直接原位RT-PCR检测人成骨肉瘤MG-63细胞株雌激素受体亚型表达

目的观察人成骨肉瘤MG63细胞株雌激素受体亚型mRNA表达特征。方法用直接原位RTPCR及激光共聚焦显微镜观察MG63细胞株在培养第6及第9天ERα、ERβmRNA表达。结果各检测点均见一定量的荧光弥散于细胞内。阳性对照点荧光较其他两点强,阴性对照点荧光明显减弱,仅见少量背景荧光。ERα的荧光强度强于ERβ。结论(1)MG63细胞株作为成骨细胞模型,存在ERα和ERβ基因的mRNA表达。(2)骨基质成熟早期阶段MG63细胞株的ERα表达比ERβ丰富,这表明两者在功能上存在差异。     

骨肉瘤miRNA基因的差异性表达

目的 筛查骨肉瘤miRNA(微小RNA)基因,建立骨肉瘤miRNA基因表达谱,分析明显上调或理调的基因.方法 提取7例骨肉瘤组织和正常骨组织的RNA,利用丹麦Exiqon公司的micm RNA芯片对其进行分析.结果 筛查出骨肉瘤中上调的基因有28个,下调的基因有26个;其中上调大于2.5倍的miRNA基因有5个:miR-381、miR-18a、miR-586、miRPlus_42780、miR-377*,表达差异最大的为miR-586,5倍;其中下调大于2.5倍的miRNAA基因有9个:miR-126、miR-146a、miR-16、miR-191、miR-142-5p、miR-106b、miR-144、miR-26b、miR-20a,其中miR-144基因下调最为明显为39倍.结论 建立了骨肉瘤的miRNA基凶表达谱,miR-586、miR-144基因为骨肉瘤表达最特异的基因.     

Antitumor effects of telomerase inhibitor TMPyP4 in osteosarcoma cell lines

Telomere studies in carcinomas have been extensively reported for prognostic utility and effective methods for targeting telomerase therapy has been described, but efficacy of telomerase inhibitor remained unknown in sarcoma cells. In this study, we investigated the effects of telomerase inhibitor cationic porphyrin TMPyP4 on telomerase activity, telomere length, cell growth, and apoptosis in osteosarcoma cell lines. TMPyP4 significantly inhibited telomerase activity in telomerase positive HOS and Saos‐2, but not in MG‐63. TMPyP4 significantly induced telomere shortening, and inhibition of the cell growth in HOS and Saos‐2 with over 17% apoptosis rates. In terms of MG‐63, TMPyP4 did not induce inhibition of both telomerase activity and cell growth, although it induced significant telomere shortening. Telomere length after treatment was 5.60 kb in HOS, 4.00 kb in Saos‐2, and 9.89 kb in MG‐63. These results may suggest that both telomerase activity loss and sufficient telomere shortening are necessary to inhibit cell growth in telomerase positive osteosarcoma cells. TMPyP4 did not induced telomere shortening but significantly inhibited the growth with 22.6% apoptosis rate in telomerase negative with extremely longer telomere‐U2OS, may indicating the antitumor effect of TMPyP4 may be related to DNA damage including telomere dysfunction through G‐quadruplex stabilization, independent on telomere length. © 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:1707–1711, 2011     

Differential expression of angiogenic cytokines by cell lines and primary cultures of human prostate cancer

Studies on angiogenic cytokines usually are initially based upon their expression by available established cell lines. Our hypothesis is that established epithelial prostate cancer (CaP) cell lines do not accurately reflect angiogenic cytokine expression as compared to epithelial and stromal components of primary cultures generated from clinical CaP specimens. Serum free and growth factor free conditioned medium (CM) was collected from PC3, LNCaP, and their orthotopic selected prostate cancer sublines. Surgically acquired and pathologically confirmed neoplastic prostate tissue was selectively grown for selection of epithelial or stromal components, and CM was also collected. CM was assayed for urokinase (u-PA), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and tumor necrosis factor alpha (TNF-alpha). u-PA was expressed only by androgen independent cell lines, but was detectable in the epithelial and stromal cultures of androgen sensitive primary cultures. bFGF was not secreted by cell lines nor epithelial primary cultures. VEGF was universally expressed, but TNF-alpha was not secreted by cells lines nor primary cultures. These data suggest that the expression of angiogenic cytokines by established epithelial CaP cell lines does not reflect epithelial and stromal primary cultures.Prostate Cancer and Prostatic Diseases (2001) 4, 106-111     

