【作者】 陈光华；

【导师】 孙颖浩；

【作者基本信息】 第二军医大学 ， 外科学， 2013， 博士

【摘要】 一、研究背景前列腺癌是欧美国家最常见的男性恶性肿瘤之一，在男性癌症死因中排在第二位。我国前列腺癌发病率虽然远低于西方国家，但近年来随着人口老龄化及生活条件的改善，发病率增长显著。前列腺癌患者经一段时间内分泌治疗后几乎全部发生雄性激素非依赖性转化。目前，在临床上雄性激素非依赖性前列腺癌尚无有效的治疗方法，其发生的具体机制仍然不清楚。雄激素受体一直是前列腺癌雄性激素性非依赖转化机制研究的重点，但其不能解释所有雄激素非依赖转化的相关现象。雄激素受体不是前列腺癌发生雄激素非依赖转化的唯一的因素。有研究已经证实，前列腺癌LNCaP细胞经缺氧培养，细胞将呈神经内分泌改变。前列腺癌组织内神经内分泌（NE）细胞的存在被认为与肿瘤的恶性进展、预后不良密切相关，而且往往提示前列腺癌从激素依赖进入激素难治阶段。前列腺癌NE细胞通过分泌各种具有促进细胞有丝分裂的神经多肽作用于邻近的前列腺癌细胞，使得这些细胞获得雄激素非依赖性生长。二、研究目的通过缺氧及相关促红细胞生成素信号通路探讨前列腺癌LNCaP细胞的神经内分泌样细胞转化，并进一步阐述缺氧诱导的前列腺癌雄激素非依赖转化的具体机制。三、研究内容1、通过缺氧环境（37℃、5%CO2、1%O2的饱和湿度）以10%FBS-含酚红RPMI1640培养液持续培养2周，构建缺氧诱导下LNCaP细胞神经内分泌样转化的细胞模型，再通过western blot的方法鉴定缺氧诱导下LNCaP细胞神经内分泌样转化细胞的标记物。2、人类前列腺癌LNcap细胞SCID鼠原位移植模型的建立。3、通过免疫组化的方法检测雄性激素依赖和雄性激素非依赖前列腺癌组织中EPO和受体（EPOR）的表达。4、通过CCK-8法绘制缺氧诱导下LNCaP细胞神经内分泌样转化的细胞的生长曲线；通过RT-PCR和Western blot的方法检测缺氧培养前后LNCaP细胞EPO、EPOR、HIF-1α的变化；siRNA沉默EPOR，通过Western blot检测缺氧诱导下LNCaP细胞神经内分泌样转化细胞的标记物的变化。四、研究结果1、通过缺氧环境（37℃、5%CO2、1%O2的饱和湿度）以10%FBS-含酚红RPMI1640培养液持续培养2周后，LNCaP细胞呈神经内分泌样改变，再通过western blot的方法发现缺氧诱导的LNCaP细胞神经内分泌样转化细胞的标记物NSE和CgA表达增加。2、常氧培养前列腺原位组：13/15小鼠的前列腺生长出肿瘤，可见大部分前列腺腺体被肿瘤细胞破坏，仅见少量正常腺体结构，大量异型性上皮细胞，细胞胞核浓染，呈多形性，可见异常分裂相，胞浆可见有泡沫样的嗜伊红物质。皮下组：9/10小鼠皮下生长出肿瘤。缺氧培养前列腺原位组：4/4小鼠前列腺生长出肿瘤，可见大部分前列腺腺体被肿瘤细胞破坏，仅见少量正常腺体结构，大量异型性上皮细胞，细胞胞核浓染，呈多形性，可见异常分裂相，胞浆可见有泡沫样的嗜伊红物质，局部可见大量坏死组织。3、穿刺标本（雄性激素依赖组织）和电切标本（雄性激素非依赖组织）均存在EPO和EPOR过表达；穿刺标本和电切标本的EPO表达评分差异有统计学意义（p<0.001），穿刺标本和电切标本的EPOR表达评分差异有统计学意义（p<0.001）4、缺氧诱导的LNCaP细胞神经内分泌样转化细胞获得雄性激素非依赖生长。缺氧培养后LNCaP细胞EPO与EPOR的mRNA和蛋白的表达持续上升，HIF-1的mRNA和蛋白的表达先上升，后出现下降。siRNA沉默EPOR再行缺氧培养2周，LNCaP细胞NSE、CgA蛋白表达的下降。五、结论1、经缺氧2周培养，LNCaP细胞获得神经内分泌样表型，提示缺氧可能促进列腺癌激素非依赖进展。2、临床研究发现CRPC与内分泌治疗前的前列腺癌组织相比，免疫组化结果EPO和EPOR表达明显增强，提示EPO和EPOR自分泌环参与前列腺癌激素非依赖进展。3、长期缺氧时，EPO、EPOR持续上升，而HIF-1α表达下降，提示EPO-EPOR自主分泌环形成可能，并可能取代HIF-1α功能，而EPO通过前列腺癌细胞表达的功能性EPOR促进细胞凋亡抵抗及缺氧应激适应。更多还原

