辛伐他汀聯(lián)合紫杉醇靶向作用Hippo信號(hào)通路對(duì)乳腺癌干細(xì)胞的影響
發(fā)布時(shí)間：2018-07-15 14:52
【摘要】：研究背景:乳腺癌是威脅全球女性健康的殺手,其術(shù)后復(fù)發(fā)和化療效果不佳,是乳腺癌死亡率高的主要原因之一。乳腺癌干細(xì)胞(Breast cancer stem cells,BCSCs)被認(rèn)為是其復(fù)發(fā)的根源,能夠抵抗放射療法且影響化療的效果。乳腺癌干細(xì)胞因其具有自我更新能力、多向分化性、高致瘤性和對(duì)放化療耐藥等特性。成為近年來乳腺癌研究的熱點(diǎn)。乳腺癌干細(xì)胞成為潛在治療靶點(diǎn)。Hippo信號(hào)通路最初被發(fā)現(xiàn)是抑制增殖和促進(jìn)凋亡來限制器官大小的信號(hào)通路。然而,許多證據(jù)表明Hippo信號(hào)通路可以調(diào)節(jié)干細(xì)胞和祖細(xì)胞自我更新和擴(kuò)增。在乳腺癌、肝癌、非小細(xì)胞肺癌和子宮內(nèi)膜癌發(fā)生進(jìn)展中起作用。Hippo信號(hào)通路經(jīng)過激酶級(jí)聯(lián)反應(yīng),最終作用其核心轉(zhuǎn)錄因子TAZ/YAP,磷酸化的TAZ/YAP與14-3-3蛋白相互作用被阻斷在細(xì)胞質(zhì)中。并被泛素依賴的蛋白酶體降解。未被磷酸化的TAZ/YAP轉(zhuǎn)移到細(xì)胞核中,與TEAD1-4或其他轉(zhuǎn)錄因子Smad、sp73等相互作用,誘導(dǎo)基因CTGF、AX1、BMP4等的表達(dá),其中,以Hippo信號(hào)通路中核心因子TAZ/YAP表現(xiàn)最為明顯。而在乳腺癌中,TAZ在影響乳腺癌干細(xì)胞成球、自我更新、分化則更加明顯。辛伐他汀是HMG-Co A抑制劑,近幾年研究表明辛伐他汀可以作為抗癌藥對(duì)腫瘤起到抑制作用。研究表明辛伐他汀可以抑制Hippo-TAZ從胞質(zhì)向胞核的轉(zhuǎn)運(yùn),從而抑制TAZ在細(xì)胞核操控下游轉(zhuǎn)錄因子發(fā)揮生物學(xué)作用。紫杉醇是一種抗微管藥物,促進(jìn)微管蛋白聚合,抑制解聚,保持微管蛋白均一穩(wěn)定,抑制癌細(xì)胞有絲分裂過程,作為乳腺癌、卵巢癌臨床化療的一線藥物已經(jīng)有很多年歷史了,近年來,其藥物效能減弱及耐藥現(xiàn)象增加,辛伐他汀是否可以協(xié)同紫杉醇,增加其對(duì)腫瘤的藥效還未曾報(bào)道。本研究選用辛伐他汀和紫杉醇對(duì)乳腺癌細(xì)胞MDA-MB-231細(xì)胞侵襲、轉(zhuǎn)移、EMT過程及對(duì)乳腺癌干細(xì)胞的影響,并探討其有關(guān)作用機(jī)制。方法:實(shí)驗(yàn)分為四組:空白處理組、辛伐他汀處理組、紫杉醇處理組、辛伐他汀+紫杉醇處理組。(1)cck8檢測(cè)腫瘤抑制率:各處理組的胰酶消化后將MDA-MB-231細(xì)胞按5×103/孔接種,24h后換用不同濃度藥物梯度培養(yǎng)基(梯度),培養(yǎng)48h,CCK8避光加入,避光37℃孵育2h,用酶標(biāo)儀測(cè)出OD值,應(yīng)用Gradpad計(jì)算藥物IC50。加入兩種藥物與之前單獨(dú)測(cè)IC50時(shí)濃度梯度相同(兩種藥物添加比例1:1)48h后,測(cè)OD值。(2)劃痕實(shí)驗(yàn)檢測(cè)MDA-MB-231細(xì)胞遷移能力的變化:各處理組的MDA-MB-231細(xì)胞無血清培養(yǎng)48h,分別于0h以及48h時(shí)取樣拍照,觀察細(xì)胞遷移距離。采用Transwell實(shí)驗(yàn)檢測(cè)各組MDA-MB-231細(xì)胞侵襲和轉(zhuǎn)移能力的變化:各處理組細(xì)胞分別培養(yǎng)24h后取樣拍照,并計(jì)數(shù)小室膜細(xì)胞數(shù)量。采用明膠酶譜檢測(cè)各處理組MDA-MB-231細(xì)胞內(nèi)MMP-2表達(dá)水平的變化。(3)Western blot技術(shù)檢測(cè)MDA-MB-231細(xì)胞內(nèi)蛋白水平的變化。檢測(cè)指標(biāo)包括:Hippo信號(hào)通路關(guān)鍵因子TAZ,干細(xì)胞標(biāo)志物-OCT4、ALDH1。金屬基質(zhì)蛋白酶-2—MMP-2,E-鈣粘素—E-cadherin,波形蛋白—Vimentin,凋亡通路相關(guān)蛋白—Bax、P53、Caspsase3、Bcl-2。免疫細(xì)胞化學(xué)技術(shù)觀察細(xì)胞內(nèi)TAZ、OCT4及E-cadherin、Vimentin蛋白表達(dá)情況。(4)流式細(xì)胞儀檢測(cè)CD44+/CD24-染色檢測(cè)各處理組干細(xì)胞比例。(5)干細(xì)胞成球?qū)嶒?yàn)檢測(cè)各處理組成球能力及數(shù)量。(6)各組細(xì)胞藥物處理48h后,每孔種植相同數(shù)量細(xì)胞培養(yǎng),兩周后,結(jié)晶紫色,觀察克隆細(xì)胞群大小和數(shù)量。結(jié)果:(1)劃痕及侵襲實(shí)驗(yàn)結(jié)果顯示:辛伐他汀聯(lián)合紫杉醇可以效地抑制其遷移距離及侵襲及轉(zhuǎn)移數(shù)量。Western blot技術(shù)及明膠酶譜技術(shù)檢測(cè)MMP-2的表達(dá)情況,結(jié)果顯示:與空白對(duì)照組和單獨(dú)加藥組相比,辛伐他汀聯(lián)合紫杉醇處理組MMP-2表達(dá)更顯著下降。(2)通過對(duì)EMT標(biāo)記物的檢測(cè)發(fā)現(xiàn):與空白對(duì)照組和單獨(dú)加藥組相比,辛伐他汀聯(lián)合紫杉醇組升高E-cadherin的蛋白含量,降低細(xì)胞內(nèi)Vimentin的蛋白含量。(3)凋亡相關(guān)信號(hào)通路的標(biāo)記物檢測(cè)結(jié)果顯示:與空白對(duì)照組和單獨(dú)加藥組相比,辛伐他汀聯(lián)合紫杉醇組,P53、Bcl-2、Caspsase3表達(dá)明顯下降,Bax表達(dá)明顯升高,克隆形成實(shí)驗(yàn):(4)Western blot技術(shù)及免疫熒光化學(xué)技術(shù)檢測(cè)結(jié)果顯示:與空白對(duì)照組和單獨(dú)加藥組相比,辛伐他汀聯(lián)合紫杉醇組,TAZ、OCT4、ALDH1蛋白表達(dá)明顯下降,(5)流式細(xì)胞儀CD44+/CD24-染色檢測(cè)干細(xì)胞比例及干細(xì)胞成球?qū)嶒?yàn)結(jié)果顯示:空白對(duì)照組和單獨(dú)加藥組相比,辛伐他汀聯(lián)合紫杉醇組更明顯抑制乳腺癌干細(xì)胞比例及其成球能力。結(jié)論:辛伐他汀與紫杉醇聯(lián)用可以抑制MDA-MB-231轉(zhuǎn)移、侵襲,促進(jìn)凋亡、抑制增殖。辛伐他汀與紫杉醇聯(lián)用對(duì)乳腺癌干細(xì)胞通過下調(diào)Hippo-TAZ及其下游靶點(diǎn)Oct4對(duì)增殖與自我更新能力均有影響,或許可以對(duì)臨床化療提供新思路。