CD-147在骨肉瘤中的表达及其与临床病理指标的相关性实验研究

[目的]验证CD-147在骨肉瘤组织中的表达及与临床病理指标的相关性.[方法]SABC法免疫组化分析55例骨肉瘤组织中CD-147基因的表达,并对其与骨肉瘤临床病理的关系进行统计学分析.[结果]作者的研究提示CD-147基因在骨肉瘤组织有高表达,并且与其外科分级具有明显相关性.[结论]骨肉瘤CD-147蛋白的表达与肿瘤恶性程度密切相关,对骨肉瘤诊断和预后评估具有重要价值.     

人骨肉瘤中S100A6蛋白的表达及其意义

目的观察人骨肉瘤中S100A6蛋白的表达,探讨其与肿瘤转移的关系以及对预后的影响。方法采用免疫组织化学技术对30例骨软骨瘤和60例骨肉瘤石蜡包埋标本进行检测,对9例骨软骨瘤和7例骨肉瘤泳冻标本进行Western-blot检测,分析其表达与转移和预后的关系。结果与骨软骨瘤（10％）相比,S100A6蛋白显著表达于人骨肉瘤（85％）中（P〈0．01）,其表达与性别、肿瘤的分期、分型无关（P〉0.05）,而与肿瘤的转移（P〈0．05）和术后生存时间（P〈0．01）密切相关。结论S100A6蛋白选择表达于骨肉瘤中,可作为肿瘤的早期诊断标志之一,也是判断骨肉瘤转移和预后的重要指标。     

Stimulation of human prostate cancer cell lines by factors present in human osteoblast-like cells but not in bone marrow

Secondary deposits of prostate tumours are frequently found in the skeleton where they produce osteoblastic lesions. In this study both osteoblast-like cells and bone marrow from the proximal femur have been cultured to determine whether or not they can release factors which could support the growth of secondary prostate tumours. Media conditioned by both osteoblast-like cells (OBCM) and bone marrow were examined for their potential to stimulate prostate carcinoma cell lines. Whilst the results obtained demonstrated that OBCM could enhance the growth of both the hormone sensitive (LNCaP) and hormone unresponsive (PC-3 and DU-145) prostate carcinoma cell lines, no proliferative effect could be shown on cell lines derived from cancers of the breast, bladder, and liver. Significantly, media conditioned by either bone marrow or human skin fibroblasts also had no effect on the growth of prostate carcinoma cell lines. This study supports the possibility that the proliferation of prostate cancer cells at secondary skeletal sites, in vivo, may be due to osteoblast derived factors. © 1995 Wiley-Liss, Inc.     

骨肉瘤抑癌基因表达及其增殖活性的临床意义

目的 检测ｐ１６,ｐ５３基因蛋白及增殖细胞核抗原在骨肉瘤中的存在情况,探讨其与骨肉瘤发生,发展和预后的关系。方法 采用改良的免疫组化,ＡＢＣ法,对３２例骨肉瘤手术标本和１５例正常骨组织标本进行ｐ１６,ｐ５３基因蛋白,ＰＣＮＡ的检测。结果 ｐ１６,ｐ５３基因蛋白,ＰＣＮＡ的阳性表达分别为３７．５％,７５．０％,１００％,焉正常对照差异有显著性;     

骨肉瘤中Survivin蛋白表达及其与P-糖蛋白、bcl-2蛋白表达的相关性

目的探讨Survivin蛋白在骨肉瘤中的表达及其与P-糖蛋白、bcl-2蛋白表达的相关性。方法采用免疫组化S—P法检测凋亡抑制基因Survivin在骨肉瘤、骨软骨瘤和正常骨组织中的表达及P-糖蛋白、bcl-2蛋白在骨肉瘤中的表达。结果Survivin蛋白在骨肉瘤中的阳性表达率为63．15％,在骨软骨瘤和正常骨组织中未见表达;Survivin阳性表达与患者的年龄、性别及肿瘤部位无关,与Enneking分期及WHO组织学分型有相关性;P-糖蛋白和bcl-2蛋白在骨肉瘤中的阳性表达率分别为47．36％和55．26％。在骨肉瘤中．Survivin的表达与P-糖蛋白、bcl-2蛋白表达密切相关。结论①Survivin在骨肉瘤中呈高表达,与临床耐药密切相关．有望成为骨肉瘤靶向治疗的一个新靶点。②Survivin与bcl-2蛋白表达密切相关．二者协同发挥抗凋亡效应。     

