本文選題：上皮細胞粘附分子 + 雄激素抵抗性前列腺癌�。� 參考：《第四軍醫(yī)大學》2014年博士論文

【摘要】：雄激素抵抗性前列腺癌是前列腺癌致死的主要原因。關于雄激素抵抗性前列腺癌發(fā)病機制的研究目前已經成為前列腺癌研究的熱點之一。上皮細胞黏附分子(Epithelial cell adhesion molecule，EpCAM)是一類I型跨膜糖蛋白，其在正常組織中只局限性的表達在上皮組織的基底膜處，但在快速增殖的腫瘤細胞中則大量表達。EpCAM在腫瘤組織中的過度表達與腫瘤的發(fā)生、發(fā)展及預后密切相關，已經成為腫瘤診斷和靶向治療重要的候選分子。關于EpCAM在前列腺癌組織中的表達與雄激素抵抗性前列腺癌的發(fā)生相關性的研究目前還鮮有報道。 本研究利用免疫組織化學和前列腺癌組織芯片技術在6例正常前列腺組織、24例前列腺增生組織及210例前列腺癌組織中檢測了EpCAM蛋白的表達差異。結果顯示：EpCAM蛋白在正常前列腺組織表達量較低且主要局限性表達在前列腺管腔上皮細胞的基底膜處，在管腔上皮的細胞間連接處僅有極少量表達。在前列腺增生組織中，EpCAM的表達量增高，其表達不僅在管腔上皮組織的基底膜處且在管腔上皮細胞間連接處表達量也較高。在前列腺癌組織中，EpCAM的表達量顯著升高，且彌散的表達在前列腺管腔上皮細胞的各個部分，除了上皮細胞的基底部和細胞間連接處有大量的表達以外，在管腔上皮細胞的頂端也有大量的表達。Western-blot在前列腺癌組織及相應的癌旁組織中檢測EpCAM的表達,結果進一步肯定了其在前列腺癌組織中的表達量顯著高于癌旁組織。統(tǒng)計學分析結果顯示：EpCAM蛋白的高表達與前列腺癌的臨床病理分級、淋巴結轉移、遠處轉移以及手術前血清中總的前列腺特異性抗原（Prostatespecific antigen，PSA）水平等具有明顯相關性，且EpCAM高表達的前列腺癌患者，其無轉移生存期和總生存期均顯著短于EpCAM低表達的前列腺癌患者。 Western-blot檢測四種前列腺癌細胞系DU-145，PC-3，LNCaP及VCaP中EpCAM的表達，結果顯示：LNCaP細胞中EpCAM的表達量最高，其次為PC-3細胞、DU-145細胞和VCaP細胞。采用shRNA干擾技術即利用EpCAM-shRNA慢病毒顆粒及相應的慢病毒shRNA載體顆粒感染前列腺癌LNCaP細胞并建立穩(wěn)定轉染的細胞系EpCAM-shRNA5和對照細胞系Vector3。在EpCAM-shRNA5和Vector3細胞中分別檢測雄激素受體（Androgen receptor，AR）及PSA的表達水平，結果顯示：EpCAM-shRNA5細胞中，AR和PSA的表達水平明顯低于Vector3細胞。ELISA法檢測EpCAM-shRNA5細胞培養(yǎng)上清中的PSA水平同樣低于Vector3細胞。MTT法、TUNEL法、Annexin V/PI雙染法、細胞劃痕實驗以及Transwell實驗分別用來在體外檢測EpCAM-shRNA5和Vector3細胞的增殖、抗凋亡、遷移以及侵襲能力，結果顯示EpCAM-shRNA5細胞的增殖、抗凋亡、遷移以及侵襲能力均顯著低于對照組Vector3細胞。采用小鼠體內腫瘤種植模型進一步在體內檢測兩種細胞的腫瘤形成能力，結果顯示EpCAM-shRNA5細胞的體內成瘤能力明顯低于Vector3細胞。我們的研究結果提示：EpCAM在前列腺癌組織中的過度表達可能通過促進前列腺癌細胞中AR的表達來使得前列腺癌細胞能夠適應去勢雄激素水平而繼續(xù)存活。 對EpCAM-shRNA5和Vector3細胞進行體外培養(yǎng)時可以觀察到Vector3細胞與LNCaP細胞相比形態(tài)變化不大，即均為紡錘狀，并有細絲狀的偽足，細胞間連接幾乎不存在。而EpCAM-shRNA5細胞形態(tài)則由細長的紡錘狀變?yōu)檩^規(guī)則的卵石狀，細絲狀偽足消失，且細胞間出現了比較稀疏的細胞間連接。這些形態(tài)變化提示EpCAM的表達可能促進上皮-間質轉化（Epithelial-mesenchymal transition，，EMT）。進一步在EpCAM-shRNA5和Vector3細胞中檢測EMT發(fā)生相關的蛋白標志物,結果顯示：與Vector3細胞相比EpCAM-shRNA5細胞中上皮細胞特異性蛋白E-cadherin和Cytokeratin的表達增高而間質細胞特異性蛋白Vimentin和Fibronectin表達降低。利用免疫熒光染色法進一步肯定了EpCAM可促進EMT的發(fā)生。由于EMT的發(fā)生會賦予細胞干細胞的特性，同時EpCAM在一些腫瘤組織中也被看作干細胞的標記物。因此，我們進一步驗證EpCAM-shRNA5和Vector3細胞是否具有干細胞方面的特性。首先，利用平板克隆形成實驗檢測兩種細胞的克隆形成能力,結果顯示：EpCAM-shRNA5細胞的克隆形成能力顯著低于Vector3。進一步檢測干細胞相關的轉錄因子OCT4, SOX2, NANOG的表達,結果顯示：EpCAM-shRNA5細胞中三種干細胞相關轉錄因子的表達水平明顯低于Vector3細胞。提示EpCAM不僅可以通過上調AR水平來提高前列腺癌細胞對去勢水平雄激素的耐受，同時還可以通過促進EMT的發(fā)生及腫瘤細胞的干細胞特性的表達來提高前列腺癌細胞在去勢水雄激素平下的生存能力。 綜上所述，我們的研究首次將EpCAM在前列腺組織中的過表達與雄激素抵抗性前列腺癌的發(fā)生相聯系起來。證實EpCAM可通過促進AR的表達，EMT的發(fā)生及干細胞特性的形成等來促進前列腺癌細胞在去勢雄激素水平下的生存能力，從而促進雄激素抵抗性前列腺癌的發(fā)生。我們的實驗數據為雄激素抵抗性前列腺癌的免疫治療提供了重要的候選靶分子。
[Abstract]:Androgen resistance to prostate cancer is the main cause of the death of prostate cancer. Research on the mechanism of androgen resistance to prostate cancer is now one of the hotspots in the research of prostate cancer. Epithelial cell adhesion molecule (EpCAM) is a class of I type transmembrane glycoprotein, which is only in normal tissues. Limited expression is located at the basal membrane of epithelial tissue, but the excessive expression of.EpCAM in the tumor cells in the rapidly proliferating tumor cells is closely related to the occurrence, development and prognosis of tumor. It has become an important candidate for the diagnosis of tumor and target therapy. The expression of EpCAM in the prostate cancer tissue and the male excitation There is little report on the correlation between prostate cancer and prostate cancer.
