P16INK4a和Fas在椎间盘组织细胞中的表达及意义

目的：分析衰老关键基因p16^INK4a及凋亡相关基因Fas在人类椎间盘退变过程中的表达变化。方法：分别取正常人和腰椎间盘退变患者的髓核及纤维环组织块制作石蜡切片，利用免疫组织化学及免疫荧光法检测p16^INK4a和Fas的表达情况；提取总蛋白及总RNA，利用Western blot及RT-PCR对p16^INK4a和Fas的表达以及视网膜母细胞瘤蛋白（pRb）的磷酸化状态进行分析。结果：p16m在正常人椎间盘髓核及纤维环组织中的表达阳性率分别为6．6％、4．7％，在内破裂椎间盘（IDD）及突出椎间盘（LIDP）组织中的表达分别为44．1％、38．9％和56．1％、46．7％，较正常人椎间盘明显升高（P〈0．05），尤以LIDP中的表达上调显著（P〈0．05）；Fas在正常椎间盘与IDD的髓核及纤维环组织中的表达阳性率均较低。分别为9．9％、8．1％和10．2％、10．9％，在LIDP组织中有相对较高表达阳性率。分别为25．2％和22．0％；Western blot及RT-PCR分析显示，p16^INK4a与Fas在各自蛋白及相应mRNA水平上的表达具有相同的变化趋势：p16^INK4a与Fas极少在同一个椎间盘细胞内表达；随着p16^INK4a表达的升高磷酸化pRb也逐渐减少。结论：p16^INK4a可能参与了椎间盘细胞的衰老过程，是导致椎间盘退变发生和发展的原因之一；Fas表达升高可能是突出椎间盘细胞中的一种继发改变。     

肝硬化组织中p16INK4a和Rb的基因甲基化状态和蛋白表达

目的：探讨肝硬化组织中p16INK4a和视网膜母细胞瘤易感基因（Rb基因）启动子区域的甲基化状态与其蛋白的表达的关系。方法：分别采用PCR和免疫组织化学方法测定65例肝硬化患者和20例正常人肝组织中p16INK4a和Rb启动子区域甲基化状况和蛋白的表达。结果：20例正常肝组织（对照组肝组织）中均未检测到p16INK4a和Rb甲基化异常,肝硬化组肝组织中p16INK4a和Rb基因甲基化率分别为40.0%（26/65）和36.9%（24/65）;p16INK4a和Rb蛋白表达的积分光密度（IOD）在正常肝组织为225.7±27.4和254.7±34.8,在肝硬化组肝组织中为32.4±7.5和45.2±6.4,差异均有统计学意义（均P<0.05）。结论：肝硬化组织中存在p16INK4a和Rb异常甲基化和p16INK4a和Rb蛋白的表达降低,由此导致的细胞周期失控可能参与了肝硬化的发生发展过程。     

Foxo3a和p27kip1在大鼠坐骨神经损伤后背根神经节中的表达

目的 探讨坐骨神经夹伤后Foxo3a和p27kip1在腰段背根神经节(DRG)中表达变化及其意义.方法 将成年SD大鼠随机分为正常对照组和实验组.对实验组实行坐骨神经夹伤术.运用蛋白质印迹、免疫荧光双标,研究大鼠坐骨神经夹伤后,腰段DRG中Foxo3a和p27kip1的表达、分布以及细胞增殖和轴突再生情况.结果 Foxo3a在坐骨神经夹伤后1 d表达值(7.0±3.5)开始明显下降,2 d表达值(6.0±3.8)达到最低值,随之逐渐升高,p27kip1在坐骨神经夹伤后2 d表达值(29.0±3.5)表达开始明显下降,7 d表达值(21.0±3.0)达到最低值,随之逐渐升高;Foxo3a和p27kip1分布于神经元和神经胶质细胞内且在坐骨神经夹伤后2d DRG中,Foxo3a和p27kip1在神经元细胞[(37.8±5.7)%、(43.3±4.3)%]和神经胶质细胞[(22.4±3.9)%、(13.8±3.2)%]中表达较在正常组神经元细胞[(73.6±2.5)%、(84.1±3.7)%]和神经胶质细胞[(61.3±4.4)%、(68.7±5.6)%]减少;细胞核增殖抗原(proliferating cell nuclear antigen,PCNA)和神经元生长相关蛋白(GAP-43)在坐骨神经夹伤后2 d DRG中表达值[12±2.6,15±1.9]均开始上调,PCNA在7 d表达值(25.0±3.2)达到最高点,GAP-43则保持较高水平;此外,PCNA与神经胶质细胞共定位明显,与神经元细胞几乎无共定位.结论 大鼠坐骨神经损伤后,Foxo3a和p27kip1在腰段DRG中的表达减少与神经胶质细胞增殖和轴突再生密切相关.     

