【摘要】：前列腺癌(Prostate Cancer,PCa)作為世界上男性第二大發(fā)病的腫瘤性疾病,已嚴(yán)重影響全球男性的身心健康與生活質(zhì)量。前列腺癌轉(zhuǎn)移發(fā)生率高,許多患者診斷時(shí)已伴有骨轉(zhuǎn)移等。對(duì)于轉(zhuǎn)移性激素敏感性前列腺癌(metastatic hormone-sensitive prostate cancer,mHSPC),治療首選雄激素剝奪治療(androgen deprivation therapy,ADT),其次有化療、免疫治療等。盡管ADT治療有效率高達(dá)80%,但絕大多數(shù)患者在治療后的一到兩年內(nèi)疾病會(huì)繼續(xù)惡化,進(jìn)展為轉(zhuǎn)移性去勢(shì)抵抗性前列腺癌(metastatic castration-resistant prostate cancer,mCRPC)。多西他賽(docetaxel)作為mCRPC的一線化療藥,因可顯著延長(zhǎng)患者的生存率,而被廣泛應(yīng)用。然而,幾乎全部mCRPC患者在接受多西他賽化療后均會(huì)出現(xiàn)抵抗性,從而形成多西他賽抵抗性前列腺癌(docetaxel resistant prostate cancer,DRPC)。深入研究DRPC的形成機(jī)制,對(duì)于患者的治療至關(guān)重要。本實(shí)驗(yàn)室其他研究小組發(fā)現(xiàn)在DRPC細(xì)胞中,AR的表達(dá)明顯增強(qiáng),其AR信號(hào)通路相關(guān)基因的表達(dá)也發(fā)生了相應(yīng)改變。選取本實(shí)驗(yàn)室已有的高表達(dá)AR的PC3-AR9細(xì)胞與AR陰性的PC3細(xì)胞,在不同梯度濃度的多西他賽作用下,PC3-AR9細(xì)胞的死亡率顯著低于PC3細(xì)胞(P0.05)。從而證明,AR信號(hào)通路的激活參與DRPC的形成。PC3-AR9細(xì)胞與PC3細(xì)胞在不同濃度的多西他賽處理后,應(yīng)用碘化丙啶(Propidium iodide,PI)與赫斯特?zé)晒馊玖?3258(Hoechst33258)行熒光染色,共聚焦顯微鏡下發(fā)現(xiàn)PC3細(xì)胞會(huì)發(fā)生壞死性凋亡(Necroptosis),且壞死性凋亡率隨藥物處理濃度增加而升高;而PC3-AR9細(xì)胞的壞死性凋亡率均顯著低于PC3細(xì)胞(P0.0001)。PC3細(xì)胞內(nèi)壞死復(fù)合體(Necrosome)核心蛋白混合譜系激酶結(jié)構(gòu)區(qū)域樣蛋白(Mixed Lineage Kinase Domain-like Protein,MLKL)的總表達(dá)量與磷酸化表達(dá)量,會(huì)隨多西他賽處理濃度的增加而增加;而PC3-AR9細(xì)胞的MLKL的總表達(dá)量與磷酸化表達(dá)量則不隨多西他賽處理濃度變化,且表達(dá)量均低于PC3細(xì)胞�？偨Y(jié)數(shù)據(jù)得出,多西他賽可誘導(dǎo)前列腺癌細(xì)胞系發(fā)生壞死性凋亡,且AR可抑制多西他賽對(duì)壞死性凋亡的誘導(dǎo)作用。在TNF-α+Smac+Z-VAD-fmk(TSZ)聯(lián)合誘導(dǎo)下建立壞死性凋亡細(xì)胞模型;在ShAR質(zhì)粒轉(zhuǎn)染下,建立敲減AR的LNCaP與C4-2細(xì)胞模型。在TSZ誘導(dǎo)下,PC3、C4-2-Sh AR與LNCaP-ShAR細(xì)胞的死亡率分別高于PC3-AR9(P0.01)、C4-2-Scramble(P0.01)與LNCa P-Scramble(P0.05)細(xì)胞。在TSZ誘導(dǎo)后應(yīng)用PI與Hoechst33258行熒光染色,可見(jiàn)PC3細(xì)胞發(fā)生了壞死性凋亡,且其壞死率顯著高于PC3-AR9細(xì)胞。同理驗(yàn)證,發(fā)現(xiàn)C4-2-ShAR與LNCaP-ShAR細(xì)胞也發(fā)生壞死性凋亡,且壞死率均分別高于C4-2-Scramble與LNCaP-Scramble細(xì)胞。表明AR的激活可抑制壞死性凋亡的發(fā)生,且隨AR表達(dá)量的升高其對(duì)壞死性凋亡的抑制作用也在增強(qiáng)。后提取TSZ處理后的PC3與PC3-AR9細(xì)胞蛋白,Western Blot檢測(cè)后證實(shí)在AR存在的PC3-AR9細(xì)胞中MLKL的總表達(dá)量與磷酸化表達(dá)量均明顯低于PC3細(xì)胞�？傻弥�,AR通過(guò)抑制MLKL的表達(dá)及磷酸化而抑制壞死性凋亡。后應(yīng)用MLKL第86位半胱氨酸共價(jià)調(diào)節(jié)劑Necrosulfonamide(NSA)與TSZ共同處理,可逆轉(zhuǎn)C4-2-ShAR與LNCaP-ShAR細(xì)胞的壞死性凋亡發(fā)生。TSZ聯(lián)合NSA共同處理與TSZ單獨(dú)處理相比,C4-2-Scramble與LNCaP-Scramble細(xì)胞的壞死性凋亡率無(wú)明顯變化。同樣處理?xiàng)l件下,熒光染色觀察C42-shAR與C42-Scramble細(xì)胞的差異,發(fā)現(xiàn)NSA可抑制細(xì)胞壞死性凋亡的發(fā)生,且AR的抑制作用與NSA相似。這也間接證實(shí),AR對(duì)壞死性凋亡的抑制作用是通過(guò)抑制MLKL的表達(dá)與磷酸化而實(shí)現(xiàn)的。選取42例原發(fā)性前列腺癌組織標(biāo)本行免疫組化分析,基于AR表達(dá)含量的不同,分為低表達(dá)組(Low)與高表達(dá)組(High),發(fā)現(xiàn)隨AR表達(dá)的增強(qiáng),RIP3(R=-0.919,P0.01)與MLKL(R=-0.909,P0.01)的表達(dá)均減弱。從而體內(nèi)證實(shí),PCa中AR的激活抑制了壞死性凋亡的發(fā)生,且隨AR表達(dá)量的升高其對(duì)壞死性凋亡的抑制作用也在增強(qiáng)。上述結(jié)果顯示,多西他賽通過(guò)促進(jìn)PCa細(xì)胞發(fā)生壞死性凋亡而對(duì)mCRPC起到治療作用,但在治療過(guò)程中,由于AR信號(hào)通路的重新建立,AR表達(dá)水平逆轉(zhuǎn)性升高,抑制了MLKL的表達(dá)與磷酸化,轉(zhuǎn)而抑制壞死性凋亡,且隨AR表達(dá)量的增加其對(duì)壞死性凋亡的抑制作用也在增強(qiáng),最終導(dǎo)致mCRPC對(duì)多西他賽出現(xiàn)抵抗性,發(fā)展為DRPC。對(duì)于mCRPC的患者,早期多西他賽化療結(jié)合ADT治療方案可能是新的治療策略。
