二硫代氨基甲酸吡咯烷提高甲氨蝶呤对人骨肉瘤MG-63细胞增殖活性和凋亡的影响

Objective To detect the effect of pyirolidine dithiocarbamate (PDTC) and methotrexate (MTX) on apoptosis and expression of livin of human osteosarcoma MG-63 cells. Methods MG-63 cells were cultured in vitro. At 24, 48 and 72 h after treatment of PDTC and MTX at different concentrations (0, 50, 100, 200 and 400 μmol/L) , MTT assay was used to observe the growth inhibition of MG-63 cells. The apoptosis was assessed by flow cytometry. The protein expression of livin was detected by Westem blotting. Results When PDTC and MTX were added, growth inhibition and increased apoptosis of MG-63 cells were detected. The protein expression level of livin was down-regulated obviously ( P ＜0. 05). Conclusion PDTC can promote the apoptosis of MTX-treated MG-63 cells, which may be correlated with down-regulation of the livin expression.     

NF-kB抑制剂PDTC对人骨肉瘤MG-63细胞凋亡及Livin和Caspase-9表达的影响

[目的]观察NF-kB抑制剂PDTC对人骨肉瘤MG-63细胞凋亡及Livin和Caspase-9表达的影响.[方法]体外培养人骨肉瘤MG -63细胞系,用50,100,200和400 RmoV L的PDTC作用于骨肉瘤MG-63细胞24,48,72 h后,用MTT法测各组骨肉瘤MG-63细胞存活率,用流式细胞仪测细胞的凋亡率.用Western Blot方法检测不同浓度PDTC作用于骨肉瘤MG-63细胞24 h后,各组细胞的Livin和Caspase-9蛋白的表达水平.[结果]随PDTC浓度增加,骨肉瘤MG-63细胞后其增殖活性呈下降趋势,作用时间越长细胞的增殖活性越低,而细胞的凋亡率呈上升趋势(P<0.05).随PDTC浓度增加各组细胞中Livin蛋白的表达呈下降趋势,而Caspase9蛋白表达呈上升趋势(P<0.05).[结论]PDTC可诱导人骨肉瘤MG-63细胞凋亡,其机制可能与PDTC抑制NF-kB传导通路,下调Livin表达继而上调Caspase-9表达有关.     

艾拉莫德、吡咯烷二硫代氨基甲酸盐单用和联合治疗恶病质

目的 观察艾拉莫德(T614)、吡咯烷二硫代氨基甲酸盐(PDTC)联合应用对小鼠癌症恶病质的治疗作用.方法 于雄性BALB/C小鼠接种小鼠结肠腺癌Colon26(C26)细胞,9 d后恶病质模型基本建立.用药7 d后,记录小鼠左侧腓肠肌重量和去瘤体质量,检测血生化指标、血清白细胞介素6(IL-6)、肿瘤坏死因子-α(TNF-α)水平并用免疫组织化学法检测肿瘤组织核因子-κB(NF-κB)表达.结果 恶病质组小鼠血清IL-6、TNF-α浓度为(93.72 ±11.20)、(128.70±33.41)ng/L.T614组、PDTC组、T614+PDTC组IL-6浓度为(81.11±+11.08)、(79.25±9.91)、(53.60±10.5)ng/L,TNF-α浓度为(90.16±11.57)、(99.51±15.25)、(75.45±12.48)ng/L,均低于恶病质组(P＜0.05),各生化指标亦有不同程度的改善(P＜0.05).结论 通过抑制NF-κB可以改善恶病质.T614和PDTC联合应用治疗效果优于单种药物.