纳米氧化锆强韧化高孔隙率磷酸钙人工骨细胞支架生物学评价

目的：对自行研制的纳米氧化锆强韧化高孔隙率磷酸钙人工成骨细胞支架[hyper-porosity Ca3(PO4)：bone cells frame／NM ZrO2 reinforced以下简称支架]的体外生物相容性进行评价。方法：采用细胞毒性试验、溶血试验和急性全身中毒试验3种方法。结果：(1)培养的L929成骨细胞经支架浸提液处理后形态良好，增值旺盛，材料毒性评级为0级；(2)溶血率为2．23％(小于5％)，符合溶血试验标准要求；(3)无急性全身中毒反应。结论：支架复合物具有良好的生物相容性。     

Role of the CDK Inhibitor p27 (Kip1) in Mammary Development and Carcinogenesis: Insights from Knockout Mice

The cyclin-dependent kinase inhibitor p27 (Kip1) is an important cell cycle regulatory gene in breast cancer, and decreased p27 expression is associated with poor prognosis. Some investigations of its role in mammary development have demonstrated reduced cyclin D1 expression and consequent lack of lobuloalveolar development, but others have found increased cyclin E-Cdk2 activity and increased proliferation balanced by increased apoptosis. It is unclear at present why these apparently divergent results have been obtained. Mice with reduced p27 gene dosage alone do not develop mammary carcinomas but do display substantially shorter tumor latency upon overexpression of erbB2, consistent with a role for p27 as a mammary tumor suppressor gene. In this review we summarize these and other data addressing the role of p27 in normal mammary epithelium and experimental models of mammary carcinogenesis.     

Effects of Omega-3 Fatty Acids on Serum Lipids,Lipoprotein (a), and Hematologic Factors in Hemodialysis Patients

Abstract

Background: Lipid abnormalities, especially high serum lipoprotein (a) [Lp (a)] concentration, and anemia are two major causes of cardiovascular diseases (CVDs) in hemodialysis patients. Therefore, this study was designed to investigate the effects of marine omega-3 fatty acids on serum lipids, Lp (a), and hematologic factors in hemodialysis patients. Methods: Thirty-four hemodialysis patients were randomly assigned to either omega-3 fatty acid supplement or placebo group. Patients in the omega-3 fatty acids group received 2080 mg marine omega-3 fatty acids, daily for 10 weeks, whereas the placebo group received a corresponding placebo. At baseline and the end of week 10, 7 mL blood was collected after a 12- to 14-h fast and serum triglyceride, total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), Lp (a), blood hemoglobin, hematocrit, red blood cells (RBCs), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were measured. Results: Serum triglyceride decreased significantly in the omega-3 fatty acids group at the end of week 10 compared with baseline (p < 0.05) and this reduction was significant in comparison with the placebo group (p < 0.01). No significant differences were observed between the two groups in mean changes of serum total cholesterol, LDL-C, HDL-C, Lp (a), blood hemoglobin, hematocrit, RBC, MCV, MCH, and MCHC. Conclusion: The results of our study indicate that marine omega-3 fatty acids can reduce serum triglyceride, as a risk factor for CVD, but it does not affect other serum lipids, Lp (a), and hematologic factors in hemodialysis patients.     