[Abstract]:Prostate Cancer (PCa), as the second most common oncology disease in the world, has seriously affected the physical and mental health and quality of life of men worldwide. The incidence of metastatic prostate cancer is high, and many patients have been diagnosed with bone metastases at the time of diagnosis. For metastatic hormone sensitive prostate cancer (metastatic hormone-sensitive prostat) E cancer, mHSPC), the first choice for androgen deprivation therapy (androgen deprivation therapy, ADT), followed by chemotherapy and immunotherapy. Although the effective rate of ADT therapy is up to 80%, the overwhelming majority of patients will continue to deteriorate within one to two years after treatment, progressing to metastatic castration resistant prostate cancer (metastatic castration-resistan). T prostate cancer, mCRPC). As a first-line chemotherapeutic agent for mCRPC, docetaxel is widely used because it can significantly prolong the survival rate of patients. However, almost all patients with mCRPC are resistant to chemotherapy after docetaxel, thus forming a poly (docetaxel resistant prostate cancer, D), D (cancer, D). RPC). In-depth study of the formation mechanism of DRPC is very important for the treatment of patients. Other research teams in this laboratory have found that the expression of AR is obviously enhanced in DRPC cells, and the expression of the AR signaling pathway related genes also changes. The selection of PC3-AR9 cells with high expression of AR in our laboratory and AR negative PC3 cells are different. With the gradient concentration of docetaxel, the death rate of PC3-AR9 cells was significantly lower than that of PC3 cells (P0.05). Thus, the activation of AR signaling pathway was involved in DRPC formation of.PC3-AR9 cells and PC3 cells at different concentrations of docetaxel, and the use of propidium iodide (Propidium iodide, PI) and Hearst fluorescent dye 33258 (Hoechst33258) The necrotizing apoptosis (Necroptosis) was found in PC3 cells under confocal microscopy, and the necrotic apoptosis rate increased with the increase of drug concentration, while the necrotic apoptosis rate of PC3-AR9 cells was significantly lower than that of the PC3 cell (P0.0001).PC3 cell necrosis complex (Necrosome) core protein mixed pedigree kinase structure. The total expression and phosphorylation of protein (Mixed Lineage Kinase Domain-like Protein, MLKL) increased with the concentration of docetaxel, while the total expression and phosphorylation of MLKL in PC3-AR9 cells did not change with the concentration of docetaxel, and the expression was lower than that of PC3 cells. The cell line can induce necrotic apoptosis in the prostate cancer cell line, and AR can inhibit the inducing effect of docetaxel on necrotic apoptosis. The necrotizing apoptotic cell model is established under the joint induction of TNF- alpha +Smac+Z-VAD-fmk (TSZ); the LNCaP and C4-2 cell model of the knockout AR is established under the ShAR plasmid. PC3, C4-2-Sh AR and PC3 under the induction of TSZ The mortality of AR cells was higher than that of PC3-AR9 (P0.01), C4-2-Scramble (P0.01) and LNCa P-Scramble (P0.05) cells. The necrotizing apoptosis was observed in PI and Hoechst33258 line after TSZ induction, and the necrosis rate of the PC3 cells was significantly higher than that of the cells. Apoptosis and necrosis rate were higher than that of C4-2-Scramble and LNCaP-Scramble cells respectively. The results showed that the activation of AR inhibited the occurrence of necrotic apoptosis, and the inhibitory effect on necrotic apoptosis was also enhanced with the increase of AR expression. Then, PC3 and PC3-AR9 cell protein after TSZ treatment were extracted, and Western Blot detected PC3-AR9 thin AR after Western Blot detection. The total expression and phosphorylation of MLKL in the cell were significantly lower than that of PC3 cells. It was found that AR inhibited the necrotic apoptosis by inhibiting the expression of MLKL and phosphorylation. Then the MLKL eighty-sixth cysteine covalent modifier Necrosulfonamide (NSA) was treated with TSZ, which could reverse the necrotic apoptosis of C4-2-ShAR and LNCaP-ShAR cells. Compared with the combined treatment of NSA and TSZ alone, the necrotic apoptosis rate of C4-2-Scramble and LNCaP-Scramble cells was not significantly changed. Under the same treatment conditions, the difference between C42-shAR and C42-Scramble cells was observed by fluorescence staining. It was found that NSA inhibited the occurrence of necrotic apoptosis of cells, and the inhibition of AR was similar to NSA, which was also indirectly confirmed. The inhibitory effect of AR on necrotic apoptosis was achieved by inhibiting the expression and phosphorylation of MLKL. 42 cases of primary prostate cancer specimens were selected by immunohistochemical analysis. Based on the difference of AR expression, they were divided into low expression group (Low) and high expression group (High). The enhancement of AR expression, RIP3 (R=-0.919, P0.01) and MLKL (R=-0.909, P0.01) were found. The expression of AR was weakened in vivo. In vivo, the activation of PCa inhibited the occurrence of necrotic apoptosis and increased the inhibitory effect on necrotic apoptosis with the increase of AR expression. The results showed that docetaxel could play a therapeutic role in mCRPC by promoting necrotic apoptosis in PCa cells, but in the course of treatment, because AR The reestablishment of the signal pathway, the elevation of AR expression level, the inhibition of the expression and phosphorylation of MLKL, and the inhibition of necrotic apoptosis, and the increase of the inhibitory effect on necrotic apoptosis with the increase of AR expression, eventually lead to the resistance of mCRPC to docetaxel, and the development of DRPC. to mCRPC patients and early docetaxel. Chemotherapy combined with ADT regimen may be a new therapeutic strategy.
【學(xué)位授予單位】：天津醫(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)】：R737.25

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