本文選題：上皮細(xì)胞粘附分子 + 雄激素抵抗性前列腺癌��； 參考：《第四軍醫(yī)大學(xué)》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(guān)于雄激素抵抗性前列腺癌發(fā)病機(jī)制的研究目前已經(jīng)成為前列腺癌研究的熱點(diǎn)之一。上皮細(xì)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(dá)在上皮組織的基底膜處，但在快速增殖的腫瘤細(xì)胞中則大量表達(dá)。EpCAM在腫瘤組織中的過度表達(dá)與腫瘤的發(fā)生、發(fā)展及預(yù)后密切相關(guān)，已經(jīng)成為腫瘤診斷和靶向治療重要的候選分子。關(guān)于EpCAM在前列腺癌組織中的表達(dá)與雄激素抵抗性前列腺癌的發(fā)生相關(guān)性的研究目前還鮮有報(bào)道。 本研究利用免疫組織化學(xué)和前列腺癌組織芯片技術(shù)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(dá)差異。結(jié)果顯示：EpCAM蛋白在正常前列腺組織表達(dá)量較低且主要局限性表達(dá)在前列腺管腔上皮細(xì)胞的基底膜處，在管腔上皮的細(xì)胞間連接處僅有極少量表達(dá)。在前列腺增生組織中，EpCAM的表達(dá)量增高，其表達(dá)不僅在管腔上皮組織的基底膜處且在管腔上皮細(xì)胞間連接處表達(dá)量也較高。在前列腺癌組織中，EpCAM的表達(dá)量顯著升高，且彌散的表達(dá)在前列腺管腔上皮細(xì)胞的各個(gè)部分，除了上皮細(xì)胞的基底部和細(xì)胞間連接處有大量的表達(dá)以外，在管腔上皮細(xì)胞的頂端也有大量的表達(dá)。Western-blot在前列腺癌組織及相應(yīng)的癌旁組織中檢測EpCAM的表達(dá),結(jié)果進(jìn)一步肯定了其在前列腺癌組織中的表達(dá)量顯著高于癌旁組織。統(tǒng)計(jì)學(xué)分析結(jié)果顯示：EpCAM蛋白的高表達(dá)與前列腺癌的臨床病理分級(jí)、淋巴結(jié)轉(zhuǎn)移、遠(yuǎn)處轉(zhuǎn)移以及手術(shù)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(guān)性，且EpCAM高表達(dá)的前列腺癌患者，其無轉(zhuǎn)移生存期和總生存期均顯著短于EpCAM低表達(dá)的前列腺癌患者。 Western-blot檢測四種前列腺癌細(xì)胞系DU-145，PC-3，LNCaP及VCaP中EpCAM的表達(dá)，結(jié)果顯示：LNCaP細(xì)胞中EpCAM的表達(dá)量最高，其次為PC-3細(xì)胞、DU-145細(xì)胞和VCaP細(xì)胞。采用shRNA干擾技術(shù)即利用EpCAM-shRNA慢病毒顆粒及相應(yīng)的慢病毒shRNA載體顆粒感染前列腺癌LNCaP細(xì)胞并建立穩(wěn)定轉(zhuǎn)染的細(xì)胞系EpCAM-shRNA5和對(duì)照細(xì)胞系Vector3。在EpCAM-shRNA5和Vector3細(xì)胞中分別檢測雄激素受體（Androgen receptor，AR）及PSA的表達(dá)水平，結(jié)果顯示：EpCAM-shRNA5細(xì)胞中，AR和PSA的表達(dá)水平明顯低于Vector3細(xì)胞。ELISA法檢測EpCAM-shRNA5細(xì)胞培養(yǎng)上清中的PSA水平同樣低于Vector3細(xì)胞。MTT法、TUNEL法、Annexin V/PI雙染法、細(xì)胞劃痕實(shí)驗(yàn)以及Transwell實(shí)驗(yàn)分別用來在體外檢測EpCAM-shRNA5和Vector3細(xì)胞的增殖、抗凋亡、遷移以及侵襲能力，結(jié)果顯示EpCAM-shRNA5細(xì)胞的增殖、抗凋亡、遷移以及侵襲能力均顯著低于對(duì)照組Vector3細(xì)胞。