Usefulness of PCNA labeling index and p53 expression in determining prognosis in osteosarcoma

Immunohistochemical staining of osteosarcoma specimens was performed with anti-proliferating cell nuclear antigen (PCNA) monoclonal antibody (PC10, Novocastra) and anti-p53 antibody (DO7, DAKO), using the streptavidinbiotin (SAB) method. The average PCNA labeling index was 55.3% and all patients with a labeling index higher than 68% died of diesease. p53 expression was 40.9% and the labeling index was 33.3% in patients who were continuously disease-free, but the index was 37.9% (average) in patients who died of disease; all patients with an index higher than 50% died of disese. However, in patients with metastasis, the p53 index was low in patients who survived after resection of the metastasis, suggesting that this profile may reflect the metastatic behavior of the tumor. We often perform preoperative chemotherapy for osteosarcoma and we consider that markers of the effect of chemotherapy are important in predicting survival. It seems that an increase in the PCNA labeling index and an increase in the p53 labeling index or in p53 expression after chemotherapy indicates a good prognosis.     

CD44v6在骨肉瘤组织中的表达及其临床意义

目的探讨骨肉瘤中CD44v6的表达与其临床及生物学行为的关系.方法应用流式细胞免疫学方法检测40例骨肉瘤、10例骨巨细胞瘤、10例骨软骨瘤石蜡标本的CD44v6表达情况,综合分析检测结果对骨肉瘤侵袭转移及预后的影响.结果 CD44v6在骨肉瘤中高表达,明显高于骨巨细胞瘤及骨软骨瘤.CD44v6表达与骨肉瘤患者的性别、年龄、病程及肿瘤发生部位无关;在Enneking外科分期≥ⅡB期(ⅡB、Ⅲ期)、局部复发、伴有肺转移的骨肉瘤中,其CD44v6表达值明显高于＜ⅡB期(ⅡA期)、局部未复发、无肺转移的骨肉瘤.结论 CD44v6可作为骨肿瘤恶性表型的标志和诊断的辅助指标.CD44v6阳性可能预示骨肉瘤复发或转移的倾向,具有一定的临床价值.     

Endogenous bone morphogenetic proteins mediate 1α, 25‐dihydroxyvitamin D3‐induced expression of osteoblast differentiation markers in human dermal fibroblasts

Human dermal fibroblasts are generally considered to be restricted to a fibroblastic lineage. Although dermal fibroblasts do not typically express markers of osteoblastic differentiation, they have previously been shown to undergo osteoinduction when stimulated with bone morphogenetic proteins (BMPs) or vitamin D₃. However, involvement of BMP signaling in vitamin D₃‐mediated osteoinduction has not been reported. In this study, human dermal fibroblasts were cultured in chemically defined medium containing vitamin D₃, in the presence of the BMP antagonist noggin or neutralizing antibodies specific for BMP‐4 or BMP‐6, and characterized for markers of osteoblastic differentiation. Treatment of dermal fibroblasts with vitamin D₃ induced expression of BMP‐4 (1.2 ± 0.2, 1.7 ± 0.2, and 1.8 ± 0.2 relative fold increase) and BMP‐6 (9.1 ± 0.3, 23.3 ± 2.1, and 30.4 ± 3.0 relative fold increase) at 3, 14, and 21 days, respectively. Vitamin D₃ was also shown to induce the expression of the osteoblast‐specific markers, alkaline phosphatase and osteocalcin, in a dose‐dependent manner in human dermal fibroblasts. Addition of noggin, BMP‐4 antibodies, and BMP‐6 antibodies resulted in a downregulation of alkaline phosphatase activity (by 42%, 22%, and 20%, respectively) and secreted osteocalcin (by 20%, 31%, and 49%, respectively) after 21 days in culture. However, blocking BMP signaling did not result in complete recovery of a fibroblastic phenotype. Taken together, these results suggest that BMP signaling plays a role in the induction of an osteoblastic phenotype in human dermal fibroblasts in response to vitamin D₃ stimulation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:162–168, 2009     