[Abstract]:Background: breast cancer is a killer that threatens the health of women in the world. The recurrence and chemotherapy of breast cancer are one of the main causes of high mortality in breast cancer. Breast cancer stem cells (BCSCs) is considered to be the root cause of its recurrence. It can resist radiation therapy and affect the effect of chemotherapy. The stem cells of breast cancer are caused by the cancer stem cells. It has the characteristics of self renewal, multidifferentiation, high tumorigenicity, and chemoradiotherapy. It has become a hot spot in the research of breast cancer in recent years. The.Hippo signal pathway of breast cancer stem cells has been found to be a signal pathway that inhibits proliferation and promotes apoptosis to limit the size of the organ. However, a lot of evidence suggests that Hippo signals are used. The pathway regulates the self renewal and amplification of stem cells and progenitor cells. In the progression of breast cancer, liver cancer, non-small cell lung cancer and endometrial cancer, the.Hippo signaling pathway is cascaded through kinase cascade, which ultimately acts as the core transcription factor TAZ/YAP, and the interaction of phosphorylated TAZ/YAP and 14-3-3 protein is blocked in the cytoplasm. The ubiquitin dependent proteasome is degraded. The non phosphorylated TAZ/YAP is transferred to the nucleus, interacting with TEAD1-4 or other transcription factors Smad, sp73 and so on, inducing gene CTGF, AX1, BMP4 and so on. Among them, the core factor TAZ/YAP in Hippo signaling pathway is most obvious. In breast cancer, TAZ is affecting the stem cells of breast cancer. The ball, self-renewal, and differentiation is more obvious. Simvastatin is a HMG-Co A inhibitor. Recent studies have shown that simvastatin can inhibit the tumor as an anticancer drug. The study shows that simvastatin can inhibit the transport of Hippo-TAZ from the cytoplasm to the nucleus, thus inhibiting the biological role of TAZ in the nuclear control of the downstream transcription factors. Paclitaxel is an anti microtubule drug that promotes microtubule polymerization, inhibits depolymerization, keeps microtubule protein stable and inhibits the process of mitosis in cancer cells. As breast cancer, the clinical chemotherapy of ovarian cancer has a history of many years. In recent years, its drug efficacy and drug resistance have increased. The effects of simvastatin and paclitaxel on the invasion, metastasis, EMT process and the effect on breast cancer stem cells of breast cancer cell MDA-MB-231 were selected in this study. Methods: the experiment was divided into four groups: the blank treatment group, the simvastatin treatment group, the paclitaxel treatment group. Simvastatin + paclitaxel treatment group. (1) CCK8 detection of tumor inhibition rate: after trypsin digestion, MDA-MB-231 cells were inoculated after trypsin digestion in each treatment group, and MDA-MB-231 cells were inoculated at 5 x 103/ holes. After 24h, different concentrations of drug gradient medium (gradient) were changed, 48h, CCK8 were incubated to avoid light, and 2H was incubated at 37 degrees, and two species were added with Gradpad to calculate IC50.. After the same concentration gradient (two medications added to 1:1) 48h before IC50, the drug was measured. (2) the change of migration ability of MDA-MB-231 cells was detected by scratch test: the MDA-MB-231 cells in each treatment group were serum-free to culture 48h, and were taken and photographed at 0h and 48h, respectively, to observe the migration distance of the cells. Transwell test was used to detect the MD of each group. Changes in the invasion and