Performance of the Prostate Cancer Antigen 3 (PCA3) Gene and Prostate-Specific Antigen in Prescreened Men: Exploring the Value of PCA3 for a First-line Diagnostic Test

Background

The performance characteristics of serum prostate-specific antigen (PSA) as a diagnostic test for prostate cancer (PCa) are poor. The performance of the PCa antigen 3 (PCA3) gene as a primary diagnostic is unknown.

Objective

Assess the value of PCA3 as a first-line diagnostic test.

Design, setting and participants

Participants included men aged 63–75 who were invited for rescreening in the period from September 2007 to February 2009 within the European Randomised Study of Screening for Prostate Cancer, Rotterdam section.

Interventions

Lateral sextant biopsies were performed if the serum PSA value was ≥3.0 ng/ml and/or the PCA3 score was ≥10.

Measurements

Measurements included distribution and correlation of PSA value and PCA3 score and their relation to the number of cases and the characteristics of PCa detected. Additional value of PCA3 was included in men with previous negative biopsy and/or PSA <3.0 ng/ml.

Results and limitations

In 721 men, all biopsied, 122 PCa cases (16.9%) were detected. Correlation between PSA and PCA3 is poor (Spearman rank correlation: ρ = 0.14; p < 0.0001). A PSA ≥3.0 ng/ml misses 64.7% of the total PCa that can be detected with the sextant biopsy technique and 57.9% of serious PCa (T2a or higher and/or Gleason grade ≥4, n = 19), and 68.2% of biopsies could have been avoided; the respective data for PCA3 ≥35 are 32%, 26.3%, and 51.7%. Performance of PCA3 in men with low PSA (area under the curve [AUC]: 0.63) and/or previous negative biopsy (AUC: 0.68) is unclear but has limited reliability due to small numbers.

Conclusions

PCA3 as a first-line screening test shows improvement of the performance characteristics and identification of serious disease compared with PSA in this prescreened population.     

Refined Genomic Localization of the Genetic Lesion in the Osteopetrosis (op) Rat and Exclusion of Three Positional and Functional Candidate Genes, Clcn7, Atp6v0c, and Slc9a3r2

Osteopetrosis is a disease characterised by a generalized skeletal sclerosis resulting from a reduced osteoclast-mediated bone resorption. Several spontaneous mutations lead to osteopetrotic phenotypes in animals. Moutier et al. (1974) discovered the osteopetrosis (op) rat as a spontaneous, lethal, autosomal recessive mutant. op rats have large nonfunctioning osteoclasts and severe osteopetrosis. Dobbins et al. (2002) localized the disease-causing gene to a 1.5-cM genetic interval on rat chromosome 10, which we confirm in the present report. We also refined the genomic localization of the disease gene and provide statistical evidence for a disease-causing gene in a small region of rat chromosome 10. Three strong functional candidate genes are within the delineated region. Clcn7 was previously shown to underlie different forms of osteopetrosis, in both human and mice. ATP6v0c encodes a subunit of the vacuolar H(+)-ATPase or proton pump. Mutations in TCIRG1, another subunit of the proton pump, are known to cause a severe form of osteopetrosis. Given the critical role of proton pumping in bone resorption, the Slc9a3r2 gene, a sodium/hydrogen exchanger, was also considered as a candidate for the op mutation. RT-PCR showed that all 3 genes are expressed in osteoclasts, but sequencing found no mutations either in the coding regions or in intron splice junctions. Our ongoing mutation analysis of other genes in the candidate region will lead to the discovery of a novel osteopetrosis gene and further insights into osteoclast functioning.     

