【摘要】：第一部分TGFβ1對(duì)三陰性乳腺癌肝臟轉(zhuǎn)移的作用研究研究背景:我們課題組前期的研究發(fā)現(xiàn)TGFβ1在三陰性乳腺癌中特異性的高表達(dá),但是這種高表達(dá)是否參與對(duì)三陰性乳腺癌的調(diào)控研究仍較少。本實(shí)驗(yàn),我們主要在體探討了 TGFβ1對(duì)三陰性乳腺癌體內(nèi)成瘤、局部侵襲及肝臟轉(zhuǎn)移能力的影響。研究方法:將對(duì)照組及經(jīng)TGFβ1反復(fù)處理的三陰性乳腺癌MDA細(xì)胞注入3-4周大的雌性裸鼠乳腺脂肪墊中建立乳腺癌原位模型。觀察原位腫瘤的生長情況及肝臟轉(zhuǎn)移情況。4-5周左右處死小鼠,HE染色觀察對(duì)照組及TGFβ1組乳腺癌原位腫瘤及肝臟轉(zhuǎn)移灶。利用免疫組化染色對(duì)照組及TGFβ1組原位及肝臟腫瘤灶Ki67、α-MSA及KRT8等相關(guān)乳腺癌免疫組化指標(biāo),以判斷轉(zhuǎn)移灶來源。研究結(jié)果:與對(duì)照組相比,TGFβ1處理組三陰性乳腺癌細(xì)胞裸鼠原位成瘤能力增加,HE染色顯示TGFβ1預(yù)處理增加了三陰性乳腺癌局部侵襲能力。同時(shí),肝臟大體觀及HE染色結(jié)果顯示TGFβ1預(yù)處理增加了三陰性乳腺癌肝轉(zhuǎn)移的能力。免疫組化結(jié)果顯示肝臟轉(zhuǎn)移灶來源于原位腫瘤灶。研究結(jié)論:我們的結(jié)果提示TGFβ1預(yù)處理能夠增加三陰性乳腺癌體內(nèi)成瘤、局部侵襲及肝臟轉(zhuǎn)移能力。第二部分TGFβ1對(duì)三陰性乳腺癌干細(xì)胞特性的作用研究研究背景:研究普遍認(rèn)為干細(xì)胞是腫瘤轉(zhuǎn)移的根源。近年來研究發(fā)現(xiàn),TGFβ配體和它的信號(hào)元件在人乳腺癌干細(xì)胞的調(diào)控中起到了極其重要的作用。我們的前期研究也發(fā)現(xiàn)TGFβ1在三陰性乳腺癌細(xì)胞中特異性表達(dá)升高,高度提示TGFβ1可能參與三陰性乳腺癌干細(xì)胞的調(diào)控。因此,本實(shí)驗(yàn)中,我們主要探討TGFβ1對(duì)三陰性乳腺癌干細(xì)胞及其特性的影響。研究方法:首先,利用流式細(xì)胞術(shù)分析TGFβ1對(duì)乳腺癌干細(xì)胞樣CD44+/CD24-表型的作用;其次,通過RT-PCR檢測乳腺癌細(xì)胞腺上皮標(biāo)志物(KRT8、KRT18)和間質(zhì)/肌上皮標(biāo)志物(KRT14、ACTA2)的表達(dá),從而研究TGFβ1對(duì)乳腺癌干細(xì)胞樣間質(zhì)細(xì)胞表型的作用影響;然后,RT-PCR檢測TGFβ1對(duì)乳腺癌干細(xì)胞球的干性相關(guān)轉(zhuǎn)錄因子OCT4、SOX2、NANOG表達(dá)的作用;之后,成球?qū)嶒?yàn)及成球效率評(píng)估TGFβ1對(duì)乳腺癌干細(xì)胞自我更新能力的影響;隨后,利用TGFβ1受體阻斷劑研究TGFβ1對(duì)乳腺癌干細(xì)胞自我更新能力的影響是否通過其受體發(fā)揮作用的;最后,3D培養(yǎng)檢測TGFβ1對(duì)乳腺癌干細(xì)胞球侵襲能力的作用。研究結(jié)果:我們的結(jié)果顯示:TGFβ1增加三陰性乳腺癌中CD44high/CD24-/low干細(xì)胞群比例;TGFβ1增加三陰性乳腺癌干細(xì)胞樣間質(zhì)細(xì)胞表型;TGFβ1促進(jìn)干細(xì)胞成球并增加干性標(biāo)志物的表達(dá);TGFβ1增強(qiáng)三陰性乳腺癌干細(xì)胞的自我更新能力;TGFβ1增強(qiáng)三陰性乳腺癌干細(xì)胞的自我更新能力能夠被其受體抑制劑阻斷;TGFβ1增強(qiáng)三陰性乳腺癌干細(xì)胞的侵襲能力。研究結(jié)論:我們的結(jié)果提示TGFβ1能夠增強(qiáng)三陰性乳腺癌的自我更新能力等干細(xì)胞特性。第三部分CDK4參與TGFβ1對(duì)三陰性乳腺癌干細(xì)胞的調(diào)控研究研究背景:細(xì)胞周期蛋白依賴性激酶4(CDK4)為細(xì)胞周期蛋白D1(cyclinD1)的配體,通常與cyclinD1結(jié)合并被激活來發(fā)揮細(xì)胞周期調(diào)節(jié)的作用。研究發(fā)現(xiàn)CDK4促進(jìn)胚胎干細(xì)胞、造血干細(xì)胞、神經(jīng)干細(xì)胞及乳腺前提細(xì)胞增殖并抑制其分化。提示CDK4可能也在乳腺癌干細(xì)胞的增殖及轉(zhuǎn)移中發(fā)揮了重要的作用。前期我們的研究也發(fā)現(xiàn)TGFβ1可以提高三陰性乳腺癌中CyclinD1/CDK4的表達(dá),猜測CDK4可能在TGFβ1參與的三陰性乳腺癌干細(xì)胞調(diào)控中發(fā)揮重要作用。因此,本部分實(shí)驗(yàn)主要探討CDK4是否作為TGFβ1的下游底物,介導(dǎo)了其對(duì)三陰性乳腺癌干細(xì)胞的調(diào)控。研究方法:流式細(xì)胞術(shù)檢測對(duì)照組、CDK4蛋白沉默組及CDK4激酶抑制劑組SUM159中CD44+/CD24-干細(xì)胞比例;成球?qū)嶒?yàn)及成球效率檢測不同組(對(duì)照組、CDK4蛋白沉默組、CDK4激酶抑制劑組、TGFβ1組、CDK4激酶抑制劑+TGFβ1組)SUM159干細(xì)胞自我更新能力,RT-PCR檢測不同組導(dǎo)管上皮細(xì)胞標(biāo)志物(KRT8,KRT18)及間質(zhì)細(xì)胞標(biāo)志物(ACTA2,KRT14)的表達(dá)。免疫共沉淀(Co-IP)及Western blot技術(shù)檢測對(duì)照組及TGFβ1組CyclinD1、CDK4、p-RB等蛋白的表達(dá)。研究結(jié)果:沉默CDK4蛋白或使用CDK4激酶抑制劑能夠降低三陰性乳腺癌中CD44+/CD24-干細(xì)胞比例及自我更新能力,抑制三陰性乳腺癌干細(xì)胞樣間質(zhì)細(xì)胞表型。TGFβ1上調(diào)CDK4蛋白表達(dá)及激酶活性,CDK4激酶抑制劑可以阻斷TGFβ1增強(qiáng)三陰性乳腺癌干細(xì)胞自我更新能力及維持其間質(zhì)樣表型的作用。研究結(jié)論:這些結(jié)果提示CDK4蛋白本身在維持三陰性乳腺癌干細(xì)胞自我更新及間質(zhì)樣表型中發(fā)揮重要的作用,TGFβ1可能是通過上調(diào)CDK4蛋白及其激酶活性來維持三陰性乳腺癌干細(xì)胞自我更新能力及干細(xì)胞樣間質(zhì)細(xì)胞表型的。第四部分腫瘤干細(xì)胞與三陰性乳腺癌肝轉(zhuǎn)移及預(yù)后相關(guān)分析研究背景:近年來,很多臨床數(shù)據(jù)庫的開發(fā)與建立為多種不同來源的腫瘤研究提供了有效的生物信息學(xué)分析。因此,本部分研究主要利用Kaplan Meier Plotter、TCGA Cancer Browser以及GOBO geneset數(shù)據(jù)庫對(duì)三陰性乳腺癌中腫瘤標(biāo)志物,TGFβ1和CDK4做相關(guān)生物信息學(xué)分析。