metastasis ability of A-MB-231 cells: the cells in each treatment group were cultured for 24h to take pictures and count the number of small cell membrane cells. The changes in the level of MMP-2 expression in the MDA-MB-231 cells of each treatment group were detected by gelatinase spectrum. (3) the Western blot technique was used to detect the changes in the level of the intracellular protein in the MDA-MB-231 cells. The detection index included: Hippo Key signal pathway factor TAZ, stem cell marker -OCT4, ALDH1. metal matrix proteinase -2 MMP-2, E- cadherin E-cadherin, vimentin Vimentin, apoptotic pathway related proteins - Bax, P53, Caspsase3, Bcl-2. immunocytochemical technique observation cell expression. (4) flow cytometry The ratio of stem cells in each treatment group was measured by CD44+/CD24- staining. (5) the ability and quantity of each treatment was detected by the stem cell formation test. (6) after the treatment of 48h, the cells were cultivated for the same number of cells in each hole. After two weeks, the size and quantity of the cloned cell group were observed. Results: (1) the results of scratch and invasion test showed octyl. Statins combined paclitaxel could effectively inhibit the migration distance, invasion and metastasis number.Western blot technique and gelatinase spectrum technique to detect the expression of MMP-2. The results showed that compared with the blank control group and the single addition group, the expression of MMP-2 in the combined paclitaxel treatment group was significantly decreased. (2) the detection of EMT markers was detected. It was found that: compared with the blank control group and the single addition group, simvastatin combined paclitaxel group increased the protein content of E-cadherin and decreased the protein content of Vimentin in the cell. (3) the detection results of apoptosis related signal pathway markers showed that compared with the blank control group and the single addition group, the simvastatin combined paclitaxel group, P53, Bcl-2, Caspsa The expression of Se3 was obviously decreased, the expression of Bax was obviously increased and the cloning experiment was found. (4) the results of Western blot and immunofluorescence chemical technique showed that compared with the blank control group and the single addition group, the expression of TAZ, OCT4, ALDH1 protein in the group of simvastatin combined paclitaxel, TAZ, OCT4 and ALDH1 was markedly decreased, and (5) flow cytometry CD44+/CD24- staining was used to detect the proportion of stem cells The results of the stem cell formation test showed that the ratio of simvastatin combined with paclitaxel group was more obviously inhibited by the combination of simvastatin and paclitaxel than in the single addition group. Conclusion: the combined use of simvastatin and paclitaxel can inhibit MDA-MB-231 metastasis, invasion, apoptosis and inhibition of proliferation. Breast cancer stem cells may influence the proliferation and self-renewal ability by downregulating Hippo-TAZ and its downstream target Oct4, which may provide new ideas for clinical chemotherapy.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.9
本文編號(hào)：2124440
