前列腺干细胞抗原（PSCA）在肿瘤诊断及治疗中的应用*

前列腺干细胞抗原(PSCA)是最早发现于前列腺癌的GPI锚定的细胞膜蛋白,PSCA的在正常或肿瘤细胞中的具体的病理生理功能还不清楚。PSCA在前列腺癌、胰腺癌、膀胱癌等肿瘤中表达增加,相反在食管癌和胃癌中表达降低可能在胃上皮中发挥肿瘤抑制功能,PSCA发挥致瘤和抑瘤两种截然不同的作用与细胞所处的环境不同有关。除此之外,PSCA作为肿瘤的免疫治疗的靶点也显示出其良好的临床应用潜力。因此PSCA不仅成为肿瘤诊断和预后判断的生物学标记也是肿瘤免疫治疗重要的候选靶蛋白。本文对PSCA的功能和在前列腺癌以及其他肿瘤的临床诊断、预后判断以及治疗应用中的研究进展进行了综述,并讨论了未来PSCA的研究前景。     

以前列腺干细胞抗原为靶点的前列腺癌免疫治疗研究进展

前列腺干细胞抗原(PSCA)为细胞膜表面抗原,在正常前列腺组织中低表达,在雄激素依赖性和非依赖性前列腺癌组织中高表达,有较高的组织特异性,是前列腺癌治疗的理想靶标,近年来以PSCA为靶点的前列腺癌治疗性疫苗的研究已成为热点。我们简要综述以PSCA为靶点治疗前列腺癌的研究进展。     

前列腺干细胞抗原(PSCA)的表达及其特异结合肽的筛选

通过反转录 PCR从人前列腺癌细胞中克隆了前列腺干细胞抗原 (PSCA)基因 ,在大肠杆菌中利用pQE30载体对截断型PSCA基因进行了可溶性表达。蛋白纯化后 ,利用噬菌体随机展示 12肽库筛选了PSCA蛋白的特异结合肽 ,通过与EGFP蛋白的耦联表达验证了结合肽的特异性。此特异结合肽的获得 ,为进一步研究针对PSCA的前列腺癌靶向免疫治疗奠定了基础     

超抗原SEA的GPI锚定修饰和在CHO-dhfr~-细胞膜上的表达

将GPI锚定修饰的免疫分子直接转移到肿瘤细胞膜上,是研究治疗性肿瘤疫苗的一种有潜力的新策略。通过将从衰变加速因子(DAF)来源的GPI修饰性信号序列与SEA嵌合,获得了GPI修饰的SEA分子,构建的真核表达载体pCIGPI-SEA,用脂质体方法转染到CHOdhfr-细胞中,并用氨甲喋呤(MTX)进行筛选,细胞免疫荧光分析证实,SEA能够在细胞膜上表达。上述GPI锚定修饰的SEA,可用于进一步研究治疗性肿瘤疫苗。     

靶向前列腺癌CAR-NK细胞的抗肿瘤活性评价

目的：探索基于DAP12共刺激信号和靶向前列腺干细胞抗原(PSCA)的嵌合抗原受体NK细胞(CAR-NK)对前列腺肿瘤细胞的杀伤作用。方法：使用慢病毒转染系统构建CAR-NK细胞,使用流式细胞术检测前列腺癌细胞DU145 PSCA的表达水平和CAR-NK细胞的阳性率,并经细胞和动物模型评价CAR-NK细胞的抗肿瘤活性。结果：前列腺癌细胞DU145高表达PSCA,阳性率约为98.50%。流式细胞术检测显示CAR-NK细胞CAR分子表达阳性率为(64.07±3.01)%。细胞毒性实验发现,与对照NK细胞相比,携带DAP12共刺激信号的CAR-NK细胞具有较强抗前列腺肿瘤作用,杀伤率提升了约1.5倍。ELISA结果显示,与对照NK细胞相比,CAR-NK细胞杀伤DU145细胞时释放的TNF-α、IFN-γ、CD107α、Granzyme B和Perforin-1等因子水平显著提高。动物实验表明,CAR-NK细胞相对于对照NK细胞,更能有效抑制肿瘤增殖,两者之间有显著性统计学差异(P<0.000 1)。结论：靶向PSCA并提供DAP12共刺激信号的CAR-NK细胞具有较强杀伤前列腺肿瘤的作...     

