)間(24h、48h、72h)，以加入DAPT的溶劑0.375%DMSO為對(duì)照組。MTT法檢測(cè)DAPT對(duì)細(xì)胞生長(zhǎng)的影響，篩選出藥物作用的最適濃度及時(shí)間。 4蛋白印跡法（Western blotting）：檢測(cè)SKOV3細(xì)胞及不同濃度的DAPT(0，25，50，75μmol/L)作用于SKOV3/DDP細(xì)胞48h后，上皮標(biāo)記物E-cadherin和間質(zhì)標(biāo)記物vimentin的表達(dá)水平。 5劃痕實(shí)驗(yàn)：常規(guī)培養(yǎng)SKOV3/DDP細(xì)胞使其融合形成單細(xì)胞層，無血清培養(yǎng)基同步化24h后，按實(shí)驗(yàn)分組加入不同干預(yù)（實(shí)驗(yàn)組：50μmol/LDAPT；對(duì)照組：0.25%DMSO），在培養(yǎng)板底部中央呈“一”字形劃痕，分別于給藥后0h、24h后倒置顯微鏡（40×）下觀察照相，計(jì)算24h內(nèi)劃痕愈合的面積評(píng)估細(xì)胞的遷移能力。 6Transwell實(shí)驗(yàn)：以50μmol/L DAPT作用SKOV3/DDP細(xì)胞為實(shí)驗(yàn)組，以加入0.25%DMSO為對(duì)照組，連續(xù)培養(yǎng)24小時(shí)后取出Transwell侵襲小室，用95%乙醇固定，經(jīng)結(jié)晶紫染色，在顯微鏡（200×）下拍照并計(jì)數(shù)移至微孔膜下層的細(xì)胞評(píng)估細(xì)胞的遷移和侵襲能力。 7應(yīng)用SPSS16.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析。 結(jié)果： 1SKOV3細(xì)胞與SKOV3/DDP細(xì)胞：倒置顯微鏡下觀察，SKOV3細(xì)胞呈橢圓形、圓形或立方形，呈鋪路石樣排列，胞間連接緊密，為典型上皮樣細(xì)胞形態(tài)；SKOV3/DDP細(xì)胞呈長(zhǎng)梭形，細(xì)胞排列較松散，胞間連接不緊密，呈間質(zhì)細(xì)胞樣形態(tài)；Western blotting檢測(cè)結(jié)果示：SKOV3/DDP細(xì)胞的E-cadherin表達(dá)量低于SKOV3細(xì)胞（0.054±0.012VS0.718±0.125），而Vimentin表達(dá)量高于SKOV3細(xì)胞（3.232±0.267VS0.517±0.059），差別具有統(tǒng)計(jì)學(xué)意義（P＜0.01）； RT-PCR結(jié)果示SKOV3/DDP細(xì)胞Notch1mRNA的表達(dá)量為SKOV3細(xì)胞的3.952倍。 2MTT結(jié)果示，隨著DAPT濃度增加和作用時(shí)間的延長(zhǎng)，SKOV3/DPP細(xì)胞的生長(zhǎng)抑制率明顯升高，差異均有統(tǒng)計(jì)學(xué)意義（均P＜0.01）。 3Western blotting結(jié)果示不同濃度的DAPT（25、50、75μmol/L）作用于SKOV3/DDP細(xì)胞48h后，其上皮標(biāo)記物E-cadherin表達(dá)較對(duì)照組明顯增加（0.207±0.020、0.295±0.011、0.429±0.024VS0.054±0.012）（P＜0.01），且隨DAPT作用濃度增加而升高，各組間差異顯著（P＜0.01）；間質(zhì)標(biāo)記物vimentin表達(dá)較對(duì)照組明顯降低（2.502±0.170、1.873±0.177、1.624±0.057VS3.232±0.266）（P＜0.01），但50μmol/L與75μmol/L濃度組表達(dá)差異無統(tǒng)計(jì)學(xué)意義（P＞0.05）。 4以濃度為50μmol/L的DAPT作用于SKOV3/DDP細(xì)胞24h后，劃痕試驗(yàn)結(jié)果：處理組的劃痕愈合面積顯著小于對(duì)照組（14.31±3.59VS52.91±10.59）（P＜0.01）；Transwell實(shí)驗(yàn)結(jié)果示處理組穿膜細(xì)胞個(gè)數(shù)明顯少于對(duì)照組（遷移實(shí)驗(yàn)13±2VS37±2，侵襲實(shí)驗(yàn)9±1VS27±3），差異均有統(tǒng)計(jì)學(xué)意義（P＜0.01）。 結(jié)論： SKOV3/DDP細(xì)胞化療耐藥過程中獲得了EMT表型，γ-分泌酶抑制劑DAPT可通過靶向作用于Notch信號(hào)通路，部分逆轉(zhuǎn)EMT，抑制人卵巢癌順鉑耐藥細(xì)胞的增殖、粘附及侵襲轉(zhuǎn)移能力，有望成為有效治療耐藥復(fù)發(fā)轉(zhuǎn)移EOC的新選擇。
[Abstract]:Objective: Epithelial ovarian cancer (EOC) is the most common malignant tumor of the ovary, which is difficult to be diagnosed early, has high recurrence and mortality rate. The 5-year survival rate of patients with ovarian cancer has been hovering around 30% because metastasis is an important cause of treatment failure and death of patients with ovarian cancer.
Recent studies have shown that epithelial-mesenchymal transition (EMT) is not only involved in embryonic development and wound repair, but also plays an important role in tumor invasion and metastasis. EMT is associated with a variety of cytokines, transcription factors and signaling pathways. The process of EMT is accompanied by the activation of Notch, Wnt, Hedgehog and other signaling pathways. The expression of E-cadherin was down-regulated and that of vimentin was up-regulated. In addition, tumor cells with EMT could acquire stem cell-like characteristics such as self-renewal, which may be related to chemotherapeutic resistance of tumor cells.
Recent studies have shown that Notch signaling pathway is abnormally expressed in a variety of solid tumors and hematological tumors, and is closely related to the occurrence and development of tumors. In this study, the expression of Notch1 mRNA in human ovarian serous cystadenocarcinoma cell line SKOV3 and its cisplatin-resistant strain SKOV3/DDP was detected, and the expression of E-cadherin and vimentin in SKOV3/DDP cells treated with gamma-secretase inhibitor DAPT was further investigated. The purpose of this study is to investigate whether DAPT can inhibit the invasion and metastasis of chemotherapeutic resistant EOC by inhibiting Notch signaling pathway at molecular level, and to provide experimental evidence for the treatment strategy of chemotherapeutic resistant EOC.
METHODS: Human ovarian serous cystadenocarcinoma cell line SKOV3 and its cisplatin-resistant strain SKOV3/DDP were cultured in RMPI-1640 medium containing 10% fetal bovine serum in a constant temperature incubator with saturated humidity of 5% CO2 at 37 C.
