本文選題：激素依賴性前列腺癌 + 激素非依賴性前列腺癌��； 參考：《吉林大學》2015年博士論文

【摘要】：前列腺癌是歐美國家男性最常見的惡性腫瘤，死亡率居男性腫瘤的第二位。我國前列腺癌的發(fā)病率雖然低于西方國家，但近年來呈顯著增長且年輕化趨勢。 前列腺癌的持續(xù)和擴散依賴于雄激素受體傳導信號，因此雄激素去勢治療是除外科手術(shù)與放射治療外的標準前列腺癌治療方法，多數(shù)前列腺癌患者在首次接受雄激素去勢治療后都有顯著療效。但經(jīng)過短暫的緩解期后，幾乎所有患者都會復發(fā)并由雄激素依賴性前列腺癌發(fā)展成高度惡化且廣泛轉(zhuǎn)移的雄激素非依賴性前列腺癌，，使常規(guī)的雄激素去勢治療失去效果。轉(zhuǎn)變后的前列腺癌治療非常棘手，死亡率極高，因此成為危害老年男性人群健康的重大疾病。 對AIPC的轉(zhuǎn)變機制的研究，是目前國內(nèi)外前列腺癌研究領(lǐng)域的熱點，主要包括：雄激素受體基因的擴增、過表達以及突變，AR信號通路異常等�；虮磉_的改變及信號通路異常，原癌基因、抑癌基因以及生長因子調(diào)控失調(diào)被認為是ADPC發(fā)展為AIPC的主要原因，研究人員運用各種技術(shù)發(fā)現(xiàn)了大量前列腺癌的相關(guān)基因及信號通路。但是前列腺癌的發(fā)生發(fā)展過程十分復雜，呈現(xiàn)多步驟、多基因復合的特點。另外，多數(shù)AIPC研究都是以激素非依賴性細胞PC3、DU145作為模型，因其不表達內(nèi)源性雄激素受體而產(chǎn)生雄激素抵抗。但前列腺癌的發(fā)生發(fā)展，以及從激素依賴到非依賴的轉(zhuǎn)變，都不可避免的經(jīng)歷激素與受體的結(jié)合，經(jīng)雄激素-雄激素受體信號通路，將信號傳遞至細胞內(nèi)特異靶基因而發(fā)揮作用。因此，以上理論都不能合理地闡述AIPC的發(fā)生機制，雄激素非依賴性前列腺癌發(fā)病仍需進一步深入研究。 本研究選擇保留了人前列腺癌功能分化特征，同時具有很強雄激素依賴性的LNCaP細胞系作為對象，建立能模擬臨床前列腺癌由雄激素依賴轉(zhuǎn)變?yōu)樾奂に胤且蕾囘^程的細胞模型，采用現(xiàn)代細胞生物學、分子生物學、生物信息學技術(shù)探索人前列腺癌基因表達改變，進一步揭示雄激素依賴性前列腺癌向雄激素非依賴性前列腺癌轉(zhuǎn)化過程中可能的發(fā)生機制，有望為雄激素非依賴性前列腺癌的治療提供新的理論依據(jù)。 1.雄激素非依賴性前列腺癌亞細胞模型的建立 選擇保留了人前列腺癌功能分化特征的LNCaP細胞為基礎(chǔ)，以激素遞減法誘導建立雄激素非依賴性前列腺癌細胞模型。LNCaP細胞分別經(jīng)過去雄激素培養(yǎng)液和去雄激素+氟他胺環(huán)境連續(xù)傳代培養(yǎng)后，觀察細胞形態(tài)學改變并檢測細胞增殖能力、侵襲和遷移能力、PSA分泌等生物學活性變化。 結(jié)果可見，經(jīng)去雄激素誘導并連續(xù)培養(yǎng)30代的LNCaP-A細胞形態(tài)發(fā)生明顯變化：細胞胞體變小，細胞形態(tài)由三角形、長梭型、圓形變?yōu)椴灰?guī)則；細胞不再均勻分布，多見單個細胞獨立生長，同時出現(xiàn)聚集生長現(xiàn)象。細胞增殖能力增高，可以在去雄激素環(huán)境快速生長，并對雄激素競爭性抑制劑氟他胺產(chǎn)生抵抗。細胞的侵襲性及遷移能力也大大增強，在無激素環(huán)境下仍能恢復部分PSA的分泌，且隨時間的延長而增加。說明該細胞模型從形態(tài)學以及生物學行為上都很接近臨床內(nèi)分泌治療形成的AIPC過程，可以用來進行前列腺癌在細胞學以及分子生物學上的發(fā)生機制的初探。 2.雄激素非依賴性前列腺癌基因表達變化 以基因芯片技術(shù)對雄激素依賴性LNCaP細胞和雄激素非依賴亞細胞模型LNCaP-A進行全基因表達譜掃描，篩選AIPC形成過程中差異表達基因，同時整合KEGG和PANTHER的在線分析以及MAS等生物信息學工具，對AIPC相關(guān)基因進行生物信息通路、基因調(diào)控網(wǎng)絡及生物學功能等多方面分析，以了解ADPC轉(zhuǎn)變成AIPC后的基因表達變化，并在此基礎(chǔ)上篩選ADPC轉(zhuǎn)變成AIPC的關(guān)鍵基因及可能信號通路。 