阿霉素和长春新碱对pRb-胃癌细胞株的影响

目的：探索一种对Rb基因表达异常之胃癌细胞的化疗方案。方法：用Rb-CMV质粒转染Rb表达阴性的胃癌AGS细胞株，然后将25ng/ml阿霉素和100ng/ml长春新碱序贯加入两组细胞，应用MTT、流式细胞技术来测定转染前后此细胞株对序贯用药后的活性及细胞周期变化。结果：预先作用的阿霉素使Rb阳性胃癌AGS细胞阻滞于细胞周期的G1／S期，而不影响Rb阴性胃癌AGS细胞增殖。阿霉素和长春新碱序贯用药对Rb阴性胃癌AGS细胞有显著细胞毒性作用，而对Rb阳性胃癌AGS细胞的毒性作用轻微。结论：由于绝大多数正常组织的Rb是正常表达，而在胃癌中表达大多是阴性，故此联合用药有望用于Rb表达阴性胃癌的治疗。     

下调miR-150-5p调控PI3K/AKT信号通路抑制体外糖尿病肾病足细胞模型凋亡的机制研究

目的:探讨下调miR-150-5p调控PI3K/AKT信号通路对体外糖尿病肾病足细胞模型凋亡的影响和机制。方法:以小鼠足细胞MPC5作为研究对象,分成Control(空白对照)、Model(高糖处理)、Anti-NC(转染inhibitor control后高糖处理)、Anti-miR-150-5p组(转染miR-150-5p inhibitor后高糖处理),Realtime PCR方法测定细胞中miR-150-5p表达,MTT方法测定细胞增殖变化,PI单染法检测细胞周期变化,Annexin V-FITC/PI双染法检测细胞凋亡变化,Western blot法检测细胞中C-Caspase-3、cyclinD1、p-AKT、p-PI3K蛋白表达。PI3K/AKT抑制剂LY294002处理转染miR-150-5p inhibitor后的足细胞,给予高糖处理,检测细胞增殖、周期和凋亡变化。结果:与Control组比较,Model组细胞中miR-150-5p表达水平升高,细胞增殖能力下降,细胞凋亡率升高,细胞G1期比例升高,细胞中C-Caspase-3蛋白表达水平升高,cyclinD1蛋白表达水平下降,p-AKT/AKT、p-PI3K/PI3K表达水平下降。与Anti-NC组比较,Anti-miR-150-5p组细胞中miR-150-5p表达水平降低,细胞增殖能力升高,细胞凋亡率降低,细胞G1期比例降低,细胞中C-Caspase-3蛋白表达水平降低,cyclinD1蛋白表达水平升高,p-AKT/AKT、p-PI3K/PI3K表达水平升高。PI3K/AKT抑制剂LY294002逆转miR-150-5p inhibitor对高糖条件下足细胞增殖、周期和凋亡影响。结论:下调miR-150-5p通过促进PI3K/AKT信号通路激活减少体外糖尿病肾病足细胞模型细胞凋亡。     

黏附分子T-cadherin抑制C6胶质母细胞瘤增殖与p21表达相关

目的探讨T-cadherin分子表达抑制胶质母细胞瘤C6细胞增殖的分子机制。方法利用脂质体将pcDNA3和pcDNA3．T-cadherin表达质粒转染C6细胞，筛选出表达T-cadherin的C6克隆3、4和5以及转染空载体后不表达T-cadherin的C6克隆1和2，以Westerrl Bolt检测各克隆的p21和p27的表达；同时，以pcDNA3和pcDNA3．T-cadherin表达质粒分别转染野生型（p21＋／＋）和p21缺失突变型（p21-／-）成纤维细胞，研究T-cadherin分子介导的细胞增殖抑制是否依赖细胞周期蛋白p21的表达。结果转染pcDNA3．T-cadherin质粒后表达T-cadherin分子的C6克隆3、4和5的p21表达水平明显升高，而p27的表达与C6细胞是否表达T-cadherin无关。另外。转染pcDNA3．T-cadherin质粒的野生型表达p21的成纤维细胞的增殖显著受抑。而转染pcDNA3．T-cadherin质粒对p21缺失突变的成纤维细胞的增殖不产生抑制作用。结论T-cadherin分子抑制胶质母细胞瘤C6细胞的增殖与p21表达密切相关，且T-cadherin分子对成纤维细胞的增殖抑制作用依赖于p21的表达。     

