TGF-β1在乳腺癌微环境诱导肿瘤相关成纤维细胞形成的机制研究

发布时间：2018-05-18 11:36

本文选题：转化生长因子(TGF-β1) + 自噬　；参考：《暨南大学》2016年博士论文

【摘要】：肿瘤微环境已经成为肿瘤治疗的重要靶点。对于许多实体瘤而言,尤其是癌组织,他们的微环境由肿瘤细胞本身、内皮细胞、免疫细胞和成纤维细胞组成,通过分泌细胞因子或者细胞之间互相作用促进肿瘤的发生。尤其是肿瘤相关成纤维细胞(CAFs),是肿瘤微环境中激活的成纤维细胞,在肿瘤的发展进程和转移中起着重要作用。TGF-β1作为一个转化生长因子,在肿瘤微环境起着重要的作用,既可以促进肿瘤的生长,也可抑制肿瘤的生长。但是,TGF-β1在肿瘤微环境中的作用机制尚不明确。而且,我们的临床结果显示在乳腺癌病人的肿瘤组织中TGF-β1的表达与CAFs的表型标志蛋白α-SMA的表达成正相关关系,说明TGF-β1的表达与CAFs的形成有关,但是其机制有待进一步探讨。首先,本课题将小鼠胚胎成纤维细胞NIH3T3血清饥饿24 h,建立体外营养剥夺模型,也是为了模拟肿瘤微环境中营养剥夺的条件。并考察TGF-β1在一定浓度下(2.5 ng/ml)对血清饥饿的NIH3T3细胞的作用。实验结果显示TGF-β1不仅缓解了饥饿导致的生长抑制、线粒体损伤和细胞凋亡,而且TGF-β1能够诱导饥饿的成纤维细胞NIH3T3的CAFs表型的形成。实验结果还显示,TGF-β1能够增强经饥饿处理NIH3T3细胞的MDC阳性率、自噬相关基因和蛋白的表达,而自噬抑制剂3-MA却阻断了TGF-β1的作用,降低了自噬的发生。共聚焦和电镜的结果进一步发现TGF-β1增强经饥饿处理的NIH3T3细胞自噬的发生。其次,自噬是应激条件下修复细胞损伤的主要机制之一,并且自噬与肿瘤的发生和发展也密切相关。因此,本课题以TGF-β1增强血清饥饿状态下NIH3T3细胞自噬功能的角度研究和探讨了TGF-β1缓解血清饥饿导致的细胞损伤和诱导NIH3T3细胞CAFs的表型形成的作用机理。为此,我们依次进行了以下实验:(1)采用自噬促进剂(Rapa)和自噬抑制剂(3-MA)探讨了自噬是否参与了TGF-β1诱导的细胞保护作用和CAFs的转变作用,实验结果显示自噬促进剂进一步增强TGF-β1诱导的细胞增殖和线粒体功能,而自噬抑制剂则抑制了TGF-β1的作用,阻断了TGF-β1的抗凋亡作用和CAFs转变作用。(2)采用siRNA技术干扰Atg5后检测自噬水平、细胞凋亡和坏死情况以及CAFs表型蛋白α-SMA和FAP-α的表达。实验结果显示Atg5 siRNA不仅阻断了TGF-β1诱导的自噬的发生,而且废除了TGF-β1对经饥饿处理NIH3T3细胞的作用,提示自噬是TGF-β1缓解血清饥饿状态下NIH3T3细胞损伤和诱导CAFs表型形成的作用靶点。(3)我们采用TGF-βR1/ALK5抑制剂LY-2157299探讨了TGF-β1介导的自噬缓解血清饥饿状态下NIH3T3细胞损伤和诱导CAFs表型形成的具体分子机制。实验结果发现LY-2157299能够阻断TGF-β1的经典通路-Smad通路,而且进一步阻断了TGF-β1诱导的自噬相关蛋白的表达和CAFs表型标志蛋白的表达。综合以上所有结果,我们推断出TGF-β可以通过激活Smad通路提高线粒体自噬的水平,在营养剥夺状态下清除受损的线粒体和降低细胞凋亡水平,从而一定程度上修复细胞损伤;并且TGF-β可通过Smad/自噬通路在营养剥夺状态下诱导成纤维细胞CAFs表型的形成。最后,为了进一步验证TGF-β1介导的自噬在营养剥夺状态下能够修复成纤维细胞的损伤和诱导肿瘤微环境中CAFs表型的形成,我们采用小鼠混合移植瘤模型,即小鼠乳腺癌细胞4T1和小鼠成纤维细胞NIH3T3以1:2的比例混合接入Balb/c小鼠内,从体内去探讨TGF-β1诱导的自噬对肿瘤微环境中成纤维细胞的作用以及其对肿瘤发展进程实验的影响。实验结果显示,与正常组相比,血清饥饿显著提高了肿瘤组织的自噬水平,但是却抑制了肿瘤的生长,增加了肿瘤组织的凋亡和坏死水平。而TGF-β1能够显著增强肿瘤组织的自噬水平和诱导CAFs表型的形成,并且促进了肿瘤的生长,也缓解了肿瘤组织的凋亡和坏死情况。而自噬抑制剂3-MA则抑制了TGF-β1诱导的自噬,废除了TGF-β1诱导的CAFs转变作用,从而抑制了肿瘤的生长。综上所述,TGF-β1诱导的自噬促进了肿瘤微环境中的营养剥夺状态下成纤维细胞的存活,并且促进了CAFs表型的形成,从而促进了肿瘤的生长。阐明了TGF-β1在肿瘤微环境中诱导CAFs转变以促进肿瘤生长的分子机制。本实验研究结果为研究和开发抗乳腺恶性肿瘤药物提供了新的作用靶点。
[Abstract]:Tumor microenvironment has become an important target for cancer treatment. For many solid tumors, especially cancer tissues, their microenvironment is composed of tumor cells, endothelial cells, immune cells, and fibroblasts, promoting the occurrence of tumors by secreting cytokines or cells, especially tumor related fibroblasts. Cell (CAFs), an active fibroblast in the tumor microenvironment, plays an important role in the progression and metastasis of tumor,.TGF- beta 1 as a transforming growth factor, plays an important role in tumor microenvironment, which can promote the growth of tumor and inhibit the growth of tumor. However, the role of TGF- beta 1 in the tumor microenvironment The system is not clear. Moreover, our clinical results show that the expression of TGF- beta 1 in the tumor tissues of the breast cancer patients is positively related to the expression of the phenotypic marker protein alpha -SMA of CAFs, indicating that the expression of TGF- beta 1 is related to the formation of CAFs, but the mechanism needs to be further explored. First, the subject will make the mouse embryonic fibroblast cells NIH3T3 Serum starvation was 24 h and an in vitro nutrient deprivation model was established to simulate the conditions of nutritional deprivation in the tumor microenvironment. The effect of TGF- beta 1 on serum starved NIH3T3 cells at a certain concentration (2.5 ng/ml) was investigated. The results showed that TGF- beta 1 not only alleviated the growth inhibition, mitochondrial damage and apoptosis, but also the apoptosis, and TGF - beta 1 could induce the formation of CAFs phenotypes of starved fibroblast NIH3T3. The results also showed that TGF- beta 1 enhanced the MDC positive rate of NIH3T3 cells in starvation treatment, the expression of autophagy related genes and proteins, while the autophagy inhibitor 3-MA blocked the role of TGF- beta 1 and reduced the occurrence of autophagy. Confocal and electron microscopy results entered into one. TGF- beta 1 enhanced the autophagy of NIH3T3 cells treated by starvation. Secondly, autophagy