對基因芯片測得的原始數(shù)據(jù)進行分析處理，共比對表達基因12207個。以差異倍數(shù)＞2.0為標準，得到雄激素非依賴LNCaP-A細胞與對照組LNCaP細胞差異表達基因347個，其中表達上調(diào)基因156個，表達下調(diào)基因191個，表明激素非依賴性的前列腺癌細胞在基因表達上發(fā)生了明顯變化。而對比雄激素非依賴LNCaP-A細胞與去雄激素后再以氟他胺處理的LNCaP-A+F細胞，兩組細胞基因表達則無明顯差異，這也說明經(jīng)去雄激素誘導產(chǎn)生的激素非依賴性的前列腺癌細胞生長不再受到雄激素抑制劑的影響，對雄激素抑制劑產(chǎn)生抵抗。 應用ToppGene工具對差異表達基因進行歸類分析，結(jié)果顯示下調(diào)的基因主要涉及膜蛋白、跨膜蛋白、離子結(jié)合、細胞間信號傳導、基因表達調(diào)控等功能，上調(diào)基因的基因功能主要涉及細胞內(nèi)部基因調(diào)控、表達、代謝、可變剪切等。參與細胞內(nèi)部基因調(diào)控、表達、新陳代謝等功能的基因表達水平升高，參與外源性物質(zhì)進入細胞內(nèi)部的膜蛋白、信號傳導，以及細胞間識別粘附等作用的基因表達水平降低，這也與非依賴性前列腺癌細胞不再受外部去激素調(diào)控的實驗現(xiàn)象吻合。 結(jié)合差異表達基因的功能、生物學過程和已發(fā)表文獻，對該過程中的關(guān)鍵基因及可能涉及的信號通路進行初步分析，表達上調(diào)基因中主要有4個基因涉及了4種類型的信號通路，而表達下調(diào)基因中有7個基因涉及了多種類型的信號通路。這些基因及相關(guān)信號通路的提出為進一步研究AIPC發(fā)生機制提供了方向。 3.雄激素非依賴性前列腺癌機制的初步探討 采用RT-PCR及western blot方法對TLR2介導的凋亡作用途徑、PI3K/AKT途徑、MAPK途徑等信號通路關(guān)鍵蛋白進行驗證，探討其在激素依賴性前列腺癌向激素非依賴性轉(zhuǎn)化過程中的可能作用。 Western結(jié)果可見，caspase3、FADD蛋白表達水平均無明顯變化，且均未見激活caspase3條帶，提示toll-like信號通路中的凋亡作用途徑?jīng)]有參與非激素依賴性前列腺癌的轉(zhuǎn)變。AKT、p-AKT表達均無明顯增高，但NF-κB蛋白表達上調(diào)，IκBα蛋白表達下降。提示NF-κB可能為信號通路中一個關(guān)鍵位點，但在LNCaP-A細胞系中不是通過PI3K/AKT通路激活，可能存在著其他激活通路，抑制蛋白IκBα表達的下調(diào)可能影響了NF-κB表達變化。 對MAPK信號通路相關(guān)蛋白進行檢測，ERK、MEK、JNK蛋白表達均無顯著變化，而p38、HSP27、IL-8表達顯著升高，提示在LNCaP細胞轉(zhuǎn)變?yōu)長NCaP-A細胞的過程中，ERK途徑、JNK途徑?jīng)]有參與，但MAPK/p38信號通路發(fā)揮了重要作用，IL-8、HSP70都可能是該途徑中重要的信息傳遞分子。 有研究表明MAPK/p38在乳腺癌、卵巢癌、子宮內(nèi)膜癌耐藥機制中發(fā)揮著重要作用，但在前列腺癌激素耐藥中尚無報道。這為AIPC機制的研究以及AIPC藥物作用靶點的發(fā)掘提供了新的理論基礎(chǔ)。
[Abstract]:Prostate cancer is the most common malignant tumor in the European and American countries, with the death rate of the second male tumor. Although the incidence of prostate cancer in China is lower than that of the western countries, it has been increasing and young in recent years.