Rb在骨肿瘤细胞中的作用及其对转录因子E2F1的影响

目的观察Rb基因及其产物在骨肉瘤细胞中的作用及其对转录因子E2F1的影响。方法转染pRb质粒进入骨肉瘤细胞系UMR-106，用噻唑蓝（MTT）法检测转染前后细胞增殖的变化，用逆转录-聚合酶链反应（RT-PCR）检测转染前后E2F1mRNA的表达变化，用Western blot检测转染前后E2F1蛋白的表达变化。结果转染pRb质粒的骨肉瘤细胞UMR-106与未转染pRb质粒者相比细胞增殖受到抑制。转染pRb质粒的骨肉瘤细胞UMR-106与未转染者相比，转录因子E2F1mRNA和蛋白的表达均减弱。结论Rb能抑制骨肉瘤细胞的增殖，这种增殖抑制作用与Rb对转录因子E2F1的表达抑制有关。     

短发卡RNA对人胃癌细胞STAT3基因的沉默作用

目的:探讨STAT3基因小发夹RNA（shRNA）表达质粒对胃癌MKN-45细胞STAT3基因的干扰作用。方法:根据STAT3 mRNA 编码序列，设计RNA干扰靶点，构建STAT3基因的特异性小RNA干扰质粒（psiRNA-H1/STAT3），使用脂质体转染人胃癌细胞系（MKN-45细胞）。实验分为对照（A）组，psiRNA-H1转染（B）组和psiRNA-H1/STAT3转染（C）组。通过RT-PCR和Western Blot检测STAT3特异性小RNA干扰基因对胃癌细胞STAT3基因mRNA和蛋白表达的影响。结果:psiRNA-H1/STAT3经限制性酶切及部分序列分析证明基因插入正确，并经测序证实。将其成功转染MKN-45细胞后，该细胞的STAT3 mRNA和蛋白表达均明显下降(P<0.05)。 结论:将成功构建的针对STAT3基因的shRNA表达载体转染MKN-45细胞，能有效抑制该细胞的STAT3 mRNA和蛋白表达，为STAT3基因靶向治疗提供一定的实验依据。     

CO2气腹对胃癌细胞周期及细胞周期蛋白的影响

目的 通过检测不同压力CO2气腹环境下胃癌细胞周期及其相关蛋白表达的变化,探讨CO2气腹对胃癌细胞生长增殖的影响.方法 将胃癌MKN-45细胞置于0、10、12和15 mm Hg CO2气腹环境下培养4 h,然后用流式细胞法检测胃癌细胞周期的变化,再用Western blot方法检测细胞周期蛋白Cyclin D1、CDK4、Rb和pRb的表达,最后用免疫沉淀法检测细胞Cyclin D1/CDK4结合力.结果 0、10、12 mm Hg CO2气腹组胃癌细胞增殖指数与对照组比较差异均无统计学意义(P>0.05),15 mm Hg CO2气腹组胃癌细胞增殖指数(27.4%4±3.7%)与对照组(36.4%±3.3%)比较显著下降(P<0.05).0、10、12 mm Hg CO2气腹组CDK4、Cyclin D1、pRb蛋白表达以及Cyclin D1和CDK4的结合能力与对照组比较差异均无统计学意义(P>0.05),15 mm Hg CO2气腹组CDK4、Cyclin D1、pRb蛋白表达以及Cyclin D1和CDK4的结合能力(0.71%±0.12%、0.93%±0.21%、0.54%±0.11%、0.18%±0.02%)与对照组(1.05%±0.16%、1.40%±0.24%、0.75%±0.14%、0.31%±0.02%)比较显著下降(P<0.05).0、10、12、15 mm Hg CO2气腹组Rb蛋白表达与对照组比较差异无统计学意义(P>0.05).结论 临床常用压力的CO2气腹对胃癌细胞周期无显著影响,15 mm Hg CO2气腹抑制细胞增殖,其原因可能与Cyclin D1和CDK4的表达下调有关.     