[Abstract]:Background: breast cancer is a killer that threatens the health of women in the world. The recurrence and chemotherapy of breast cancer are one of the main causes of high mortality in breast cancer. Breast cancer stem cells (BCSCs) is considered to be the root cause of its recurrence. It can resist radiation therapy and affect the effect of chemotherapy. The stem cells of breast cancer are caused by the cancer stem cells. It has the characteristics of self renewal, multidifferentiation, high tumorigenicity, and chemoradiotherapy. It has become a hot spot in the research of breast cancer in recent years. The.Hippo signal pathway of breast cancer stem cells has been found to be a signal pathway that inhibits proliferation and promotes apoptosis to limit the size of the organ. However, a lot of evidence suggests that Hippo signals are used. The pathway regulates the self renewal and amplification of stem cells and progenitor cells. In the progression of breast cancer, liver cancer, non-small cell lung cancer and endometrial cancer, the.Hippo signaling pathway is cascaded through kinase cascade, which ultimately acts as the core transcription factor TAZ/YAP, and the interaction of phosphorylated TAZ/YAP and 14-3-3 protein is blocked in the cytoplasm. The ubiquitin dependent proteasome is degraded. The non phosphorylated TAZ/YAP is transferred to the nucleus, interacting with TEAD1-4 or other transcription factors Smad, sp73 and so on, inducing gene CTGF, AX1, BMP4 and so on. Among them, the core factor TAZ/YAP in Hippo signaling pathway is most obvious. In breast cancer, TAZ is affecting the stem cells of breast cancer. The ball, self-renewal, and differentiation is more obvious. Simvastatin is a HMG-Co A inhibitor. Recent studies have shown that simvastatin can inhibit the tumor as an anticancer drug. The study shows that simvastatin can inhibit the transport of Hippo-TAZ from the cytoplasm to the nucleus, thus inhibiting the biological role of TAZ in the nuclear control of the downstream transcription factors. Paclitaxel is an anti microtubule drug that promotes microtubule polymerization, inhibits depolymerization, keeps microtubule protein stable and inhibits the process of mitosis in cancer cells. As breast cancer, the clinical chemotherapy of ovarian cancer has a history of many years. In recent years, its drug efficacy and drug resistance have increased. The effects of simvastatin and paclitaxel on the invasion, metastasis, EMT process and the effect on breast cancer stem cells of breast cancer cell MDA-MB-231 were selected in this study. Methods: the experiment was divided into four groups: the blank treatment group, the simvastatin treatment group, the paclitaxel treatment group. Simvastatin + paclitaxel treatment group. (1) CCK8 detection of tumor inhibition rate: after trypsin digestion, MDA-MB-231 cells were inoculated after trypsin digestion in each treatment group, and MDA-MB-231 cells were inoculated at 5 x 103/ holes. After 24h, different concentrations of drug gradient medium (gradient) were changed, 48h, CCK8 were incubated to avoid light, and 2H was incubated at 37 degrees, and two species were added with Gradpad to calculate IC50.. After the same concentration gradient (two medications added to 1:1) 48h before IC50, the drug was measured. (2) the change of migration ability of MDA-MB-231 cells was detected by scratch test: the MDA-MB-231 cells in each treatment group were serum-free to culture 48h, and were taken and photographed at 0h and 48h, respectively, to observe the migration distance of the cells. Transwell test was used to detect the MD of each group. Changes in the invasion