小鼠GM-CSF的基因克隆和锚定修饰

将糖基化磷脂酰肌醇(GPI）锚定修饰的细胞因子直接转移到肿瘤细胞膜上,是研究治疗性肿瘤疫苗的一种有潜力的新策略。克隆了小鼠GM-CSF基因,井通过将从衰变加速因子（DAF）来源的GPI修饰性信号序列与mGM—CSF、嵌合,获得了GPI修饰的mGM-CSF分子,构建并鉴定了GPI锚定修饰的真核表达裁体pCI／GPI-mGM-CSF,为进一步研究表面工程治疗性肿瘤疫苗奠定了基础。     

多拷贝异种化前列腺干细胞抗原融合基因片段的构建及真核表达

目的：构建一系列含有人前列腺干细胞抗原（PSCA）主要T细胞表位的多拷贝异种化融合基因片段,并分别在人胚肾293T细胞中表达。方法：通过重叠延伸PCR法合成单拷贝异种化PSCA基因片段PSCA1,随即应用同尾酶法将该片段串联形成2、3、4拷贝异种化PSCA基因片段PSCA2、PSCA3和PSCA4,并将上述4种基因片段分别插入真核表达载体pCI-Fc-GPI中,构建最终目的片段1～4拷贝异种化PSCA-Fc-GPI（即PSCA1-Fc-GPI～PSCA4-Fc-GPI）,随即分别将重组质粒pCI-PSCA1-Fc-GPI～PSCA4-Fc-GPI体外转染293T细胞,利用间接免疫荧光和流式细胞仪检测其表达情况。结果：测序证实PSCA1片段与设计一致,酶切鉴定证明目的基因片段PSCA1-Fc-GPI～PSCA4-Fc-GPI构建成功;间接免疫荧光和流式细胞仪的检测结果显示,在293T细胞中1～4拷贝异种化PSCA融合基因片段均获得较好表达。结论：构建了目的基因片段PSCA1-Fc-GPI～PSCA4-Fc-GPI,为以PSCA为靶抗原的抗前列腺癌DNA疫苗的构建及功能研究奠定了重要基础。     

前列腺癌患者组织中RPL6的表达及临床意义

目的:探讨前列腺癌患者组织中核糖体蛋白L6(RPL6)表达,分析其对肿瘤恶性程度的评估作用。方法:收集2013年12月-2015年12月在本院接受治疗的前列腺疾病患者117例,根据病理结果分为前列腺炎组63例、前列腺癌组54例;另取同期进行健康体检的健康者80例作为正常对照组。采用Western-blot法检测三组研究对象的前列腺组织RPL6表达,采用酶联免疫吸附法(ELISA)测定血清肿瘤标志物含量,采用RT-PCR法检测前列腺组织增殖基因、侵袭基因mRNA表达,采用Pearson检验分析RPL6表达量与肿瘤恶性程度的相关关系。结果:前列腺癌组患者的前列腺组织RPL6表达量高于前列腺炎组及正常对照组(P0.05);前列腺癌组患者的血清前列腺特异性抗原(PSA)、前列腺特异性酸性磷酶(PSAP)、尿激酶型纤溶酶原激活剂(u PA)、硫氧还蛋白(TRX)含量高于前列腺炎组及正常对照组(P0.05);前列腺癌组患者的前列腺组织增殖基因URG11、PTEN mRNA表达量高于前列腺炎组及正常对照组,PRDM5 mRNA表达量低于前列腺炎组及正常对照组,侵袭基因IgGHG1、TMPRSS2-ER、PIK3C2B mRNA表达量高于前列腺炎组及正常对照组(P0.05)。前列腺癌患者的组织RPL6表达量与肿瘤标志物含量、增殖及侵袭基因活性均呈直接相关关系(P0.05)。结论:高表达的RPL6是前列腺癌早期诊断的可靠指标,且与肿瘤恶性程度直接相关,有望成为前列腺癌辅助诊断及预后评估的重要手段。     

