目的：探讨氧化苦参碱( oxymatrine，OM)抑制人结肠癌LoVo细胞增殖和诱导凋亡的分子作用机制.方法：采用流式细胞仪检测LoVo细胞凋亡率以及细胞周期分布；采 用荧光定量PCR法检测0.25，0.5 g·L-1 OM对LoVo细胞增殖相关基因c -myc，蛋白酶调解因子9(PSMD9)，CDK4的基因表达的影响.结果：0.5 g·L-1以下浓度的OM作用结肠癌LoVo细胞48 h，对细胞凋亡无明显影响.0.25 g·L-1 OM作用48 h时可明显抑制人结肠癌LoVo细胞c-myc基因表达(P＜0.05).0.5g·L-1 OM作用48 h时可明显抑制LoVo细胞c-myc，CDK4的基因表达(P ＜0.01，P＜0.01，).药物作用时间为96 h时，0.5g·L-1 OM可明显抑制c-myc，PSM D9，CDK4基因表达(P＜0.05，或P＜0.01).结论：较低剂量OM显著抑制人结肠癌LoVo细胞增殖的作用机制，可能与下调LoVo细胞c- myc，PSM D9，CDK4表达有关.
第一部分血清ST-PSMD蛋 白检测系统的建立及其在肝癌诊断中的应用和意义研究背景原发性肝癌(hepatocellular carcinoma， HCC)是我国最常见恶性肿瘤之一，每年新发病例约占全球50%。由于早期症状不典型，出现症状时多属中晚期，临床确诊的肝癌多数为肝癌的三、四期，肿瘤 切除后复发、转移率高。最近，肝癌的治疗手段不断发展，但肝癌患者的预后并无明显提高，复发和转移率仍较高。因此早发现、早诊断、早治疗是肿瘤诊治的基本 策略，而肿瘤标志物常常是发现早期肿瘤的线索之一。临床上，血清甲胎蛋白(a-fetoprotein， AFP)是目前唯一广泛应用的HCC标志物。但是近年研究报道，AFP诊断HCC的阳性率仅为60%-70%，在小肝癌中敏感性更低，容易造成漏诊；其次 在一些良性肝病(慢性肝炎、肝硬化)、生殖性畸胎瘤等患者中也可有升高，故AFP在肝癌的诊断上有一定的假阳性和误诊率。目前尚无任何肿瘤标志物可完全准 确的诊断肝癌，所以必须结合多种手段联合检测以提高肝癌的早期诊断率，因此发现新的高敏感性和特异性的血清肿瘤标志物意义重大。因此，临床上需要高敏感和 特异性的肝癌血清标志物来鉴别肝脏的良恶性病变，同时为高危人群的筛查提供帮助。HCC血清标志物的研究一直是科学家们的研究的重点，但大多数研究成果难 以满足实际应用的需要。探索高敏感及特异性的血清学指标尤为重要。ST-PSMD基因，编码蛋白为人26S蛋白酶体非ATP酶亚基，是蛋白酶体的组分之 一。ST-PSMD作为蛋白降解途径中的重要分子，可以选择性的结合到被泛素化标记的蛋白，从而参加和介导蛋白质的降解。其表达异常与多种免疫性疾病相 关。我们课题组的前期研究发现ST-PSMD在癌旁和正常肝组织中不表达或表达很低，但在临床肝癌样本中mRNA水平和蛋白水平的表达均较高；同时ST- PSMD也可促进肝癌细胞的恶性生物学表型，并且可以作为肝癌预后评估的标志物；提示ST-PSMD在肝癌的发生发展中发挥着重要的作用，而ST- PSMD作为肝癌血清学诊断标志物的价值仍未得到证实。有研究证实ST-PSMD蛋白在多种实体肿瘤中高表达。我们希望通过本课题的研究，阐明ST- PSMD在肝癌血清学诊断中的应用价值，为临床肝癌的诊断提供指导。研究目的通过对肝癌病人血清中ST-PSMD蛋白水平的检测，深入探讨ST-PSMD 在肝癌血清学诊断中的应用价值，为肝癌的诊断提供潜在的标志物。实验方法通过建立特异针对ST-PSMD的ELISA检测系统，研究正常人群，肝硬化病人 以及肝癌病人血清中ST-PSMD蛋白的表达情况，进一步明确其在不同人群血清样本中的表达趋势，与AFP血清水平的表达情况进行比较和分析：同时与临床 资料作联合分析；比较分析ST-PSMD和AFP在肝癌血清学诊断中的敏感性及特异性；进一步评估联合血清ST-PSMD和AFP在肝癌血清学诊断中的应 用价值。实验结果成功建立人血清学ST-PSMD蛋白的酶联免疫吸附检测方法，用此方法成功检测肝癌病人(100例)，正常人群(50例)，肝硬化患者 (20例)血清中ST-PSMD的表达情况。结果显示肝癌病人血清中ST-PSMD的表达明显高于正常人群，差异有统计学意义(P0.001)；同时显著 高于肝硬化患者血清学水平，差异有统计学意义(P0.001)。Youden指数最大的数值作为鉴别肝癌、肝硬化患者与正常人群的最佳临界值 (Cutoff)。 ST-PSMD诊断肝癌的Cutoff值为0.288ng/ml时的敏感性和特异性分别为71%，84.2%。而我们选用临床上血清学AFP检测常规 Cutoff值(20ng/ml)，肝癌诊断的敏感性为60%，特异性为84.2%，提示血清ST-PSMD有望作为新的肝癌血清学诊断标志物;ST- PSMD与AFP联合使用时肝癌诊断的敏感性为80%，特异性为84.2%，可进一步提高肝癌血清学诊断的敏感性。在肝癌组AFP阴性的患者中有 67.5%的患者血清ST-PSMD呈阳性，其在AFP阴性的肝癌患者中诊断敏感性为60%，特异性为84.3%；提示ST-PSMD可提高AFP阴性肝 癌患者的诊断率，有助于减少漏诊率。有肝硬化背景的肝癌患者血清中ST-PSMD水平明显高于肝硬化组，差异有统计学意义(P0.001)；结果提示 ST-PSMD有望成为肝脏良恶性疾病鉴别诊断的血清学标志物。结论本课题成功建立了ST-PSMD蛋白的酶联免疫吸附检测系统，检测了ST-PSMD在 不同人群中的血清表达水平。