Abstract：

Objective To observe the effect of the combined use of iguratimod (T614) and pyrrolidine dithiocarbamate (PDTC) on animal cancer cachexia model. Methods Male BALB/C mice bearing colon 26 adenocarcinoma for 9 days served as models of cancer cachexia. Seven days after the treatment,body weight and gastrocnemius muscle were documented. Biochemical indicators, serum interleukin-6 (IL-6) , and tumor necrosis factor-α (TNF-α) levels were evaluated. The expression of nuclear factor-κB ( NF-κB ) in tumor was detected by using immunohistochemistry. Results The IL-6 levels in cachexia group, T614 group, PDTC group and T614 + PDTC group were (93. 72 ± 11. 20), (81.11 ± +11.08),(79. 25 ±9. 91) and (53. 60 ± 10. 5) ng/L, and those of TNF-a were (128. 70 ± 33. 41) , (90. 16 ±11. 57) , (99. 51 ± 15. 25) and (75. 45 ± 12. 48) ng/L, respectively. The IL-6 and TNF-α levels in T614 group, PDTC group and T614 + PDTC group were significantly lower than in cachexia group ( all P ＜0. 05). The expression of NF-kB was significantly down-regulated in T614 group, PDTC group and T614 +PDTC group as compared with cachexia group (P ＜ 0. 05). Conclusion Inhibition of NF-kB can attenuate the development of cachexia in colon 26 tumor bearing mice. These findings suggest that a therapeutic approach combining T614 with PDTC does have complementary effect in improving outcomes of cancer cachexia.     

甲氨蝶呤对人骨肉瘤MG-63细胞凋亡及28Livin和Caspase-3表达的影响

目的 观察甲氨蝶呤(MTx)对人骨肉瘤MG-63细胞凋亡及Livin和Caspase-3表达的影响.方法 体外培养骨肉瘤MG-63细胞株,用0、50、100、200和400μmol/L的MTX作用于骨肉瘤MG-63细胞24、48、72 h后,用噻唑蓝(MTT)比色法检测骨肉瘤MG-63细胞的增殖活性,用流式细胞仪测细胞的凋亡率,用Western blot检测不同浓度MTX作用于骨肉瘤MG-63细胞24 h后各组细胞的Livin和Caspase-3蛋白表达水平.结果 随MTX浓度增加骨肉瘤MG-63细胞的增殖活性降低,同时细胞的凋亡率增加(P＜0.05),随MTX浓度增加各组细胞中Livin蛋白的表达降低,Caspase-3蛋白表达增加(P＜0.05).结论 MTX诱导人骨肉瘤MG-63细胞凋亡,其机制可能与下调Livin表达继而上调Caspase-3表达有关.

Abstract：

Objective To observe the effect of Methotrexate (MTX) on apoptosis and expression of Livin and Caspase-3 in human osteosarcoma cell line MG-63. Methods After treatment of MG-63 cells with MTX at different concentrations (0, 50, 100,200,400 μmol/L) for 24, 48 and 72 h, methyl thiazol tetrazolium(MTT) assay was used to observe the growth inhibition of MG-63. The apoptosis was assessed by flow cytometry. The protein expression of Livin and Caspase-3 was detected by Western blotting. Results When MTX was added, growth inhibition and increased apoptosis of MG-63 cells were detected,which was showed in a dose- and time-dependent manner. MTX also down-regulated the level of the protein expression of Livin (P＜0.05), and elevated the protein expression of Caspase-3 (P＜0.05). Conclusion MTX can induce apoptosis of MG-63 cells, by down-regulating Livin expression and subsequently up-regulating Caspase-3 expression.     

艾拉莫德、吡咯烷二硫代氨基甲酸盐单用和联合治疗恶病质

Objective To observe the effect of the combined use of iguratimod (T614) and pyrrolidine dithiocarbamate (PDTC) on animal cancer cachexia model. Methods Male BALB/C mice bearing colon 26 adenocarcinoma for 9 days served as models of cancer cachexia. Seven days after the treatment,body weight and gastrocnemius muscle were documented. Biochemical indicators, serum interleukin-6 (IL-6) , and tumor necrosis factor-α (TNF-α) levels were evaluated. The expression of nuclear factor-κB ( NF-κB ) in tumor was detected by using immunohistochemistry. Results The IL-6 levels in cachexia group, T614 group, PDTC group and T614 + PDTC group were (93. 72 ± 11. 20), (81.11 ± +11.08),(79. 25 ±9. 91) and (53. 60 ± 10. 5) ng/L, and those of TNF-a were (128. 70 ± 33. 41) , (90. 16 ±11. 57) , (99. 51 ± 15. 25) and (75. 45 ± 12. 48) ng/L, respectively. The IL-6 and TNF-α levels in T614 group, PDTC group and T614 + PDTC group were significantly lower than in cachexia group ( all P ＜0. 05). The expression of NF-kB was significantly down-regulated in T614 group, PDTC group and T614 +PDTC group as compared with cachexia group (P ＜ 0. 05). Conclusion Inhibition of NF-kB can attenuate the development of cachexia in colon 26 tumor bearing mice. These findings suggest that a therapeutic approach combining T614 with PDTC does have complementary effect in improving outcomes of cancer cachexia.     