PSCA和Mesothelin在膀胱尿路上皮癌中的异常表达及临床意义

目的探讨膀胱尿路上皮癌组织中前列腺干细胞抗原(ProstateStem Cel1 Antigen,PSCA)和间皮素(Me-sothelin)的表达与侵袭转移的关系。方法收集武汉大学人民医院病理科2000-2006年有完整临床和病理资料的膀胱尿路上皮癌存档蜡块50例和5例癌旁组织,采用免疫组织化学S-P法检测50例膀胱尿路上皮癌和5例癌旁组织中PSCA和Mesothelin的表达水平,并分析他们与临床病理特征的相关性。采用HPIAS-1000高清晰度彩色病理图文报告管理系统,对PSCA和Mesothelin的表达进行定量分析,并用SPSS13.0软件对各组免疫组织化学反应阳性颗粒的平均光密度、阳性面积率做单因素方差分析和SNK(q)检验。结果 (1)PSCA和Mesothelin在膀胱尿路上皮癌中呈高表达,癌旁组织中呈低表达。膀胱尿路上皮癌与癌旁组织相比,差异有显著性(P0.05);(2)PSCA和Mesothelin的表达与膀胱尿路上皮癌的临床分期及淋巴结转移有关(P0.05)。(3)PSCA和Mesothelin的表达与膀胱尿路上皮癌患者的性别、年龄、肿瘤大小、病理类型等因素无关(P0.05)。结论 PSCA和Mesothelin的高表达在膀胱尿路上皮癌的生长浸润及转移过程中发挥重要作用,可以作为判断尿路上皮肿瘤生物学特性的重要指标。     

糖基化磷脂酰肌醇锚定型EGFP真核表达质粒的构建及表达

构建与增强型绿色荧光蛋白基因相连的糖基化磷脂酰肌醇(glycosyl phosphatidylinositol,GPI)序列的真核表达质粒,并检测其在A549细胞中的表达.分离人外周血淋巴细胞,提取总RNA,以RT-PCR法扩增CD24基因的243 bp GPI锚定序列,双酶切后定向克隆入pEGFP-C1质粒中,构建并鉴定pEGFP-C1-GPI质粒.经脂质体介导转染A549细胞后,在荧光显微镜下观察目的蛋白在真核细胞内的表达情况.经酶切和测序鉴定证实,所克隆的CD24 GPI序列正确,荧光显微镜观察pEGFP-C1-GPI质粒转染A549细胞可见围绕细胞膜的强绿色荧光,而对照pEGFP-C1质粒转染A549细胞仅见胞内均匀荧光.成功构建与EGFP相连的GPI真核表达质粒,且能在A549细胞膜上锚定表达EGFP-GPI融合蛋白,为构建锚定表达型肿瘤疫苗奠定基础.     

Prostate Tissue Metal Levels and Prostate Cancer Recurrence in Smokers

Although smoking is not associated with prostate cancer risk overall, smoking is associated with prostate cancer recurrence and mortality. Increased cadmium (Cd) exposure from smoking may play a role in progression of the disease. In this study, inductively coupled plasma mass spectrometry was used to determine Cd, arsenic (As), lead (Pb), and zinc (Zn) levels in formalin-fixed paraffin embedded tumor and tumor-adjacent non-neoplastic tissue of never- and ever-smokers with prostate cancer. In smokers, metal levels were also evaluated with regard to biochemical and distant recurrence of disease. Smokers (N?=?25) had significantly higher Cd (median ppb, p?=?0.03) and lower Zn (p?=?0.002) in non-neoplastic tissue than never-smokers (N?=?21). Metal levels were not significantly different in tumor tissue of smokers and non-smokers. Among smokers, Cd level did not differ by recurrence status. However, the ratio of Cd ppb to Pb ppb was significantly higher in both tumor and adjacent tissue of cases with distant recurrence when compared with cases without distant recurrence (tumor tissue Cd/Pb, 6.36 vs. 1.19, p?=?0.009, adjacent non-neoplastic tissue Cd/Pb, 6.36 vs. 1.02, p?=?0.038). Tissue Zn levels were also higher in smokers with distant recurrence (tumor, p?=?0.039 and adjacent non-neoplastic, p?=?0.028). These initial findings suggest that prostate tissue metal levels may differ in smokers with and without recurrence. If these findings are confirmed in larger studies, additional work will be needed to determine whether variations in metal levels are drivers of disease progression or are simply passengers of the disease process.     