采用小鼠體內(nèi)腫瘤種植模型進(jìn)一步在體內(nèi)檢測兩種細(xì)胞的腫瘤形成能力，結(jié)果顯示EpCAM-shRNA5細(xì)胞的體內(nèi)成瘤能力明顯低于Vector3細(xì)胞。我們的研究結(jié)果提示：EpCAM在前列腺癌組織中的過度表達(dá)可能通過促進(jìn)前列腺癌細(xì)胞中AR的表達(dá)來使得前列腺癌細(xì)胞能夠適應(yīng)去勢雄激素水平而繼續(xù)存活。 對(duì)EpCAM-shRNA5和Vector3細(xì)胞進(jìn)行體外培養(yǎng)時(shí)可以觀察到Vector3細(xì)胞與LNCaP細(xì)胞相比形態(tài)變化不大，即均為紡錘狀，并有細(xì)絲狀的偽足，細(xì)胞間連接幾乎不存在。而EpCAM-shRNA5細(xì)胞形態(tài)則由細(xì)長的紡錘狀變?yōu)檩^規(guī)則的卵石狀，細(xì)絲狀偽足消失，且細(xì)胞間出現(xiàn)了比較稀疏的細(xì)胞間連接。這些形態(tài)變化提示EpCAM的表達(dá)可能促進(jìn)上皮-間質(zhì)轉(zhuǎn)化（Epithelial-mesenchymal transition，，EMT）。進(jìn)一步在EpCAM-shRNA5和Vector3細(xì)胞中檢測EMT發(fā)生相關(guān)的蛋白標(biāo)志物,結(jié)果顯示：與Vector3細(xì)胞相比EpCAM-shRNA5細(xì)胞中上皮細(xì)胞特異性蛋白E-cadherin和Cytokeratin的表達(dá)增高而間質(zhì)細(xì)胞特異性蛋白Vimentin和Fibronectin表達(dá)降低。利用免疫熒光染色法進(jìn)一步肯定了EpCAM可促進(jìn)EMT的發(fā)生。由于EMT的發(fā)生會(huì)賦予細(xì)胞干細(xì)胞的特性，同時(shí)EpCAM在一些腫瘤組織中也被看作干細(xì)胞的標(biāo)記物。因此，我們進(jìn)一步驗(yàn)證EpCAM-shRNA5和Vector3細(xì)胞是否具有干細(xì)胞方面的特性。首先，利用平板克隆形成實(shí)驗(yàn)檢測兩種細(xì)胞的克隆形成能力,結(jié)果顯示：EpCAM-shRNA5細(xì)胞的克隆形成能力顯著低于Vector3。進(jìn)一步檢測干細(xì)胞相關(guān)的轉(zhuǎn)錄因子OCT4, SOX2, NANOG的表達(dá),結(jié)果顯示：EpCAM-shRNA5細(xì)胞中三種干細(xì)胞相關(guān)轉(zhuǎn)錄因子的表達(dá)水平明顯低于Vector3細(xì)胞。提示EpCAM不僅可以通過上調(diào)AR水平來提高前列腺癌細(xì)胞對(duì)去勢水平雄激素的耐受，同時(shí)還可以通過促進(jìn)EMT的發(fā)生及腫瘤細(xì)胞的干細(xì)胞特性的表達(dá)來提高前列腺癌細(xì)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(dá)與雄激素抵抗性前列腺癌的發(fā)生相聯(lián)系起來。證實(shí)EpCAM可通過促進(jìn)AR的表達(dá)，EMT的發(fā)生及干細(xì)胞特性的形成等來促進(jìn)前列腺癌細(xì)胞在去勢雄激素水平下的生存能力，從而促進(jìn)雄激素抵抗性前列腺癌的發(fā)生。我們的實(shí)驗(yàn)數(shù)據(jù)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(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.25

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

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

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