艾拉莫德、吡咯烷二硫代氨基甲酸盐单用和联合治疗恶病质

Objective To observe the effect of the combined use of iguratimod (T614) and pyrrolidine dithiocarbamate (PDTC) on animal cancer cachexia model. Methods Male BALB/C mice bearing colon 26 adenocarcinoma for 9 days served as models of cancer cachexia. Seven days after the treatment,body weight and gastrocnemius muscle were documented. Biochemical indicators, serum interleukin-6 (IL-6) , and tumor necrosis factor-α (TNF-α) levels were evaluated. The expression of nuclear factor-κB ( NF-κB ) in tumor was detected by using immunohistochemistry. Results The IL-6 levels in cachexia group, T614 group, PDTC group and T614 + PDTC group were (93. 72 ± 11. 20), (81.11 ± +11.08),(79. 25 ±9. 91) and (53. 60 ± 10. 5) ng/L, and those of TNF-a were (128. 70 ± 33. 41) , (90. 16 ±11. 57) , (99. 51 ± 15. 25) and (75. 45 ± 12. 48) ng/L, respectively. The IL-6 and TNF-α levels in T614 group, PDTC group and T614 + PDTC group were significantly lower than in cachexia group ( all P ＜0. 05). The expression of NF-kB was significantly down-regulated in T614 group, PDTC group and T614 +PDTC group as compared with cachexia group (P ＜ 0. 05). Conclusion Inhibition of NF-kB can attenuate the development of cachexia in colon 26 tumor bearing mice. These findings suggest that a therapeutic approach combining T614 with PDTC does have complementary effect in improving outcomes of cancer cachexia.     

吡咯烷二硫代氨基甲酸盐增强肿瘤坏死因子α诱导人胃癌细胞株SGC-7901凋亡作用的研究

目的研究核转录因子-κb（NF—κb）抑制剂吡咯烷二硫代氨基甲酸盐（PDTC）对肿瘤坏死因子α（TNF—α）诱导的人胃癌细胞株SGC-7901生长抑制及凋亡的影响,并探讨其作用机制。方法应用噻唑蓝（MTr）法检测不同浓度的PDTC和TNF-α以及两者联合应用对SGC-7901细胞增殖的抑制率：采用Hoechst检测SGC-7901细胞凋亡情况：Westernblot检测SGC-7901细胞survivin和easpase-3蛋白的表达。结果PDTC在15、30、60和100μmol／L浓度时．对SGC-7901的细胞生长抑制率分别为（12．14±0．91）％、（20．00±1．11）％、（37．63±1．01）％和（41.46±1．07）％．均可抑制细胞增殖（P〈0．01）。TNF-α为25mg／L时,对SGC．7901细胞的生长抑制率为（2．38±0．67）％,与对照组（1．50±0．81）％相比,差异无统计学意义（F=28．28,P〉0．05）：而在50、100和150mg／L浓度时,对SGC-7901细胞的生长抑制率分别为（4．53±0．85）％、（4．43±0．70）％和（4．74±1．07）％,与对照组相比,差异有统计学意义（P〈0．05）。PDTC15μmol／L分别与25、50、100和150mg／L的TNF．仅联合应用时,对SGC-7901的细胞生长抑制率则分别为（18．94±1．10）％、（30．23±0．89）％、（41．55±0．94）％和（53．34±0．98）％,与单用TNF—α或单用15μmol／LPDTC比较,细胞生长抑制率增加（P〈0．01）。Hoechst检测结果显示,TNF-α100mg／L组、PDTC15μmol／L组及两者联合应用组细胞凋亡率均显著增加（P〈0．01）,且联合用药组细胞凋亡率增高最为显著（P〈0．01）。PDTC（15μmol／L）与TNF-α（100mg／L）联合用药与单用TNF—α（100mg／L）比较,细胞survivin蛋白表达明显降低（P〈0．01）,与单用PDTC（15μmol／L）比较,差异无统计学意义（P〉0．05）;但caspase-3蛋白的表达联合用药组较两者分别单用时显著增加（P〈0．01）。结论PDTC可增强TNF-α对人SGC-7901细胞的促凋亡作用,其机制可能与PDTC阻断TNF-α诱导的NF—κb活性、下调survivin表达并最终上调凋亡蛋白easpase-3的表达有关。     