骨巨细胞瘤发生与p16和Rb蛋白表达的关系

目的 探讨ｐ１６和Ｒｂ蛋白表达与骨巨细胞瘤发生的关系。方法 采用免疫组织化学链霉菌抗生物素－过氧化物酶法（ＳＰ）检测４２例骨巨细胞瘤的ｐ１６与Ｒｂ蛋白表达。研究不同病理分级的骨巨细胞瘤ｐ１６蛋白表达及Ｒｂ蛋白表达之间的关系。结果 骨巨细胞瘤ｐ１６蛋白表达缺失率为６６．７％，ｐ１６蛋白表达随骨白细胞瘤恶性程度的上升而减低。Ｒｂ蛋白表达缺失率为４２．９％。６６．７％的病例存在ｐ１６与Ｒｂ蛋白表达的倒置     

Epithelial and fibroblastoid cell lines derived from the normal canine prostate. II. Cell proliferation in response to steroid hormones

Epithelial and fibroblastoid cell lines derived from the adult canine prostate, have been used as model systems in a preliminary study of the effects of steroid hormones on isolated cell populations. The mitogenic activities of various androgens and one estrogen were assessed during logarithmic growth in replicate subcultures by measurement of population size and radiolabeled nucleotide uptake in the presence of concentration ranges of these steroids. [H³]-Leucine incorporation rate was used as an estimate of protein synthesis in the epithelial cell line during the latter phase of log growth. Of the androgens examined, elevated cell population growth rate was only demonstrated in either epithelial or fibroblastoid cell cultures supplemented with 5α-androstane-3α, 17β- and 17α-diols at physiological concentrations. No significant qualitative differences were observed between individual prostatic cell types in their proliferative response to the androgens tested. Contrasting effects were observed in the growth rates of epithelial and fibroblastoid cell types in cultures supplemented with estradiol-17β. This estrogen was significantly mitogenic in fibroblast cultures, but did not significantly affect epithelial proliferation at the concentrations studied. These results are reviewed with reference to current concepts of steroid action in the canine prostate. The importance of the characterization of culture conditions and procedures has been emphasized as a prerequisite to the study of cell proliferation, and potential mitogens, using cell culture models. The relevance of these findings in the consideration of stromal-epithelial interaction, and glandular differentiation, is discussed.     

Bak expression and cell death occur in peritumorous tissue but not in pancreatic cancer cells

Bak is a pro-apoptotic member of the Bcl-2 family whose genes are involved in regulation of programmed cell death. Using in situ hybridization, immunohistochemistry, and Northern blot analysis, we studied the expression of Bak in specimens from 12 normal pancreata and 26 primary pancreatic cancers, and correlated the findings with the clinical and histopathologic data of the patients. By comparison with normal pancreas, Northern blot analysis demonstrated a 2.5-fold increase of Bak messenger RNA expression in the tumor samples (P <0.001). Elevated levels were found in 15 of the 26 pancreatic cancer tissue specimens. In these samples Bak expression was increased 4.3 fold (P <0.001). No association was detected between Bak expression and tumor stage. In situ hybridization and immunohistochemistry revealed that the tumor cells themselves and the stroma cells expressed only low levels of Bak. In contrast, in regions adjacent to the tumor, which showed chronic inflammation, there was always high expression in the acinar and inflammatory cells, explaining the increased Bak levels found in the tumor samples by means of Northern blot analysis. In the normal pancreas the expression of Bak was generally moderate in the acinar cells and low in the ductal and islet cells. In situ analysis using the terminal deoxynucleotidyl transferase method further showed that there was extensive cell death in the peritumorous areas with chronic inflammation. Taken together, these results suggest that in pancreatic cancer Bak expression and programmed cell death are present in cells that are localized in regions of chronic inflammation surrounding the pancreatic cancer cells but not in the tumor cells themselves, a situation that may facilitate tumor growth and spread.     

Regulation of beta2-microglobulin expression in different human cell lines by proinflammatory cytokines.

