發(fā)布時(shí)間：2018-05-18 11:36

  本文選題：轉(zhuǎn)化生長(zhǎng)因子(TGF-β1) + 自噬��； 參考：《暨南大學(xué)》2016年博士論文

【摘要】：腫瘤微環(huán)境已經(jīng)成為腫瘤治療的重要靶點(diǎn)。對(duì)于許多實(shí)體瘤而言,尤其是癌組織,他們的微環(huán)境由腫瘤細(xì)胞本身、內(nèi)皮細(xì)胞、免疫細(xì)胞和成纖維細(xì)胞組成,通過(guò)分泌細(xì)胞因子或者細(xì)胞之間互相作用促進(jìn)腫瘤的發(fā)生。尤其是腫瘤相關(guān)成纖維細(xì)胞(CAFs),是腫瘤微環(huán)境中激活的成纖維細(xì)胞,在腫瘤的發(fā)展進(jìn)程和轉(zhuǎn)移中起著重要作用。TGF-β1作為一個(gè)轉(zhuǎn)化生長(zhǎng)因子,在腫瘤微環(huán)境起著重要的作用,既可以促進(jìn)腫瘤的生長(zhǎng),也可抑制腫瘤的生長(zhǎng)。但是,TGF-β1在腫瘤微環(huán)境中的作用機(jī)制尚不明確。而且,我們的臨床結(jié)果顯示在乳腺癌病人的腫瘤組織中TGF-β1的表達(dá)與CAFs的表型標(biāo)志蛋白α-SMA的表達(dá)成正相關(guān)關(guān)系,說(shuō)明TGF-β1的表達(dá)與CAFs的形成有關(guān),但是其機(jī)制有待進(jìn)一步探討。首先,本課題將小鼠胚胎成纖維細(xì)胞NIH3T3血清饑餓24 h,建立體外營(yíng)養(yǎng)剝奪模型,也是為了模擬腫瘤微環(huán)境中營(yíng)養(yǎng)剝奪的條件。并考察TGF-β1在一定濃度下(2.5 ng/ml)對(duì)血清饑餓的NIH3T3細(xì)胞的作用。實(shí)驗(yàn)結(jié)果顯示TGF-β1不僅緩解了饑餓導(dǎo)致的生長(zhǎng)抑制、線粒體損傷和細(xì)胞凋亡,而且TGF-β1能夠誘導(dǎo)饑餓的成纖維細(xì)胞NIH3T3的CAFs表型的形成。實(shí)驗(yàn)結(jié)果還顯示,TGF-β1能夠增強(qiáng)經(jīng)饑餓處理NIH3T3細(xì)胞的MDC陽(yáng)性率、自噬相關(guān)基因和蛋白的表達(dá),而自噬抑制劑3-MA卻阻斷了TGF-β1的作用,降低了自噬的發(fā)生。共聚焦和電鏡的結(jié)果進(jìn)一步發(fā)現(xiàn)TGF-β1增強(qiáng)經(jīng)饑餓處理的NIH3T3細(xì)胞自噬的發(fā)生。其次,自噬是應(yīng)激條件下修復(fù)細(xì)胞損傷的主要機(jī)制之一,并且自噬與腫瘤的發(fā)生和發(fā)展也密切相關(guān)。因此,本課題以TGF-β1增強(qiáng)血清饑餓狀態(tài)下NIH3T3細(xì)胞自噬功能的角度研究和探討了TGF-β1緩解血清饑餓導(dǎo)致的細(xì)胞損傷和誘導(dǎo)NIH3T3細(xì)胞CAFs的表型形成的作用機(jī)理。為此,我們依次進(jìn)行了以下實(shí)驗(yàn):(1)采用自噬促進(jìn)劑(Rapa)和自噬抑制劑(3-MA)探討了自噬是否參與了TGF-β1誘導(dǎo)的細(xì)胞保護(hù)作用和CAFs的轉(zhuǎn)變作用,實(shí)驗(yàn)結(jié)果顯示自噬促進(jìn)劑進(jìn)一步增強(qiáng)TGF-β1誘導(dǎo)的細(xì)胞增殖和線粒體功能,而自噬抑制劑則抑制了TGF-β1的作用,阻斷了TGF-β1的抗凋亡作用和CAFs轉(zhuǎn)變作用。(2)采用siRNA技術(shù)干擾Atg5后檢測(cè)自噬水平、細(xì)胞凋亡和壞死情況以及CAFs表型蛋白α-SMA和FAP-α的表達(dá)。