研究方法:首先利用Kaplan Meier Plotter對(duì)CD44及CD24的基因表達(dá)與249例三陰性乳腺癌患者的預(yù)后進(jìn)行生物信息學(xué)分析。然后利用UCSC Cancer Browser對(duì)TCGA數(shù)據(jù)庫1215例乳腺癌患者中三陰性乳腺癌肝轉(zhuǎn)移患者進(jìn)行CD44及CD24的基因表達(dá)的生物信息學(xué)分析。之后,利用GOBO基因分析工具對(duì)51株已報(bào)道的三陰性乳腺癌細(xì)胞系中CD44及CD24的基因表達(dá)進(jìn)行分析。最后,利用GOBO基因數(shù)據(jù)庫分析TGFβ1及CDK4在三陰性乳腺癌中的表達(dá)。研究結(jié)果:生物信息學(xué)分析提示CD44高表達(dá)/CD24低表達(dá)患者無復(fù)發(fā)生存率(RFS)較低,且三陰性乳腺癌伴肝轉(zhuǎn)移患者腫瘤干細(xì)胞表面標(biāo)志物CD44高表達(dá),而CD24低表達(dá)。TGFβ1、CDK4在三陰性乳腺癌細(xì)胞系中的表達(dá)較高。研究結(jié)論:TCGA及GOBO geneset等數(shù)據(jù)庫生物信息學(xué)分析提示三陰性乳腺癌中CD44high/CD24low/-干細(xì)胞含量較高,且TGFβ1及CDK4可能與三陰性乳腺癌干細(xì)胞調(diào)控相關(guān),與我們的研究結(jié)果TGFβ1/CDK4參與三陰性乳腺癌干細(xì)胞調(diào)控一致。
[Abstract]:The first part of the study on the role of TGF beta 1 on liver metastasis of three negative breast cancer: our previous study found that TGF beta 1 was highly expressed in three negative breast cancer, but whether this high expression is involved in the regulation of three negative breast cancer is still less. In this experiment, we mainly discussed the TGF beta 1 to three yin in vivo. The effect of the tumor formation, local invasion and liver metastasis in the body of sexual breast cancer. Methods: the control group and the three negative breast cancer cells treated with TGF beta 1 were injected into the breast fat pad of the female nude mice for 3-4 weeks, and the in situ model of breast cancer was established. The growth and liver metastasis of the tumor in situ were observed for about.4-5 weeks. Mice, HE staining and control group and TGF beta 1 group of breast cancer in situ tumor and liver metastases were observed. Immuno histochemical staining control group and TGF beta 1 group in situ and liver tumor Ki67, alpha -MSA and KRT8 related immunohistochemical indexes of breast cancer were used to determine the origin of metastatic foci. The results were compared with those in the group of TGF beta 1, three negative breast cancer cells The tumor ability in nude mice was increased in situ. HE staining showed that TGF beta 1 preconditioning increased the local invasiveness of three negative breast cancers. Meanwhile, the liver gross and HE staining results showed that TGF beta 1 preconditioning increased the ability of three negative breast cancer to liver metastasis. The results suggest that TGF beta 1 preconditioning can increase the tumor formation, local invasion and liver metastasis in three negative breast cancers. Second the research background of the role of TGF beta 1 on the characteristics of three negative breast cancer stem cells: the research is generally believed that stem cells are the root of tumor metastasis. In recent years, the research has found that TGF beta ligand and its signal components are in human The regulation of breast cancer stem cells plays an extremely important role. Our previous study also found that the specific expression of TGF beta 1 increased in three negative breast cancer cells. It is highly suggestive that TGF beta 1 may be involved in the regulation of three negative breast cancer stem cells. Therefore, in this experiment, we mainly discuss the TGF beta 1 against three negative breast cancer stem cells and their specificity. Study methods: first, flow cytometry was