【Abstract】 BackgroundProstate carcinoma (PCa) is one of the most common malignances among theurological neoplasms that happen in men, with a secondary highest lethality. Its recentincidence, though lower than western countries, is increasing significantly in Chinawhich results from the aging and the changes in life style. In clinic, patients with PCawill eventually become androgen-independent after endocrinotherapy. So far, thereare no effective treatments, and the mechanism of transition to androgen-independentis still unknown. Most of the studies on this field only focus on the important roles ofthe Androgen Receptor (AR) during the transition to androgen-independent; however,they fail to explain any of the related mechanisms, which suggest that the AR is notthe only pathway involved in this transition.It is indicated that the neuroendocrine-like (NE) cells within the prostate cancertissues are closely correlated to tumor progression, poor prognosis and the transitionto androgen-independent. The neuropeptides secreted by these NE cells have beenidentified as potent paracrine and autocrine growth factors, which instead of androgen,can promote cancer cell migration and growth that will lead to theandrogen-independent transition.ObjectiveStudy of neuroendocrine differentiation of the LNCaP cell induced by oxygen-deficient culture, and its related EPO-EPOR signal pathway during thisprogress. Analyze the mechanism of the androgen-independent transition that causedby hypoxia induce.Methods1. Construct a specific culture model of neuroendocrine-like (NE) cell by inducing theLNCaP cell with the10%FBS-phenol red RPMI1640culture medium under anoxygen-deficient culture environment (37℃、5%CO2、1%O2saturation humidity) for2weeks, then test the NE markers expressed by these cells using western blot.2. Establish a human prostate carcinoma xenograft model by orthotopicly injectingLNCaP cells into SCID mice.3. Compare the expression level of EPO and EPOR between the androgen-dependenttissue and the androgen-independent tissue by immunohistochemical analysis.4. Illustrate the cell growth curve of the induced neuroendocrine-like cells thattransformed from hypoxia treated LNCaP cells using CCK-8assay; identify theexpression levels of EPO, EPOR, and HIF-1α of the LNCaP cells both before andafter hypoxic culturing by RT-PCR and western blot; analyze the expression levels ofseveral NE markers of the induced neuroendocrine-like cells both before and afterhypoxic culturing, with the EPOR inhibitory treatment at beginning using SmallInterference RNA (siRNA) transfection.Results1. The LNCaP cells showed a neuroendocrine change after2weeks hypoxic culture.And western blot results revealed some neuron-specific markers, such asneuron-specific enolase (NSE) and chromogranin A (CgA), were increased in thesehypoxic induced LNCaP cells.2. In the LNCaP cells orthotopicly injected group, there were tumors that developed in13mice with the total of15(87％), in which most parts of the prostate wereinvaded by tumor cells. There are confluent atypia epithelial cells, some of whichhave hyperchromatic and polymorphic nuclei, frothy eosinophilic structures in thecytoplasm, and some are abnormally proceeding of cytokinesis. In the LNCaP cellssubcutaneously injected group, only9of10mouse (90%) resulted in tumor growth.In the induced LNCaP cells orthotopicly injected group, all of these four mice(100%) had tumors developed in their prostates, in which the cortices and medullaeof metastatic lymph nodes were destroyed by tumor cells, hyperchromatic andpolymorphic nuclei, frothy eosinophilic structures, and abnormal mitotic figure canbe found in these atypia epithelial cells. There were also visible necrotic tissueswithin the tumors.3. EPO and EPOR were up-regulated in both the androgen-dependent tissue and theandrogen-independent tissue. And statistic significances (p<0.001) of the EPOscores and the EPOR scores can be found between the androgen-dependent tissueand the androgen-independent tissue respectively.4. The induced neuroendocrine-like cells, which transformed from hypoxia treatedLNCaP cells, turned to be androgen-independence that show a continuouslyincreased expression of EPO and EPOR both in the mRNA and the protein level.However, HIF-1α was up-regulated at the beginning and then followed by adown-regulation. NSE and CgA in the induced neuroendocrine-like cells weredecreased when blocking EPOR by siRNA.Conclusions1. The neuroendocrine transformation of the LNCaP cells, after2weeks hypoxicculture, indicates oxygen deficit which may promote theandrogen-independent progress of the prostate carcinoma.2. Compared with androgen-dependent tissue, the up-regulation of EPO andEPOR in androgen-independent tissue suggests that EPO-EPOR autocrineloop plays an important role during tumor development andandrogen-independent progression. 3. The continuously increase of both EPO and EPOR in LNCaP cells underhypoxic culture, together with the sustained decrease of HIF-1α, indicates thatthere is formation of a EPO-EPOR autocrine loop that takes over the functionof the HIF-1α. Besides, the EPO can also regulate cell apoptosis and cellresponse to hypoxic stress by binding to the functional EPOR.更多还原