吡咯烷二硫代氨基甲酸酯治疗大鼠骨关节炎

目的 观察吡咯烷二硫代氨基甲酸酯(PDTC)对大鼠骨关节炎(OA)形态学的影响,探讨PDTC用于OA治疗的可能性.方法 通过前交叉韧带切断的方法建立大鼠OA动物模型,将20只大鼠动物模型分为实验组(10只)和对照组(10只),于造模后每隔48 h肌肉注射PDTC10 ms/ks,对照组仅肌注PDTC的载体生理盐水0.1 ml,术后6周后处死大鼠,进行大体观察和光镜观察,比较两组差异有统计学意义.结果 注射PDTC的实验组大鼠软骨的软骨大体评分明显优于对照组,实验组和对照组的Mankin's评分分别为5.70±1.70,8.20±2.10,两者间差异有统计学意义(P<0.05).结论 PDTC能有效延缓实验性OA软骨的退变过程.     

吡咯烷二硫代氨基甲酸盐对NF-κB调控基因血管内皮生长因子转录和表达的影响

血管内皮生长因子（VEGF）是最重要、最特异的一种血管发生特异性生长因子，在肿瘤血管的形成中起关键作用。活化的核转录因子NF-κB（NF-κB）可调控VEGF等与血管形成相关的基因转录，参与肿瘤的生长调控。吡咯烷二硫代氨基甲酸盐（PDTC）是一种抗氧化剂，是目前公认的NF-κB活性特异性抑制剂。我们通过PDTC抑制胃癌SGC-7901细胞中NF-κB活性，探讨NF-κB对VEGF基因转录和表达的影响，阐明NF-κB在肿瘤血管形成中的作用。     

二硫代氨基吡咯烷预先给药对内毒素诱发大鼠心肌损伤的影响

目的 评价二硫代氨基吡咯烷预先给药对LPS诱发大鼠心肌损伤的影响.方法 成年Wistar大鼠72只,体重200～250 g,雌雄各半,随机分为3组(n=24):对照组(C组)、LPS组和二硫代氨基吡咯烷组(PDTC组).LPS组腹腔注射LPS 8 mg/kg;PDTC组尾静脉注射二硫代氨基吡咯烷120 mg/kg,30 min后腹腔注射LPS 8 mg/kg;C组注射等量生理盐水.分别于注射LPS后1、3/6、12 h时,腹腔注射2%戊巴比妥钠40 mg/kg麻醉大鼠,下腔静脉取血样后处死,开胸取心脏,采用ELISA法测定血清肌钙蛋白T(cTnT)浓度,免疫组化法检测心肌组织Toll样受体4(TLR4)的表达和NF-κB p65的活性;光镜下观察心肌组织病理学结果.结果 与C组比较,LPS组和PDTC组各时点血清cTnT浓度升高,心肌组织TLR4表达上调,NF-κB p65活性升高(P＜0.01);与LPS组比较,PDTC组各时点血清cTnT浓度下降,心肌组织NF-κB p65活性降低,注射LPS后6、12 h时心肌组织TLR4表达下调(P＜0.01),病理学损伤减轻.结论 二硫代氨基吡咯烷可减轻LPS诱发大鼠心肌损伤,其机制可能与抑制心肌组织NF-κB的活性和TLR4的表达有关.     