结果显示肝癌病人血清中ST-PSMD蛋白的表达明显高于正常人群和肝硬化患者的血清学水平(P0.001)。我们的研究结果 表明，ST-PSMD蛋白有望成为新的肝癌早期血清学诊断标志物，联合使用血清ST-PSMD和AFP可进一步提高肝癌血清学诊断的敏感性，为肝癌临床诊 断提供指导；肝硬化肝癌患者血清ST-PSMD水平显著高于肝硬化患者血清水平(P0.001)，其在两者的鉴别诊断中存在较好的敏感性和特异性，提示其 可能成为鉴别肝脏良恶性疾病的血清学指标。本课题创新点及潜在应用价值首次建立了人血清中ST-PSMD的ELISA检测方法。首次将ST-PSMD运用 于肝癌病人的血清学检测。第二部分 肝脏原发性神经内分泌肿瘤的临床研究研究背景原发性肝脏神经内分泌肿瘤(Primary hepatic neuroendocrine tumor， PHNET)是一种罕见的肝脏肿瘤，大部分患者因没有明显的临床表现而导致诊治较晚。因此PHNET的诊断必须依赖于病理表现和免疫表型，同时还要经过全 面的检查和长期的随访。同原发性肝癌一样，手术切除肿瘤是目前主要的治疗手段。虽然有些原发性肝脏神经内分泌肿瘤肿块较大且部位较深，但手术切除仍可以有 效的延长病人生存期，曾有文献报道过一例巨大型且进展较快的原发性肝脏神经内分泌肿瘤在手术切除术后无病生存长达4年。对于不适合手术切除的病人可以考虑 肝脏移植手术，有文献报道过一例原发性肝脏神经内分泌肿瘤患者肝移植术后无病生存长达7年。目前，学术界尚无统一的组织学分级标准。研究目的通过对东方肝 胆外科医院17例原发性肝脏神经内分泌肿瘤(Primary hepatic neuroendocrine tumor， PHNET)的临床病理特征以及临床资料的回顾性分析，提高临床上对该病的认识和诊治水平。实验方法根据消化系统肿瘤WHO分类(2010)神经内分泌肿 瘤分级标准对我院17例病理诊断明确的原发性肝脏神经内分泌肿瘤(Primary hepatic neuroendocrine tumor， PHNET)进行分级，评价该系统对PHNET的诊断价值，并对临床资料进行回顾性分析，提高对PHNET的认识。结果17例原发性肝脏神经内分泌肿瘤 (PHNET)组织标本进行新标准分型，其中5例因标本因素未能进行分类，12例原发性肝脏神经内分泌肿瘤(PHNET)中NET G1(5例)，NET G2(7例)，NEC(0例)；17例患者均无明显类癌综合征表现，NET(G1，G2)I临床表现无差异。NET G1组术后无瘤生存率为80%，NET G2组术后无瘤生存率为57.1%。结论原发性肝脏神经内分泌肿瘤(PHNET)临床上罕见，患者多无类癌综合征表现，确诊主要依靠病理表型及分级，原发 性肝脏神经内分泌肿瘤(PHNET)的恶性程度及预后判断需要在消化系统神经内分泌肿瘤WHO分类的基础上结合肿瘤大小及有无子灶等多种因素综合判断。本 课题创新点及潜在应用价值评价了消化系统肿瘤WHO分类(2010)神经内分泌肿瘤分级标准对原发性肝脏神经内分泌肿瘤(PHNET)的诊断价值。
Objective Analysis of correlation detection of Survivin，Psmd10 and pancreatic ductal adenocarcinoma. Methods 30 cases of adenocarcinoma patients in our hospital from 2010 June to 2012 June were pancreatic duct，were retrospectively analyzed，and the expression of immunohistochemical method was used to detect pancreatic ductal adenocarcinoma tissue in patients with Survivin，Psmd10. Results for the detection of pancreatic ductal adenocarcinoma in 30 cases after the discovery，the expression of Survivin，Psmd and 10 in pancreatic ductal adenocarcinoma，and the expression of Survivin in pancreatic ductal adenocarcinoma was positively correlated with the degree of differentiation，patients with pancreatic ductal adenocarcinoma of the gender，age and tumor location was not associated with 10; Psmd expression in pancreatic ductal adenocarcinoma tumor differentiation was positively related，not related to gender，age，tumor location，tumor size; the expression of Survivin，in pancreatic ductal adenocarcinoma Psmd l0 are