1 inverted microscope was used to observe the morphology of SKOV3 cells and SKOV3/DDP cells and take pictures.
2 real-time fluorescence quantitative PCR (PT-PC) was used to detect the expression level of Notch1mRNA in SKOV3 cells and SKOV3/DDP cells.
MTT method: SKOV3/DDP cells were treated with different concentrations of DAPT (25,50,75 micromol/L) for different time (24h,48h,72h) and 0.375% DMSO as the control group. MTT method was used to detect the effect of DAPT on the growth of SKOV3/DDP cells.
Western blotting: To detect the expression of E-cadherin and vimentin in SKOV3/DDP cells 48 hours after exposure to different concentrations of DAPT (0,25,50,75 micromol/L).
5 Scratch test: SKOV3/DDP cells were cultured routinely and fused to form a single cell layer. After 24 hours of synchronization in serum-free medium, the SKOV3/DDP cells were divided into experimental group (50 micromol/LDAPT) and control group (0.25% DMSO). Scratch was observed under an inverted microscope (40 x) at the bottom of the plate 0 h and 24 h after administration. Photographs were taken to calculate the area of scratch healing within 24h to assess cell migration.
6 Transwell experiment: SKOV3/DDP cells treated with 50 micromol/L DAPT were used as experimental group, and 0.25% DMSO as control group. Transwell invasive cells were taken out after 24 hours of continuous culture, fixed with 95% ethanol, stained with crystal violet, photographed under microscope (200 *) and counted to evaluate the migration and invasiveness of the cells.
7 SPSS16.0 software was used for statistical analysis.
Result:
SKOV3 cells and SKOV3/DDP cells: under inverted microscope, SKOV3 cells were oval, round or cubic, paving stone-like arrangement, tight intercellular junction, typical epithelial-like cell morphology; SKOV3/DDP cells were spindle-shaped, loosely arranged, intercellular junction was not tight, and showed mesenchymal-like morphology; Western blotting detection of nodules; The results showed that the expression of E-cadherin in SKOV3/DDP cells was lower than that in SKOV3 cells (0.054+0.012VS 0.718+0.125), while the expression of Vimentin was higher than that in SKOV3 cells (3.232+0.267VS 0.517+0.059), the difference was statistically significant (P < 0.01); the expression of Notch1 mRNA in SKOV3/DDP cells was 3.952 times that in SKOV3 cells.
The results of 2MTT showed that the growth inhibition rate of SKOV3/DPP cells increased significantly with the increase of DAPT concentration and prolongation of action time (P < 0.01).
Western blotting showed that the expression of E-cadherin in SKOV3/DDP cells treated with different concentrations of DAPT (25,50,75 micromol/L) for 48 hours was significantly higher than that in the control group (0.207.020,0.295.011,0.429.024VS 0.054.012) (P < 0.01), and the expression of E-cadherin was significantly higher than that in the control group (P < 0.01). The expression of vimentin was significantly lower than that of the control group (2.502.170, 1.873.177, 1.624.057VS 3.232.266) (P < 0.01), but there was no significant difference between the 5065507
4 Scratch test showed that the area of scratch healing in the treatment group was significantly smaller than that in the control group (14.31 [3.59] VS 52.91 [10.59]) (P < 0.01); Transwell test showed that the number of penetrating cells in the treatment group was significantly less than that in the control group (13 [2] VS 37 [2] migration test, 9 [1] VS 27 [3] invasion test). The differences were statistically significant (P < 0.01).
Conclusion: SKOV3/DDP cells acquired EMT phenotype during chemotherapeutic resistance, and DAPT, a gamma-secretase inhibitor, can partially reverse EMT by targeting Notch signaling pathway, inhibit the proliferation, adhesion and invasion and metastasis of cisplatin-resistant human ovarian cancer cells. It is expected to be a new choice for effective treatment of drug-resistant recurrence and metastasis of EOC.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.31

【共引文獻(xiàn)】

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1 桑明;王燕;王s

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