噻莱昔布(celecoxib)对人骨肉瘤细胞MG-63增殖与凋亡的影响

目的:研究环氧化酶2(cyclooxygenase2,COX2)抑制剂celecoxib对人骨肉瘤细胞MG63抑制增殖及诱导凋亡的作用。方法:MTT比色观察celecoxib的细胞毒性作用及其浓度依赖性;Hoechst33258荧光染色、透射电子显微镜和TUNEL观察细胞凋亡的形态学变化;流式细胞仪检测细胞凋亡率及其时间依赖性。结果:celecoxib分别以10、20、40、80μmol/L作用后,细胞生长受到不同程度抑制;荧光显微镜、电镜和TUNEL观察到细胞胞浆浓缩、核凝聚、核碎裂和凋亡小体形成。流式细胞仪显示celecoxib40μmol/L作用24、48、72h后,细胞凋亡率分别与对照组比较有非常显著性差异(P<0.01)。结论:celecoxib能抑制人MG63细胞增殖,诱导细胞凋亡。     

Effect of ciprofloxacin on the proliferation of osteoblast-like MG-63 human osteosarcoma cells in vitro

Locally applied antibiotic therapy is gaining popularity for the treatment of infections associated with open fractures and posttraumatic osteomyelitis. With use of local techniques, ciprofloxacin levels as high as 1,300 μg/ml. or over 200 times the bone levels achieved with intravenous administration, have been reported. To study the possible effects of ciprofloxacin on bone, osteoblast-like cells from the MG-63 human osteosarcoma cell line were studied. The cells were grown in antibiotic-free media and exposed to concentrations of ciprofloxacin at 0, 10, 100, 200, and 1,000 μg/ml to establish an initial dose-response curve. Media containing the appropriate dose of ciprofloxacin were changed every 24 hours. Cell number and [³H]thymidine incorporation per cell were determined at 0, 24 and 72 hours. A second dose-response curve was performed at concentrations of 0, 10, 20, 40, and 80 μg/ ml. Three experiments, each with four observations, were performed. The results of this study demonstrated that ciprofloxacin caused significant decreases (p < 0.05) in cell number at 40 μg/ml at 24 hours and 20 μg/ml at 72 hours. [³H]thymidine incorporation per cell decreased significantly at levels of 80 μg/ml at 24 hours and 20 μg/ml at 72 hours. The authors conclude that reported local levels of ciprofloxacin seen in vivo inhibit the proliferation of human osteoblast-like cells in vitro.     

Livin凋亡抑制蛋白与肿瘤治疗的研究进展

凋亡抑制蛋白Livin是凋亡抑制因子(IAP)家族的新成员,在多种肿瘤细胞中高表达,参与抑制细胞凋亡,与肿瘤发生、发展及预后密切相关,是一种具有潜在价值的肿瘤标志物,可作为肿瘤早期诊断和治疗的新分子靶点.本文就Livin基因分子结构、组织表达、生物学功能及在肿瘤治疗中的应用等研究进展作一综述.     

Livin基因和Survivin基因联合靶向siRNA诱导结肠癌细胞凋亡的作用

目的探讨Livin基因和Survivin基因联合靶向小干扰（si）RNA对结肠癌细胞增殖及凋亡的影响。方法构建Livin和Survivin联合靶向的siRNA重组表达载体并转染结肠癌细胞．通过RT—PCR和蛋白质印迹方法分别检测Livin和Survivin的表达．通过MTT法检测siRNA对细胞增殖的抑制作用．利用流式细胞仪检测处理后细胞的凋亡效应。结果经酶切鉴定和测序分析证实Livin和Survivin联合靶向的siRNA重组表达载体构建成功。这种联合靶向SiRNA对Livin mRNA及蛋白表达的抑制率分别为27．9％和22．3％．对Survivin mRNA及蛋白表达的抑制率分别为32．2％和40．9％。与对照组相比,联合靶向siRNA可降低肿瘤细胞增殖率．增加细胞凋亡率,但其细胞生长抑制作用和促细胞凋亡作用均弱于单独干扰Livin或Survivin基因．结论Livin和Survivin基因联合靶向siRNA能降低结肠癌细胞中Livin和survivin基因的表达．抑制结肠癌细胞的增殖．并诱导结肠癌细胞的凋亡．但此协同抑制作用较单独干扰Livin或survivin基因为弱。     