relevant to the. Conclusion In clinic，and detect the occurrence of Survivin，Psmd10 and pancreatic ductal adenocarcinoma，in development with Survivin and Psmd10 had synergetic effect of pancreatic ductal adenocarcinoma，according to Psmd10 Survivin，suggests that the changes can do work on prognosis of patients with pancreatic ductal adenocarcinoma，is worthy to be popularized in practice.
Abstract B22: PSMD2 is a molecular marker for a poor prognosis and determines cancer stem cells traits in breast cancer
The cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSCs that is also capable of differentiating into non-self -renewing cell populations that constitute the bulk tumor mass. These cells have stem cell characteristics and may play a role in tumorigenic growth， metastasis， and therapeutic resistance. Here we interrogated the oncogenic roles of PSMD2， a subunit of the 19S regulatory complex of proteasomes， as a constituent of a signature is a candidate oncogene in breast cancer. PSMD2 is required for breast CSCs proliferation both and ， as shRNA-mediated PSMD2 silencing attenuated tumor growth. Further， PMSD2 is sufficient to induce stem cell characteristics， as forced PSMD2 expression facilitated mammosphere formation and increased the CD44breast CSC-like population. Mechanistically， the action of PSMD2 for induction of breast stemness is mediated by transcriptional regulation of the Notch signaling pathway and PSMD2 inhibition were associated with changes in epithelial-mesenchymal transition (EMT) phenotype. Knockdown of PSMD2 induced dramatic morphological changes， the typical spindle-like appearance cells was replaced with the round shape， like epithelial morphology. Moreover， treatment of TGF-β， the main inducer of EMT， lead to increased expression of PSMD2. PSMD2 silencing up-regulated E-cadherin and down-regulated expression of vimentin， N-cadherin and snail， mesenchymal markers. Clinically， PSMD2 expression was elevated in basal-like TNBC (triple negative breast cancer)， correlated with poor prognosis， insilico analysis on a dataset comprising over 2413 microarrays of the web application bc-GenExMiner. Altogether， our findings suggest that PSMD2 may be a good molecular target candidate and PSMD2 inhibitor may kills breast CSCs and ameliorates poor prognosis of breast cancer.