The persistence and diffusion of prostate cancer is dependent on the androgen receptor conduction signal, so androgen castration is a standard treatment for prostate cancer except for surgical and radiological treatment. Most of the prostate cancer patients have a significant effect after the first androgenic treatment. After a short period of remission, almost all patients are treated. The recurrent androgen dependent prostate cancer is developed into a highly deteriorating and widely metastatic androgen independent prostate cancer, which makes the routine androgenic treatment of androgen deprivation treatment out of effect. The post transition prostate cancer is very difficult to treat and has a very high mortality rate, so it has become a major disease that endangers the health of old men.
The research on the transformation mechanism of AIPC is a hot spot in the field of prostate cancer at home and abroad. It mainly includes the amplification, overexpression and mutation of androgen receptor gene, abnormal AR signaling pathway, the change of gene expression and the abnormal signal pathway. The proto oncogene, tumor suppressor base and the regulation of growth factors are considered to be the development of ADPC. The main reason for AIPC is that the researchers have used various techniques to discover a large number of genes and signaling pathways in prostate cancer. However, the development of prostate cancer is very complex and has the characteristics of multistep and multi gene recombination. In addition, most of the AIPC studies are based on the hormone non dependent PC3, DU145 as a model, because of its non expression. Androgen resistance is produced by the androgenic androgen receptor. However, the development of prostate cancer, as well as the hormone dependent to non dependent transformation, inevitably experience the combination of hormones and receptors, and the signal passes through the androgen receptor signaling pathway to the specific target genes in the cell. The pathogenesis of AIPC is discussed, and the pathogenesis of androgen independent prostate cancer still needs further study.
In this study, we chose to retain the functional differentiation of human prostate cancer, with a strong androgen dependent LNCaP cell line as an object, to establish a cell model that can simulate the transformation of prostatic cancer from androgen dependence to androgens, and to explore people with modern cell biology, molecular biology and bioinformatics. The changes in the gene expression of prostate cancer further reveal the possible mechanism of androgen dependent prostate cancer in the process of androgen independent prostate cancer transformation. It is expected to provide a new theoretical basis for the treatment of androgen independent prostate cancer.
Establishment of a subcellular model for androgen independent prostate cancer 1.
On the basis of LNCaP cells, which have retained the characteristics of human prostate cancer function differentiation, the androgen independent prostate cancer cell model.LNCaP cells were induced by hormone decreasing method, and the cell morphology changes were observed and the cell proliferation ability was detected by the continuous subculture of androgen and androgen plus flutamide. Invasion and migration, PSA secretion and other biological activities.
The results showed that the morphology of LNCaP-A cells, which were induced by androgens and continuously cultured for 30 generations, showed that the cell body became smaller and the cell morphology changed from triangle, long shuttle type and round to irregular, and the cells were no longer uniformly distributed. The androgen environment is fast growing and resists the androgen competitive inhibitor fluroamine. The cell invasiveness and migration ability is also greatly enhanced, and the secretion of partial PSA can be restored in the hormone free environment, and increases with the time. It shows that the cell model is close to the clinical endocrinology from the morphological and biological behavior. The AIPC process can be used to explore the mechanism of prostate cancer in cytology and molecular biology.