used to analyze the effect of TGF beta 1 on the CD44+/CD24- phenotype of breast cancer stem cells. Secondly, the expression of KRT8 (KRT18) and interstitial / myoepithelial markers (KRT14, ACTA2) of breast cancer cells were detected by RT-PCR, and the phenotype of TGF beta 1 on the stem cell like cell phenotype of breast cancer was studied. The effect of TGF beta 1 on the expression of OCT4, SOX2, NANOG in the stem cells of breast cancer stem cells was detected by RT-PCR. After that, the effect of TGF beta 1 on the self-renewal capacity of breast cancer stem cells was evaluated by the spherocification test and the ball efficiency. Then, the self-renewal of TGF beta 1 on breast cancer stem cells was studied by TGF beta 1 receptor blocker. The effect of the effect of capacity through its receptor; finally, 3D culture was used to detect the effect of TGF beta 1 on the invasive ability of breast cancer stem cells. Results: Our results showed that TGF beta 1 increased the proportion of CD44high/CD24-/low stem cells in three negative breast cancers; TGF beta 1 increased the phenotype of three negative breast cancer stem cell like mesenchymal cells; TGF beta 1 Promote the formation of stem cells and increase the expression of dry markers; TGF beta 1 enhanced the self-renewal capacity of three negative breast cancer stem cells; TGF beta 1 enhanced the self-renewal ability of three negative breast cancer stem cells to be blocked by its receptor inhibitors; TGF beta 1 enhanced the invasive ability of three negative breast cancer stem cells. Conclusions: Our results suggest TGF beta 1 can enhance the self renewal capacity of three negative breast cancer. Third part of CDK4 participates in the research background of TGF beta 1 on three negative breast cancer stem cells: cell cyclin dependent kinase 4 (CDK4) is a ligand of cyclin D1 (cyclinD1), which is usually combined with cyclinD1 and is activated to play cell cycle. The study found that CDK4 promotes embryonic stem cells, hematopoietic stem cells, neural stem cells, and mammary cells to proliferate and inhibit their differentiation. It suggests that CDK4 may also play an important role in the proliferation and metastasis of breast cancer stem cells. Our previous study also found that TGF beta 1 could improve the CyclinD1/C of breast cancer in negative breast cancer. The expression of DK4 suggests that CDK4 may play an important role in the regulation of three negative breast cancer stem cells involved in TGF beta 1. Therefore, this part of this experiment mainly discusses whether CDK4 is a downstream substrate of TGF beta 1 and mediates its regulation of three negative breast cancer stem cells. The proportion of CD44+/CD24- stem cells in the enzyme inhibitor group SUM159, the ball forming experiment and the detection of the ball forming efficiency in different groups (control group, CDK4 protein silencing group, CDK4 kinase inhibitor group, TGF beta 1 group, CDK4 kinase inhibitor +TGF beta 1 group) SUM159 stem cells self-renewal capacity, RT-PCR detection of different groups of ductal epithelial cell markers (KRT8, KRT18) and interstitial cell markers Expression of ACTA2 (KRT14). Immunocoprecipitation (Co-IP) and Western blot techniques for the detection of CyclinD1, CDK4, p-RB and other proteins in the TGF beta 1 groups. The results of the study: silencing CDK4 protein or using CDK4 kinase inhibitors can reduce the ratio of CD44+/CD24- stem cells and self renewal in three negative breast cancers and inhibit three negative breast cancer The phenotype of stem cell like mesenchymal cells.TGF beta 1 up-regulated the expression of CDK4 protein and kinase activity. CDK4 kinase inhibitor can block the role of TGF beta 1 to enhance the self-renewal capacity of three negative breast cancer stem cells and maintain its interstitial like phenotype. These results suggest that the CDK4 protein itself maintains the self renewal of the three negative breast cancer stem cells. An important role in interstitial like phenotype, TGF beta 1 may be to maintain the self-renewal capacity of three negative breast cancer stem cells and the phenotype of stem like mesenchymal cells by up regulation of CDK4 protein and its kinase activity. Fourth part of tumor stem cells and the correlation analysis of liver metastasis and prognosis in three negative breast cancer: many clinical trials in recent years The development and establishment of the database provide an effective bioinformatics analysis for a variety of different sources of cancer research. Therefore, this part of this study mainly uses Kaplan Meier Plotter, TCGA Cancer Browser and GOBO geneset database to analyze the tumor markers in three negative breast cancers, TGF beta 1 and CDK4, and the related bioinformatics analysis. Method: bioinformatics analysis was performed on the gene expression of CD44 and CD24 with Kaplan Meier Plotter and the prognosis of 249 cases of three negative breast cancer. Then, UCSC Cancer Browser was used to analyze the gene expression of CD44 and CD24 in three negative breast cancer patients with breast cancer in 1215 cases of breast cancer. Then, the GOBO gene analysis tool was used to analyze the expression of CD44 and CD24 in 51 reported three negative breast cancer cell lines. Finally, the expression of TGF beta 1 and CDK4 in three negative breast cancer was analyzed by GOBO gene database. The results of bioinformatics analysis suggested that the CD44 high expression of low expression of /CD24 in patients with high expression of /CD24 had no recurrence. The rate (RFS) was low, and the expression of the surface marker of the tumor stem cells was high in the three negative breast cancer patients with liver metastasis, while the expression of.TGF beta 1 was low in CD24, and the expression of CDK4 in the three negative breast cancer cell lines was higher. Conclusion: TCGA and GOBO geneset and other database bioinformatics analysis showed that CD44high/CD24low/- stem cells contained in three negative breast cancer contained CD44high/CD24low/- stem cells. High volume, and TGF beta 1 and CDK4 may be related to the regulation of three negative breast cancer stem cells. It is consistent with our findings that TGF beta 1/CDK4 participates in the regulation of three negative breast cancer stem cells.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.9

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