Abstract BACKGROUND： More than 50% of cancer patients are recommended to receive radiotherapy. Recommendations are based mainly on clinical and pathological factors and not intrinsic tumour radio-sensitivity. Use of radiotherapy according to predictive markers would potentially reduce costly over-treatment， and improve the treatment risk-benefit ratio and cancer outcomes. Tumour expression of the 26S proteasome has been reported to predict radiotherapy response： low expression was associated with higher rates of local recurrence after radiotherapy， suggesting that low proteasome expression and activity was associated with radio-resistance. However， this conclusion is at odds with the emerging use of proteasome inhibitors as radio-sensitizers. Our aim was to further analyse the relevance of 26S proteasome expression， focussing specifically on the PSMD9 subunit， in the largest clinical cohort to date， and to investigate the functional role of PSMD9 in radio-sensitivity in breast cancer cell lines. METHODS： We examined expression of PSMD9 using immunohistochemistry in a cohort of 157 breast cancer patients， including 32 cases (20.4%) that subsequently developed local recurrences. The value of expression as a prognostic or radiotherapy predictive marker was tested using Kaplan-Meier and Cox regression analyses. PSMD9 function was examined in breast cancer cell lines MCF7 and MDA-MB-231 using siRNA knock-downs and colony forming assays after irradiation. RESULTS： Low tumour PSMD9 expression was significantly associated with a reduced incidence of local recurrence in patients receiving adjuvant radiotherapy (univariate log rank p = 0.02; multivariate regression p = 0.009)， but not in those treated without radiotherapy， suggesting that low PSMD9 expression was associated with relative tumour radio-sensitivity. In support of this， reduction of PSMD9 expression using siRNA in breast cancer cell lines in vitro sensitized cells to radiotherapy. CONCLUSIONS： We conclude that PSMD9 expression may predict radiotherapy benefit， with low expression indicative of relative radio-sensitivity， the opposite of previous reports relating to 26S proteasome expression. Our conclusion is compatible with use of proteasome inhibitors as radio-sensitizers， and highlights PSMD9 as a potential target for radio-sensitizing drugs.
Diabetic neuropathy， a long-term complication of type 2 diabetes (T2D)， has a genetic inheritance component to its manifestation. We aimed at identifying whether the proteasome modulator 9 (PSMD9) gene responsible for linkage with T2D and maturity-onset diabetes of the young 3 in Italian families may play a role in the inheritance of diabetic neuropathy in T2D as well.
SUMOylation is the covalent conjugation of SUMO polypeptides to cellular target proteins. Psmd1 is a subunit of the proteasomal 19S regulatory particle that acts as a docking site for Adrm1， another proteasome subunit that recruits ubiquitinated substrates for proteolysis. Here， we show that the SUMO deconjugating enzyme xSENP1 specifically interacts with Psmd1 and that disruption of xSENP1 targeting delays mitotic exit. Psmd1 becomes SUMOylated through the action of the SUMO E3 enzyme PIASy. We mapped SUMOylation sites within Psmd1 and found that SUMOylation of a critical lysine immediately adjacent to the Adrm1-binding domain regulates the association of Adrm1 with Psmd1. Together， our findings suggest that the interaction of Psmd1 with Adrm1 is controlled by SUMOylation in a manner that may alter proteasome composition and function. These findings demonstrate a mechanism for regulation of ubiquitin-mediated protein degradation by ubiquitin-like proteins of the SUMO family.
A variant of PSMD6 is associated with the therapeutic efficacy of oral antidiabetic drugs in Chinese type 2 diabetes patients.