2. gene expression changes in androgen independent prostate cancer
Using gene chip technology to scan the full gene expression spectrum of androgen dependent LNCaP cells and androgen independent subcellular model LNCaP-A, screening the differentially expressed genes in the process of AIPC formation, and integrating the online analysis of KEGG and PANTHER and MAS and other bioinformatics tools to carry out the biological information pathway and gene modulation of the AIPC related genes. In order to understand the changes in gene expression after the transformation of ADPC into AIPC, the changes of gene expression after the transformation of ADPC into AIPC were analyzed, and on this basis, the key genes and possible signaling pathways of AIPC were screened.
The original data measured by gene chip were analyzed and compared with 12207 expression genes. The difference multiplier > 2 was used as the standard to obtain 347 differentially expressed genes of androgen independent LNCaP-A cells and control group LNCaP cells, including 156 up-regulated genes and 191 down-regulation groups, indicating that hormone non dependent prostate cancer is fine. There was a significant change in the cell gene expression, but there was no significant difference in gene expression between the two groups of androgen independent LNCaP-A cells and the LNCaP-A+F cells treated with androgens and flutamide, which also indicated that the growth of the hormone non dependent prostatic adenocarcinoma cells induced by androgens was no longer inhibited by androgens. The effect of the agent is resistant to androgen inhibitors.
The ToppGene tool is used to classify the differentially expressed genes. The results show that the down regulated genes are mainly involved in membrane protein, transmembrane protein, ion binding, intercellular signal transduction, gene expression regulation and so on. The regulation of gene regulation, expression, metabolism and variable shear are involved in the gene function of the gene. The gene expression level of gene regulation, expression, metabolism and other functions increased, and the level of gene expression in the membrane proteins involved in exogenous substances entering the cell, signal transduction, and intercellular recognition and adhesion were reduced, which also coincided with the experimental phenomenon that non dependent prostate cancer cells were no longer regulated by external hormone.
In combination with the function of differentially expressed genes, biological processes and published literature, the key genes and possible signaling pathways involved in the process are preliminarily analyzed. The main 4 genes in the up regulation genes involve 4 types of signaling pathways, while 7 genes in the down regulated genes involve a variety of signaling pathways. These genes and related signaling pathways provide a direction for further study of the pathogenesis of AIPC.
Preliminary study on the mechanism of 3. androgen independent prostate cancer
The possible role of TLR2 mediated apoptosis pathway, PI3K/AKT pathway, MAPK pathway and other key signaling pathway proteins in the process of hormone dependent prostate cancer to hormone dependent transformation was examined by RT-PCR and Western blot.
Western results showed that there was no obvious change in the expression level of Caspase3 and FADD protein, and no Caspase3 bands were activated, suggesting that the apoptotic pathway in the Toll-like signaling pathway did not participate in the transformation of.AKT in non hormone dependent prostate cancer, but the expression of p-AKT was not significantly increased, but the expression of NF- kappa B protein was up and I kappa B alpha protein expression declined. NF- kappa B may be a key site in the signal pathway, but it is not activated by the PI3K/AKT pathway in the LNCaP-A cell line, and there may be other activation pathways. The inhibition of the down regulation of the expression of I kappa B alpha may affect the expression of NF- kappa B.
There was no significant change in the expression of ERK, MEK, and JNK protein in the MAPK signaling pathway related proteins, and the expression of p38, HSP27 and IL-8 significantly increased, suggesting that ERK pathway and JNK pathway were not involved in the transformation of LNCaP cells into LNCaP-A cells, but the MAPK/p38 signaling pathway was important. Information transfer molecules.
Studies have shown that MAPK/p38 plays an important role in the mechanism of breast cancer, ovarian cancer, and endometrial cancer, but it has not been reported in the drug resistance of the prostate cancer. This provides a new theoretical basis for the study of the AIPC mechanism and the discovery of the target of the AIPC drug.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R737.25

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