STAT3基因短发卡RNA表达质粒的构建及其对胃癌细胞生长和侵袭的抑制作用

目的 构建信号传导及转录活化因子3(STAT 3)基因短发夹RNA(shRNA)表达质粒,探讨STAT 3抑制后对胃癌MKN-45细胞增殖、凋亡和侵袭的影响.方法 根据STAT 3 Mrna 编码序列,设计RNA干扰靶点,构建STAT 3基因的特异性小RNA干扰质粒(psiRNA-H1/STAT 3),使用脂质体转染MKN-45细胞,实验分为对照组、psiRNA-H1转染组和psiRNA-H1/STAT 3转染组.通过RT-PCR和Western blot检测STAT 3特异性小RNA干扰基因对胃癌细胞STAT 3 Mrna和蛋白表达的影响; MTT比色法检测细胞的生长抑制率; 采用流式细胞术检测转染后对MKN-45细胞凋亡的影响; 采用Boyden小室侵袭实验检测转染后MKN-45细胞侵袭力的变化.结果 成功转染重组体的MKN-45细胞中STAT 3 Mrna和蛋白表达明显下降(P＜0.05).psiRNA-H1/STAT 3转染组各时相均明显抑制MKN-45细胞生长,且MKN-45细胞凋亡率为34.26%,相比对照组(3.75%)和psiRNA-H1转染组(4.43%)明显升高(P＜0.01).另外,psiRNA-H1/STAT 3转染组MKN-45细胞侵袭力较另外2组明显减弱(P＜0.01).结论 成功构建了针对STAT 3基因的shRNA表达载体,通过转染MKN-45细胞,在体外能有效抑制人胃癌细胞STAT 3 Mrna和蛋白表达,细胞增殖、侵袭能力减弱并且促进细胞凋亡,为STAT 3基因靶向治疗提供一定的实验依据.     

微小RNA-30a-5p激活Wnt/β-连环蛋白信号通路对胃癌细胞增殖与侵袭的作用机制

目的探讨人微小RNA(has-miR)-30a-5p调控Wnt/β-连环蛋白(β-catenin)信号通路对胃癌细胞增殖及侵袭的影响及其作用机制。方法体外培养胃癌MKN-45细胞;在所研究胃癌细胞系中使用小干扰RNA(siRNA)转染建立miR-30a-5p的敲除和过表达模型, 胃癌MKN-45细胞分为3组:miR-30a-5p小干扰RNA(miR-30a-5p inhibitor)组, miR-30a-5p过表达RNA组和空白对照组(NC), 荧光定量聚合酶链反应(qPCR)和蛋白质印迹法(Western blot)检测Wnt/β-catenin信号通路相关标志物Cyclin D1(CCND1)基因的表达水平;双荧光素酶报告基因实验验证miR-30a-5p与Wnt信号通路下游关键基因CCND1的靶向调节关系;在培养的胃癌细胞MKN-45中加入Wnt/β-catenin信号通路抑制剂-银杏双黄酮, 分为miR-30a-5p inhibitor组、miR-30a-5p inhibitor+银杏双黄酮组、miR-30a-5p Inhibitor NC组及miR-30a-5p Inhibit...     

SEPT7对胶质瘤细胞系TJ905生物学特性的影响

目的研究SEPT7对胶质瘤细胞系TJ905的生物学特性的影响。方法SEPT7表达缺失的人脑胶质瘤细胞系TJ905转染SEPT7构建体,pcDNA3载体转染组为空载对照,应用蛋白印迹鉴定转染是否成功。采用MTT法检测转染SEPT7构建体的细胞增殖活性,流式细胞术分析细胞周期分布变化,Annexin V法评价细胞的凋亡,Martrigel三维立体培养法分析侵袭能力。结果转染SEPT7后,胶质瘤细胞增殖活性降低,细胞周期分析结果为S期细胞比例（SPF）降低、G0／G1期阻滞;蛋白印迹检测细胞周期因子显示正向调节因子cyclinD1、cyclinE、CDk2、CDk4表达下降,负向调节因子p21、p16表达升高,肿瘤细胞侵袭能力明显受到抑制,并可诱发细胞凋亡。结论SEPT7转染人胶质瘤细胞系TJ905,可使细胞SPF降低、G0／G1期出现阻滞、抑制细胞侵袭和增殖、促进凋亡。     

p16基因对大鼠肝癌生长抑制作用的研究

1 .材料与方法 :大鼠肝癌细胞系CBRH 7919来自中科院上海细胞生物研究所。Wistar大鼠 30只 ,体重 (15 0± 2 0 )g ,雌雄不限 ,购自天津市医学实验动物中心。pcDNA3 0 /p16真核表达质粒转染CBRH 7919细胞后MTT法检测细胞增殖 ;流式细胞仪检测细胞周期 ;双层软琼脂培养观察细胞克隆形成能力 ;大鼠原位肝癌基因治疗观察其体内成瘤能力。结果用统计软件SPSS行t检验。2 .结果 :(1)重组质粒转染CBRH 7919细胞P16蛋白免疫组化显示有P16蛋白表达。 (2 )流式细胞仪分析pcDNA3 0 /p16质粒转染 7919细胞细胞周期结果 (表 1)。 (3)双层软琼…     