Calcineurin/nuclear factor of activated T cells (NFAT) signaling in cobalt–chromium–molybdenum (CoCrMo) particles‐induced tumor necrosis factor‐α (TNFα) secretion in MLO‐Y4 osteocytes

Aseptic loosening is the devastating long term complication of total hip arthroplasty and orthopedic implant debris has been shown to trigger an intense inflammatory reaction leading to resorption of the bone matrix. Inflammatory cytokines, such as tumor necrosis factor‐α (TNFα), have been implicated in this process and osteocytes may play a role in its production. We previously demonstrated that cobalt–chromium–molybdenum (CoCrMo) particles upregulate TNFα production by MLO‐Y4 osteocytes in vitro, but the underlying mechanism has not been elucidated. Based on previous studies by others, we hypothesized that the calcineurin‐nuclear factor of activated T cells (NFAT) pathway mediates CoCrMo particle‐induced TNFα production in MLO‐Y4 osteocytes. MLO‐Y4 osteocytes exposed to CoCrMo particle treatment resulted in a rapid and significant increase in calcineurin activity. We also demonstrate that CoCrMo particle‐induced upregulation of TNFα is reduced to control levels with calcineurin‐NFAT inhibitors and this was also confirmed at mRNA level. Moreover, we demonstrate the localization of NFATs in MLO‐Y4 osteocytes and that NFAT1 and 2 translocate to the nucleus upon CoCrMo particle treatment. Our results suggest that calcineurin‐NFAT signaling is involved in TNFα production by MLO‐Y4 osteocytes after CoCrMo particle treatment. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1867–1873, 2011     

Sequestosome 1 Mutations in Paget's Disease of Bone in Australia: Prevalence,Genotype/Phenotype Correlation,and a Novel Non‐UBA Domain Mutation (P364S) Associated With Increased NF‐κB Signaling Without Loss of Ubiquitin Binding

Previously reported Sequestosome 1(SQSTM1)/p62 gene mutations associated with Paget's disease of bone (PDB) cluster in, or cause deletion of, the ubiquitin‐associated (UBA) domain. The aims of this study were to examine the prevalence of SQSTM1 mutations in Australian patients, genotype/phenotype correlations and the functional consequences of a novel point mutation (P364S) located upstream of the UBA. Mutation screening of the SQSTM1 gene was conducted on 49 kindreds with PDB. In addition, 194 subjects with apparently sporadic PDB were screened for the common P392L mutation by restriction enzyme digestion. HEK293 cells stably expressing RANK were co‐transfected with expression plasmids for SQSTM1 (wildtype or mutant) or empty vector and a NF‐κB luciferase reporter gene. GST‐SQSTM1 (wildtype and mutant) proteins were used in pull‐down assays to compare monoubiquitin‐binding ability. We identified SQSTM1 mutations in 12 of 49 families screened (24.5%), comprising 9 families with the P392L mutation and 1 family each with the following mutations: K378X, 390X, and a novel P364S mutation in exon 7, upstream of the UBA. The P392L mutation was found in 9 of 194 (4.6%) patients with sporadic disease. Subjects with SQSTM1 mutations had more extensive disease, but not earlier onset, compared with subjects without mutations. In functional studies, the P364S mutation increased NF‐κB activation compared with wildtype SQSTM1 but did not reduce ubiquitin binding. This suggests that increased NF‐κB signaling, but not the impairment of ubiquitin binding, may be essential in the pathogenesis of PDB associated with SQSTM1 mutations.     

Polyamines,hydrolases (PAP,LAP, SDH,plasmin) TSH,T3, T4 and C-peptide in benign hyperplasia of the prostate

Summary High concentrations of polyamines have been found in the normal human prostate. The profile of these amines appeared significantly changes in benign hypertrophy of the prostate. An increase of spermine and a fall of putrescine were always found in patients with a hypertrophied prostate weighing more than 30 g. Alterations of plasmin in these tissues seemed to reflect changes in the matrix; abnormalities of thyroid and pancreatic function documented by changes in the serum levels of TSH and c-peptide which are thought to be further evidence of a mesenchymal-epithelial interaction in the pathogenesis of benign prostate hypertrophy.Supported by DFG Du 83-2, 3