The PSMD6 variant rs831571 has been identified as a susceptibility locus for type 2 diabetes mellitus (T2DM). This study aimed to investigate the association of this variant with therapeutic effects of oral antidiabetic drugs in Chinese T2DM patients. 209 newly diagnosed T2DM patients were randomly assigned to treatment with repaglinide or rosiglitazone for 48 weeks， and the therapeutic effects were compared. In the rosiglitazone cohort， rs831571 showed significant associations with fasting plasma glucose (FPG)， 2-h glucose and decrement of glycated haemoglobin (HbA1c) levels after 24 weeks of treatment (P = 0.0368， 0.0468 and 0.0247， respectively). The C allele was significantly associated with a better attainment of FPG at 24 and 32 weeks (P = 0.0172 and 0.0257， respectively). Survival analyses showed CC homozygotes were more likely to attain a standard FPG level (P = 0.0654). In the repaglinide cohort， rs831571 was significantly associated with decreased HbA1c levels after 24 weeks of treatment， the homeostatic model assessment of insulin resistance and fasting insulin level after 48 weeks of treatment with repaglinide (P = 0.0096， 0235 and 0.0212， respectively). In conclusion， we observed that the PSMD6 variant rs831571 might be associated with the therapeutic effects of rosiglitazone and repaglinide in Chinese T2DM patients. However， these findings need to be confirmed in the future.
Microgravity environment of space can induce a serial of changes in cells， such as morphology alterations， cytoskeleton disorder and cell cycle disturbance. Our previous study of simulated-microgravity on zebrafish (Danio rerio) embryos demonstrated 26s proteasome non-ATPase regulatory subunit 8 (PSMD8) might be a microgravity sensitive gene. However， functional study on PSMD8 is very limited and it has not been cloned in zebrafish till now. In this study， we tried to clone PSMD8 gene in zebrafish， quantify its protein expression level in zebrafish embryos after simulated microgravity and identify its possible function in cell cycle regulation. A rotary cell culture system (RCCS) designed by national aeronautics and apace administration (NASA) of America was used to simulate microgravity. The full-length of psmd8 gene in zebrafish was cloned. Preliminary analysis on its sequence and phylogenetic tree construction were carried out subsequently. Quantitative analysis by western blot showed that PSMD8 protein expression levels were significantly increased 1.18 and 1.22 times after 24-48hpf and 24-72hpf simulated microgravity， respectively. Moreover， a significant delay on zebrafish embryo development was found in simulated-microgravity exposed group. Inhibition of PSMD8 protein in zebrafish embryonic cell lines ZF4 could block cell cycle in G1 phase， which indicated that PSMD8 may play a role in cell cycle regulation. Interestingly， simulated-microgravity could also block ZF4 cell in G1 phase. Whether it is PSMD8 mediated cell cycle regulation result in the zebrafish embryo development delay after simulated microgravity exposure still needs further study. Key Words： PSMD8; Simulated-microgravity; Cell cycle; ZF4 cell line
Genetic variants at PSMD3 interact with dietary fat and carbohydrate to modulate insulin resistance.
Abstract PSMD3 encodes subunit 3 of the 26S proteasome， which is involved in regulating insulin signal transduction， and dietary factors could potentially regulate the function of this gene. We aimed to investigate the associations of PSMD3 variants with glucose-related traits and the interactions of those variants with dietary fat and carbohydrate for glucose-related traits in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) study and to replicate the findings in the Boston Puerto Rican Health Study (BPRHS). Ten single nucleotide polymorphisms (SNPs) were selected， covering 90% the genetic variations in or near PSMD3. Minor allele (C) carriers of rs4065321 had higher homeostasis model assessment of insulin resistance (HOMA-IR) than noncarriers in males of both the GOLDN (P = 0.022) and BPRHS (P = 0.036). Minor allele (T) carriers of rs709592 had significantly higher HOMA-IR (P = 0.032) than C homozygotes in the GOLDN， whereas the T allele carriers of rs709592 tended to have higher HOMA-IR (P = 0.08) than C homozygotes in the BPRHS. In the GOLDN， there was an interaction between rs709592 and dietary carbohydrate on HOMA-IR (P = 0.049). Subjects carrying the T allele of rs709592 had higher HOMA-IR compared only with noncarriers with low carbohydrate intake (≤49.1% energy; P = 0.004). SNPs rs4065321 and rs709592 both significantly interacted with dietary MUFAs and carbohydrate on glucose concentrations in the GOLDN. Our study suggests that PSMD3 variants are associated with insulin resistance in populations of different ancestries and that these relationships may also be modified by dietary factors.