BACKGROUND: Proinflammatory monocytic cytokines such as interleukin-1 (IL-1), tumour necrosis factor-alpha (TNF-alpha) and IL-6 have been incriminated in the pathogenesis of elevated beta2-microglobulin (beta2M) serum concentrations in patients undergoing haemodialysis with so-called bioincompatible dialyser membranes. However, neither the source of the elevated serum beta2M nor the precise role of monocytic cytokines in the expression of the beta2M gene have been elucidated conclusively. The aim of the current study was to evaluate whether monocytic cytokines, and in particular IL-6, are regulators of beta2M gene expression in human hepatoma cells, T-lymphocytes and monocytes. METHODS: HepG2 and HuH7 human hepatoma cells, Jurkat T-cells, monocytic MonoMac6 cells, primary human monocytes and synoviocytes were stimulated with IL-1beta, IL-6, interferon-alpha (IFN-alpha), IFN-gamma or conditioned media from lipopolysaccharide (LPS)-treated monocytes. Expression of beta2M mRNA was analysed by Northern blotting, beta2M protein synthesis was determined by metabolic labelling and immunoprecipitation, and beta2M secretion was measured by an enzyme-linked immunosorbent assay (ELISA). RESULTS: In all cell types tested, IFN-gamma and, to a lesser extent, IFN-alpha stimulated gene expression of beta2M resulting in an increased synthesis and secretion of beta2M protein. Neither IL-1beta and IL-6 nor supernatants from LPS-treated monocytes were capable of inducing beta2M gene expression, with the exception of a small increase in HuH7 hepatoma cells upon IL-1beta treatment. CONCLUSIONS: The present study provides evidence that interferons are important regulators of beta2M expression. It also shows that proinflammatory monocytic cytokines do not modulate directly the expression of beta2M in cells of hepatic, monocytic and T-lymphocytic origin. Whether they influence beta2M synthesis and secretion indirectly by modulating interferon synthesis needs to be elucidated.     

Prostasin基因在前列腺细胞株中表达情况研究

目的 探讨Prostasin在前列腺癌中的功能。方法 运用RT-PCR方法检测人前列腺增生细胞株BPH-1，无转移能力前列腺癌细胞株LNCaP，及有转移能力的前列腺癌细胞株PC-3、DU-145中Prostasin基闪表达情况。结果 Prostasin基斟在BPH-l和LNCaP中正常表达，在PC-3、DU-145中低表达。BPH-l中Prostasin的表达与DU-145和PC-3比较差异有显著性，同样LNCaP中Prostasin的表达与DU-145和PC-3比较差异亦有显著性（P〈0．01）。BPH-1与LNCaP之间表达差异无显著性，DU-145和PC-3之间表达差异亦无显著性，（P〉0.05）。结论 ProStasin可能是前列腺癌的转移抑制剂。     

应用RNA干扰下调多药耐药蛋白4表达对结直肠癌细胞照射敏感性的影响

目的探讨多药耐药蛋白4（MRP4）表达对结直肠癌细胞照射敏感性的影响。方法应用慢病毒感染RNA干扰技术（RNAi）稳定下调人结直肠癌细胞株HCT116中MRP4的表达。将HCT116细胞分为未感染任何病毒的细胞株（CON）、加阴性对照病毒感染的细胞株（NC）和加RNAi靶点病毒感染的细胞株（KD）3组,应用实时定量RT-PCR和Western blot分别从RNA和蛋白水平检测MRP4表达变化,以验证RNAi的有效性。4Gy剂量照射后24h,流式细胞术检测细胞凋亡,MTT检测细胞增殖,比较RNAi后HCT116细胞株照射敏感性的差异。结果成功构建并转染慢病毒质粒,获得稳定沉默MRP4表达的HCT116-KD细胞株,HCT116-KD细胞株MRP4mRNA和蛋白表达水平较对照均明显下调（P〈0．05）。照射后24h,KD细胞株凋亡率为（71．7±0．8）％,明显高于CON组[（56．1±0．9）％]和NC组[（59．8±0．8）％]（P〈0．05）。照射后48h和72h,KD细胞增殖能力较对照组明显下降（火0．05）。结论MRP4在结直肠癌细胞中的表达水平与照射耐受显著相关,应用慢病毒感染RNA干扰下调MRP4表达可以增强结直肠癌细胞照射敏感性。MRP4有可能成为预测放疗敏感性的分子标志物。