膀胱癌患者尿液脱落细胞Livin检测的临床意义

目的探讨膀胱癌患者尿液脱落细胞中Livin的表达及临床意义。方法采用逆转录聚合酶链反应技术（RT-PCR）检测52例膀胱癌患者,18例泌尿外科非肿瘤患者以及10例健康志愿者尿液脱落细胞Livinα和β的表达,以-βactin为内参照。结果52例膀胱癌患者42例尿液脱落细胞Livinα表达阳性,阳性率86.5%,而Livinβ均表达阴性;18例泌尿外科非肿瘤患者及10例健康志愿者Livinα及β均表达阴性。结论检测膀胱癌患者尿液脱落细胞Livinα的表达,敏感性和特异性均较高,可作为临床筛选膀胱癌的理想指标。     

Osteocalcin secretion by the human osteosarcoma cell line MG-63

The human osteosarcoma cell line MG-63 has been used to study the production of the bone-specific protein, osteocalcin. In the absence of any stimuli, MG-63 cells secreted very low levels of osteocalcin. The secretion of osteocalcin started after a lag time of 10-12 h upon 1,25-(OH)2D3 treatment. Osteocalcin secretion was measured at doses as low as 0.03 nM (fourfold increase, p less than 0.05), and this activity increased further with higher doses of 1,25-(OH)2D3 to reach a plateau at 50 nM. The secretion increased transiently from very low levels in sparse cell cultures to peak values in subconfluent cultures (+/- 40%), two- to threefold above values obtained for confluent cells. Values for confluent cells average 55.9 +/- 2.0 ng/ml protein per 48 h. A similar behavior is observed for 1,25-(OH)2D3 receptor concentration under similar experimental conditions. Bmax increased transiently from sparse to subconfluent cell cultures (40-60% confluent) and reached values 50% lower in confluent cells. However, the receptor affinity was not affected by cell density. MG-63 cells also possessed an alkaline phosphatase isoenzyme of the bone-liver-kidney type that was stimulated by 1,25-(OH)2D3 treatment (two- to threefold) and inhibited by parathyroid hormone (40 nM, -25%, p less than 0.025). PTH and PGE2 increased cAMP production in a dose-dependent manner, but the cells were irresponsive to salmon calcitonin. Basal and PTH-responsive cyclic AMP production were also modulated by cell density. Dexamethasone pretreatment (100 nM, 48 h) stimulated the PTH-dependent cAMP production but failed to influence the response to PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)     

凋亡抑制基因Livin在膀胱癌中的表达及意义

目的探讨凋亡抑制基因Livin表达在膀胱癌(TCC)发生发展中的作用.方法采用RT-PCR方法检测36例TCC组织标本中Livin的表达.男31例,女5例.年龄26～82岁,平均64岁.临床分期T122例,T29例,T3 3例,T42例.病理分级Ⅰ级10例,Ⅱ级23例,Ⅲ级3例.初发25例,复发11例.单发19例,多发17例.12例正常膀胱组织标本作对照. 结果36例TCC中Livin阳性表达28例(78%),T1表达率82%(18/22),T2～T471%(10/14);Ⅰ级表达率70%(7/10),Ⅱ级83%(19/23),Ⅲ级67%(2/3);单发表达率79%(15/19),多发者77%(13/17);初发者80%(20/25),复发者73%(8/11).Livin表达与TCC临床分期和病理分级不相关,与肿瘤数量,复发次数不相关(P＞0.05).对照组12例标本中均不表达.TCC组和对照组Livin表达差异有统计学意义.结论Livin表达与TCC的发生有关,可作为TCC的分子标志物,可能成为TCC诱导凋亡治疗的新靶点.