實(shí)驗(yàn)結(jié)果顯示Atg5 siRNA不僅阻斷了TGF-β1誘導(dǎo)的自噬的發(fā)生,而且廢除了TGF-β1對(duì)經(jīng)饑餓處理NIH3T3細(xì)胞的作用,提示自噬是TGF-β1緩解血清饑餓狀態(tài)下NIH3T3細(xì)胞損傷和誘導(dǎo)CAFs表型形成的作用靶點(diǎn)。(3)我們采用TGF-βR1/ALK5抑制劑LY-2157299探討了TGF-β1介導(dǎo)的自噬緩解血清饑餓狀態(tài)下NIH3T3細(xì)胞損傷和誘導(dǎo)CAFs表型形成的具體分子機(jī)制。實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)LY-2157299能夠阻斷TGF-β1的經(jīng)典通路-Smad通路,而且進(jìn)一步阻斷了TGF-β1誘導(dǎo)的自噬相關(guān)蛋白的表達(dá)和CAFs表型標(biāo)志蛋白的表達(dá)。綜合以上所有結(jié)果,我們推斷出TGF-β可以通過(guò)激活Smad通路提高線粒體自噬的水平,在營(yíng)養(yǎng)剝奪狀態(tài)下清除受損的線粒體和降低細(xì)胞凋亡水平,從而一定程度上修復(fù)細(xì)胞損傷;并且TGF-β可通過(guò)Smad/自噬通路在營(yíng)養(yǎng)剝奪狀態(tài)下誘導(dǎo)成纖維細(xì)胞CAFs表型的形成。最后,為了進(jìn)一步驗(yàn)證TGF-β1介導(dǎo)的自噬在營(yíng)養(yǎng)剝奪狀態(tài)下能夠修復(fù)成纖維細(xì)胞的損傷和誘導(dǎo)腫瘤微環(huán)境中CAFs表型的形成,我們采用小鼠混合移植瘤模型,即小鼠乳腺癌細(xì)胞4T1和小鼠成纖維細(xì)胞NIH3T3以1:2的比例混合接入Balb/c小鼠內(nèi),從體內(nèi)去探討TGF-β1誘導(dǎo)的自噬對(duì)腫瘤微環(huán)境中成纖維細(xì)胞的作用以及其對(duì)腫瘤發(fā)展進(jìn)程實(shí)驗(yàn)的影響。實(shí)驗(yàn)結(jié)果顯示,與正常組相比,血清饑餓顯著提高了腫瘤組織的自噬水平,但是卻抑制了腫瘤的生長(zhǎng),增加了腫瘤組織的凋亡和壞死水平。而TGF-β1能夠顯著增強(qiáng)腫瘤組織的自噬水平和誘導(dǎo)CAFs表型的形成,并且促進(jìn)了腫瘤的生長(zhǎng),也緩解了腫瘤組織的凋亡和壞死情況。而自噬抑制劑3-MA則抑制了TGF-β1誘導(dǎo)的自噬,廢除了TGF-β1誘導(dǎo)的CAFs轉(zhuǎn)變作用,從而抑制了腫瘤的生長(zhǎng)。綜上所述,TGF-β1誘導(dǎo)的自噬促進(jìn)了腫瘤微環(huán)境中的營(yíng)養(yǎng)剝奪狀態(tài)下成纖維細(xì)胞的存活,并且促進(jìn)了CAFs表型的形成,從而促進(jìn)了腫瘤的生長(zhǎng)。闡明了TGF-β1在腫瘤微環(huán)境中誘導(dǎo)CAFs轉(zhuǎn)變以促進(jìn)腫瘤生長(zhǎng)的分子機(jī)制。本實(shí)驗(yàn)研究結(jié)果為研究和開(kāi)發(fā)抗乳腺惡性腫瘤藥物提供了新的作用靶點(diǎn)。
[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.
【學(xué)位授予單位】：暨南大學(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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