Reduction of Plasma Fibrinogen, Immunoglobulin G, and Immunoglobulin M Concentrations by Immunoadsorption Therapy with Tryptophan and Phenylalanine Adsorbents

Abstract Immunoadsorption (1A) therapy with tryptophan (TR-350) or phenylalanine (PH-350) adsorbents has been used to reduce the concentration of serum antibodies in human lymphocyte antigen (HLA)-immunized patients. Other forms of plasma purification have been reported to reduce the level of fibrinogen, which affects the blood properties. In this study we investigated the effects of IA therapy using both adsorbents on plasma fibrinogen and immunoglobulins G and M in 13 patients (8 patients were treated with TR-350, and 5 patients were treated with PH-350). During each session 1 plasma volume (2.8 ± 0.4 L of plasma) was processed through the immunocolumn and then returned to the patient together with the blood cells. Compared with the pretreatment values, the plasma fibrinogen, IgG, and IgM concentrations were significantly reduced after IA therapy (p < 0.01 for TR-350; p < 0.04 for PH-350). There was a positive correlation between the degree of reduction of plasma proteins and the number of IA treatments given. A nonpara-metric test (Wilcoxon's signed-rank test or the Mann-Whitney test) was used for statistical analysis. We conclude from our study that IA therapy effectively lowers the plasma levels of fibrinogen, IgG, and IgM and thus can be considered a valuable alternative to other blood purification methods.     

The multiply injured child

Blunt trauma is the principal cause of childhood death in many developed countries. This review outlines the differences between adults and children with respect to resuscitation and treatment of orthopaedic injuries in a child with polytrauma. Recent advances in techniques of fracture stabilization are reported.     

Development of Selective Low-Density Lipoprotein (LDL) Apheresis System: Immobilized Polyanion as LDL-Specific Adsorption for LDL Apheresis System

Abstract: Low-density lipoprotein (LDL) is widely recognized as one of the major risk factors for developing coronary heart diseases. Despite intensive development of LDL-lowering drugs, there still exist those patients with refractory hyperlipidemia whose plasma LDL levels are not sufficiently lowered by drugs. LDL apheresis, direct removal of plasma LDL from circulating blood, is thought to be the most promising treatment for such refractory patients. Various techniques, such as the use of an im-munoadsorbent utilizing an anti-LDL antibody, have been used in an attempt to achieve the selective removal of LDL. However, none were widely used because of complications, poor selectivity, and so forth. To establish a safe and effective LDL apheresis system, we chose a synthetic affinity adsorbent as the LDL-removing device. Synthetic polyanion compounds were used as the affinity ligands for LDL adsorbent to simulate the anion-rich sequence of LDL binding sites in the human LDL receptor. Among various polyanion compounds, those polyanions with sulfate or sulfonate groups and hydrophilic backbone were found to have strong affinity for LDL. In contrast, polyanions with carboxyl groups showed poor affinity. Dextran sulfate (DS) was selected as the affinity ligand of LDL adsorbent for its high affinity and low toxicity. The influence of its charge density and molecular weight on its affinity for LDL was suitable. The affinity rapidly increased as the charge density increased, then, reached a constant value. Little affinity was found for either the DS monomer (glucose sulfate) or DS with a molecular weight higher than 10⁴ daltons whereas DS with molecular weights in the midrange showed strong affinity. DS with a midrange molecular weight was immobilized on cellulose hard gel to give LDL adsorbent clinical application. The adsorbent demonstrated an excellent selectivity for LDL and very low density lipoprotein (VLDL) in vitro. Adsorption of high-density lipoprotein and major plasma proteins was almost negligible. Additional study of the LDL-binding mechanism revealed that DS directly interacts with positively charged sites on LDL, which demonstrates that the nature of the interaction is the same as that of LDL receptor. An LDL adsorption column (Liposorber) packed with an LDL adsorbent and polysulfone hollow-fiber plasma separator (Sulflux) was developed as an efficient LDL apheresis system. Clinical investigation proved that this system is capable of intensively lowering the plasma LDL level without affecting major plasma components.     