PSA在前列腺增生和前列腺癌中的诊断价值

目的：探讨临床上检测前列腺特异性抗原（PSA）的变化情况对前列腺增生和前列腺癌等疾病的诊断价值。方法：采用回顾性分析的方法,选取2010年6月至2012年4月在我院泌尿科接受治疗的前列腺增生患者64例定义为前列腺增生组（BPH）,前列腺癌患者83例定义为前列腺癌组（PCa）,另选取同期接受体检的健康人群137例作为对照组。分别检测三组患者入院时的游离前列腺特异性抗原和总前列腺特异性抗原的水平变化情况。对比并分析三组检测结果。结果：经检测,前列腺增生患者的血清总PSA明显高于对照组健康人群的正常值,而前列腺癌患者的血清总PSA比前列腺增生患者增高的更为明显。对照组游离PSA为（2．78±0．94）ng／mL,总PSA（1．05±0．57）ng／mL,游离PSA与总PSA的比值为,（0．38±0．61）;前列腺增生患者游离PSA为（6．36＋3．24）ng／mL,总PSA为（1．64±0．76）ng／mL,游离PSA与总PSA的比值为（0．26±0．23）;前列腺癌患者游离PSA为（12．42±4．97）ng／mL,总PSA为（1．44±0．78）ng／mL,游离PSA与总PSA的比值为（0．12±0．16）。组间比较差异明显,具有统计学意义（P〈0．05）。结论：对患者的PSA进行检测,对前列腺增生和前列腺癌的诊断具有良好的辅助作用和,临床价值。     

Prostate cancer proteomics

Proteomics has offered the hope of biomarker discovery to improve the management of prostate cancer. Markers are needed for screening and diagnosis, distinguishing latent from aggressive disease, defining the men who will benefit from therapy, differentiating localized from metastatic disease, predicting outcome and identifying new targets for therapy. There are many potential sources of proteins derived from the prostate, including urine, prostatic fluid (expressed or ejaculate), serum, and plasma or tissue, each with distinct advantages and limitations. Equally, there are many methodological platforms for proteomic studies of the prostate. Despite the promise, protoemics has yielded little of relevance to the management of prostate cancer, and most of the work that has been published is either irreproducible or of no clinical value.     

Serum Autoantibodies in Chronic Prostate Inflammation in Prostate Cancer Patients

Background

Chronic inflammation is frequently observed on histological analysis of malignant and non-malignant prostate specimens. It is a suspected supporting factor for prostate diseases and their progression and a main cause of false positive PSA tests in cancer screening. We hypothesized that inflammation induces autoantibodies, which may be useful biomarkers. We aimed to identify and validate prostate inflammation associated serum autoantibodies in prostate cancer patients and evaluate the expression of corresponding autoantigens.

Methods

Radical prostatectomy specimens of prostate cancer patients (N = 70) were classified into high and low inflammation groups according to the amount of tissue infiltrating lymphocytes. The corresponding pre-surgery blood serum samples were scrutinized for autoantibodies using a low-density protein array. Selected autoantigens were identified in prostate tissue and their expression pattern analyzed by immunohistochemistry and qPCR. The identified autoantibody profile was cross-checked in an independent sample set (N = 63) using the Luminex-bead protein array technology.

Results

Protein array screening identified 165 autoantibodies differentially abundant in the serum of high compared to low inflammation patients. The expression pattern of three corresponding antigens were established in benign and cancer tissue by immunohistochemistry and qPCR: SPAST (Spastin), STX18 (Syntaxin 18) and SPOP (speckle-type POZ protein). Of these, SPAST was significantly increased in prostate tissue with high inflammation. All three autoantigens were differentially expressed in primary and/or castration resistant prostate tumors when analyzed in an inflammation-independent tissue microarray. Cross-validation of the inflammation autoantibody profile on an independent sample set using a Luminex-bead protein array, retrieved 51 of the significantly discriminating autoantibodies. Three autoantibodies were significantly upregulated in both screens, MUT, RAB11B and CSRP2 (p>0.05), two, SPOP and ZNF671, close to statistical significance (p = 0.051 and 0.076).

Conclusions

We provide evidence of an inflammation-specific autoantibody profile and confirm the expression of corresponding autoantigens in prostate tissue. This supports evaluation of autoantibodies as non-invasive markers for prostate inflammation.