In this study, the expression of EpCAM protein was detected in 6 normal prostate tissues, 24 cases of benign prostatic hyperplasia and 210 cases of prostate cancer. The results showed that the expression of EpCAM protein in the normal prostate tissue was low and the main limitation was expressed in the prostatic cavity. In the basal membrane of the skin cells, only a very small amount of expression was found in the intercellular junction of the lumen epithelium. In the prostatic hyperplasia tissue, the expression of EpCAM increased. The expression of EpCAM was not only in the basal membrane of the epithelial tissue of the lumen but also in the intercellular junction of the lumen, and the expression of the expression was significantly increased in the adenocarcinoma of the anterior gland. The expression of diffuse expression in all parts of the epithelial cells of the prostate gland, except for the large amount of expression in the basal part of the epithelial cells and the intercellular junction, also has a large number of expressions of.Western-blot at the top of the epithelial cells of the lumen. The expression of EpCAM in the prostate cancer tissue and the corresponding para cancerous tissue is detected. The result further affirms its expression. The expression in the prostate cancer tissue was significantly higher than that in the paracancerous tissue. The statistical analysis showed that the high expression of EpCAM protein was correlated with the clinicopathological classification, lymph node metastasis, distant metastasis and the level of Prostatespecific antigen, PSA in the serum before operation, and Ep CAM patients with high expression of prostate cancer had no metastasis survival and overall survival time significantly shorter than those with low EpCAM expression.
Western-blot was used to detect the expression of EpCAM in four kinds of prostate cancer cell lines, DU-145, PC-3, LNCaP and VCaP. The results showed that the expression of EpCAM was the highest in LNCaP cells, followed by PC-3, DU-145 and VCaP cells. The expression level of androgen receptor (Androgen receptor, AR) and PSA in EpCAM-shRNA5 and Vector3 cells was detected in the cancer LNCaP cells and the stable transfected cell line EpCAM-shRNA5 and the control cell line Vector3.. The results showed that the expression level of AR and PSA was significantly lower than that in the EpCAM-shRNA5 cells. The level of PSA in NA5 cell culture supernatant was also lower than that of Vector3 cell.MTT method. TUNEL, Annexin V/PI double staining, cell scratch test and Transwell experiment were used to detect the proliferation, anti apoptosis, migration and invasion ability of EpCAM-shRNA5 and Vector3 cells in vitro, and the fruit showed the proliferation, anti apoptosis and migration of EpCAM-shRNA5 cells. Vector3 cells were significantly lower than the control group. The tumor formation ability of two cells was further detected in vivo by the tumor planting model in mice. The results showed that the tumor formation ability of EpCAM-shRNA5 cells in vivo was significantly lower than that of Vector3 cells. Our results suggest that the overexpression of EpCAM in the prostate cancer tissue It is possible that prostate cancer cells can adapt to castrated androgen levels and continue to survive by promoting the expression of AR in prostate cancer cells.