is one of the main mechanisms to repair cell damage under stressful conditions, and autophagy is closely related to the occurrence and development of tumor. Therefore, this subject studies and discusses T with the enhancement of the autophagy function of NIH3T3 cells under the starvation of serum in the TGF- beta. GF- beta 1 alleviates the cell damage induced by serum starvation and the mechanism of inducing the phenotypic formation of CAFs in NIH3T3 cells. To this end, we conducted the following experiments in sequence: (1) whether autophagy is involved in the cytoprotection and CAFs transformation induced by TGF- beta 1 by autophagy (Rapa) and autophagy inhibitor (3-MA). The autophagy enhancer further enhanced the cell proliferation and mitochondrial function induced by TGF- beta 1, while the autophagy inhibitor inhibited the effect of TGF- beta 1 and blocked the anti apoptosis and CAFs transformation effect of TGF- beta 1. (2) the level of autophagy, apoptosis and necrosis, as well as the CAFs phenotypic protein alpha -SMA and FAP- alpha, were detected by siRNA technology to interfere with Atg5. The results showed that Atg5 siRNA not only blocked the occurrence of autophagy induced by TGF- beta 1, but also abolished the effect of TGF- beta 1 on NIH3T3 cells treated by starvation, suggesting that autophagy is the target of TGF- beta 1 to alleviate NIH3T3 cell damage and induce CAFs phenotype formation under the starvation of serum. (3) we use TGF- beta R1/ALK5 inhibitor LY-2157 299 the specific molecular mechanisms of TGF- beta 1 mediated autophagy to alleviate NIH3T3 cell damage in serum starvation and to induce the formation of CAFs phenotypes were investigated. The results showed that LY-2157299 could block the classical pathway -Smad pathway of TGF- beta 1, and further blocked the expression of autophagy related proteins induced by TGF- beta 1 and the table of CAFs phenotypic markers. Together with all the above results, we infer that TGF- beta can enhance mitochondrial autophagy by activating the Smad pathway, removing damaged mitochondria and reducing cell apoptosis in nutritional deprivation, to some extent repair cell damage, and TGF- beta can be induced by the Smad/ autophagy pathway to induce fibrinolysis in the nutritional deprivation state. Finally, in order to further verify that TGF- beta 1 mediated autophagy can repair damage to fibroblasts in the state of nutritional deprivation and induce the formation of CAFs phenotypes in the tumor microenvironment, we use a murine mixed transplant tumor model, that is, the proportion of mouse breast cancer cell 4T1 and mouse fibroblast NIH3T3 to 1:2. In Balb/c mice, the effect of TGF- beta 1 induced autophagy on the tumor microenvironment fibroblasts and its effect on the tumor development experiment. The experimental results showed that the serum starvation significantly increased the autophagy level of the tumor tissue compared with the normal group, but inhibited the growth of the tumor and increased the swelling. The level of apoptosis and necrosis of the tumor tissue. TGF- beta 1 can significantly enhance the autophagy level of tumor tissue and induce the formation of CAFs phenotypes, promote the growth of the tumor and alleviate the apoptosis and necrosis of the tumor tissue, while the autophagy inhibitor 3-MA inhibits the autophagy induced by TGF- beta 1 and abolishes the CAFs transformation induced by TGF- beta 1. In conclusion, autophagy induced by TGF- beta 1 promotes the survival of fibroblasts in the nutritional deprivation state of the tumor microenvironment, promotes the formation of CAFs phenotypes and promotes the growth of the tumor. The molecular mechanism of TGF- beta 1 to induce CAFs transformation in the tumor microenvironment in order to promote the growth of the tumor is elucidated. The results of this study provide a new target for research and development of anti breast cancer drugs.
【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

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