and metastasis ability of A-MB-231 cells: the cells in each treatment group were cultured for 24h to take pictures and count the number of small cell membrane cells. The changes in the level of MMP-2 expression in the MDA-MB-231 cells of each treatment group were detected by gelatinase spectrum. (3) the Western blot technique was used to detect the changes in the level of the intracellular protein in the MDA-MB-231 cells. The detection index included: Hippo Key signal pathway factor TAZ, stem cell marker -OCT4, ALDH1. metal matrix proteinase -2 MMP-2, E- cadherin E-cadherin, vimentin Vimentin, apoptotic pathway related proteins - Bax, P53, Caspsase3, Bcl-2. immunocytochemical technique observation cell expression. (4) flow cytometry The ratio of stem cells in each treatment group was measured by CD44+/CD24- staining. (5) the ability and quantity of each treatment was detected by the stem cell formation test. (6) after the treatment of 48h, the cells were cultivated for the same number of cells in each hole. After two weeks, the size and quantity of the cloned cell group were observed. Results: (1) the results of scratch and invasion test showed octyl. Statins combined paclitaxel could effectively inhibit the migration distance, invasion and metastasis number.Western blot technique and gelatinase spectrum technique to detect the expression of MMP-2. The results showed that compared with the blank control group and the single addition group, the expression of MMP-2 in the combined paclitaxel treatment group was significantly decreased. (2) the detection of EMT markers was detected. It was found that: compared with the blank control group and the single addition group, simvastatin combined paclitaxel group increased the protein content of E-cadherin and decreased the protein content of Vimentin in the cell. (3) the detection results of apoptosis related signal pathway markers showed that compared with the blank control group and the single addition group, the simvastatin combined paclitaxel group, P53, Bcl-2, Caspsa The expression of Se3 was obviously decreased, the expression of Bax was obviously increased and the cloning experiment was found. (4) the results of Western blot and immunofluorescence chemical technique showed that compared with the blank control group and the single addition group, the expression of TAZ, OCT4, ALDH1 protein in the group of simvastatin combined paclitaxel, TAZ, OCT4 and ALDH1 was markedly decreased, and (5) flow cytometry CD44+/CD24- staining was used to detect the proportion of stem cells The results of the stem cell formation test showed that the ratio of simvastatin combined with paclitaxel group was more obviously inhibited by the combination of simvastatin and paclitaxel than in the single addition group. Conclusion: the combined use of simvastatin and paclitaxel can inhibit MDA-MB-231 metastasis, invasion, apoptosis and inhibition of proliferation. Breast cancer stem cells may influence the proliferation and self-renewal ability by downregulating Hippo-TAZ and its downstream target Oct4, which may provide new ideas for clinical chemotherapy.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.9
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 Binhua P.Zhou;;Epithelial-mesenchymal transition in breast cancer progression and metastasis[J];癌癥;2011年09期
，本文編號(hào)：2124440
本文鏈接：http://sikaile.net/shoufeilunwen/mpalunwen/2124440.html
最近更新
教材專著