In vitro culture of EpCAM-shRNA5 and Vector3 cells can be observed that Vector3 cells have little change in morphology compared with LNCaP cells, that is, spindle shaped, and filamentous pseudo feet, with almost no intercellular connections, and EpCAM-shRNA5 cells form a slender spindle shape to a more regular pebbles, and filamentous pseudo foot disappears. There are relatively sparse intercellular connections between cells. These morphological changes suggest that the expression of EpCAM may promote epithelial mesenchymal transition (Epithelial-mesenchymal transition, EMT). The protein markers associated with EMT in EpCAM-shRNA5 and Vector3 cells are further detected, and the results show that compared with Vector3 cells in EpCAM-shRNA5 cells. The expression of epithelial cell specific protein E-cadherin and Cytokeratin increased while the expression of interstitial cell specific protein Vimentin and Fibronectin decreased. Using immunofluorescence staining, it was further affirmed that EpCAM could promote the occurrence of EMT. As EMT occurs, the characteristics of cell stem cells are given, and EpCAM is also seen in some tumor tissues. As a marker of stem cells, we further verify whether EpCAM-shRNA5 and Vector3 cells have stem cell characteristics. First, the clone formation ability of two cells was detected by a flat clone formation test. The results showed that the clone formation ability of EpCAM-shRNA5 cells was significantly lower than that of Vector3. for further detection of stem cells. The expression of transcription factors, OCT4, SOX2, NANOG, showed that the expression level of three stem cell related transcription factors in EpCAM-shRNA5 cells was significantly lower than that of Vector3 cells. It suggested that EpCAM not only improve the tolerance of prostate cancer cells to the castrated androgens by up regulation of AR, but also by promoting the occurrence of EMT and the development of EMT. The expression of stem cell characteristics of cancer cells enhances the survival of prostate cancer cells in androgen.
To sum up, our study is the first to link the overexpression of EpCAM in the prostate tissue to the occurrence of androgen resistance to prostate cancer. It is confirmed that EpCAM promotes the viability of prostate cancer cells at the level of androgens by promoting the expression of AR, the occurrence of EMT and the formation of stem cell characteristics, and thus promotes male prostatic cancer. Our data provide an important candidate for immunotherapy of androgen resistant prostate cancer.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R737.25

【引證文獻】

相關期刊論文 前1條

1 邢兆輝;王齊;;前列腺上皮內瘤基底細胞變化的形態(tài)學觀察[J];黑龍江醫(yī)學;2016年10期

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