Artificial organs 2010: a year in review

In this Editor's Review, articles published in 2010 are organized by category and briefly summarized. As the official journal of The International Federation for Artificial Organs, The International Faculty for Artificial Organs, and the International Society for Rotary Blood Pumps, Artificial Organs continues in the original mission of its founders "to foster communications in the field of artificial organs on an international level."Artificial Organs continues to publish developments and clinical applications of artificial organ technologies in this broad and expanding field of organ Replacement, Recovery, and Regeneration from all over the world. We take this time also to express our gratitude to our authors for offering their work to this journal. We offer our very special thanks to our reviewers who give so generously of time and expertise to review, critique, and especially provide such meaningful suggestions to the author's work whether eventually accepted or rejected and especially to those whose native tongue is not English. Without these excellent and dedicated reviewers the quality expected from such a journal could not be possible. We also express our special thanks to our Publisher, Wiley-Blackwell, for their expert attention and support in the production and marketing of Artificial Organs. In this Editor's Review, that historically has been widely received by our readership, we aim to provide a brief reflection of the currently available worldwide knowledge that is intended to advance and better human life while providing insight for continued application of technologies and methods of organ Replacement, Recovery, and Regeneration. We look forward to recording further advances in the coming years.     

Avaliação do clube de revista de anestesiologia por meio de mudanças semânticas

Background and objectives

The interactive approach of a journal club has been described in the medical education literature. The aim of this investigation is to present an assessment of journal club as a tool to address the question whether residents read more and critically.

Methods

This study reports the performance of medical residents in anesthesiology from the Clinics Hospital – University of São Paulo Medical School. All medical residents were invited to answer five questions derived from discussed papers. The answer sheet consisted of an affirmative statement with a Likert type scale (totally disagree–disagree–not sure–agree–totally agree), each related to one of the chosen articles. The results were evaluated by means of item analysis – difficulty index and discrimination power.

Results

Residents filled one hundred and seventy three evaluations in the months of December 2011 (n = 51), July 2012 (n = 66) and December 2012 (n = 56). The first exam presented all items with straight statement, second and third exams presented mixed items. Separating “totally agree” from “agree” increased the difficulty indices, but did not improve the discrimination power.

Conclusions

The use of a journal club assessment with straight and inverted statements and by means of five points scale for agreement has been shown to increase its item difficulty and discrimination power. This may reflect involvement either with the reading or the discussion during the journal meeting.     

Lymphocytapheresis

Abstract: Leukocytapheresis has long been performed with the centrifugal method. But in 1989 in Japan, the Asahi Medical Co. developed the extracorporeal leukocyte-removal filter, Cellsorba. This filter consists of non-woven fabric, which can remove leukocytes from whole blood during extracorporeal circulation. In the incipient stage, this filter was applied to collagen diseases, rheumatoid arthritis, and systemic lupus erythematosus. During the following studies, this filter has been found to have an immunosuppressive effect. Now, it is beginning to be applied to various kinds of autoimmune diseases. Moreover, this filter has recently been recognized to be effective in inflammatory bowel diseases, ulcerative colitis, and Crohn's disease. The outline of Cellsorba and the application of this filter is described here.     

Soluble TNFα Receptors Are Increased in Chronic Renal Insufficiency and Hemodialysis and Inhibit Neutrophil Priming by TNFα

Abstract: The oxidative burst of neutrophils from azotemic patients is refractory to priming by tumor necrosis factor-α (TNFα). Soluble TNFα binding proteins (TNFR) accumulate in the plasma of azotemic patients. To test the hypothesis that these increased sTNFR concentrations inhibit TNFa priming of oxidative burst activity, we measured plasma sTNFR concentrations in nondialyzed azotemic patients, hemodialysis patients, and normal subjects, and determined TNFa priming of fMet-Leu-Phe-stimulated superoxide production in neutrophils incubated in plasma with differing levels of sTNFR. These sTNFR concentrations increased significantly as creatinine clearance decreased and were significantly greater in hemodialysis patients than could be accounted for by loss of renal function alone. TNFα primed superoxide production by normal neutrophils in normal plasma, but this effect was significantly reduced in plasma with increased concentrations of sTNFR. Neutrophils from azotemic and hemodialysis patients were refractory to priming by TNFα in autologous plasma, and incubation in normal plasma only partially corrected this defect. We conclude that sTNFR accumulate as a result of the loss of renal function and hemodialysis and inhibit TNFα priming of neutrophils in azotemic and hemodialysis patients, but that these cells also have an intrinsic functional defect.