微小RNA-29b靶向卷曲蛋白6参与急性髓系白血病细胞柔红霉素耐药的分子机制

目的 探究微小RNA-29b(miR-29b)靶向卷曲蛋白6(FZD6)参与急性髓系白血病(AML)细胞柔红霉素(DNR)耐药的分子机制。方法 CCK-8检测AML细胞系在不同DNR浓度下的生存能力，实时定量聚合酶链反应(qRT-PCR)检测AML细胞系中miR-29b的表达。选取THP-1细胞为研究对象，分为Control组(正常培养)、miR-NC mimics组(转染miR-NC mimics)、miR-29b mimics组(转染miR-29b mimics)、miR-29b mimics+pcDNA组(转染miR-29b mimics和pcDNA)、miR-29b mimics+pcDNA-FZD6组(转染miR-29b mimics和pcDNA-FZD6),转染24 h后使用10 mM DNR处理培养24 h。qRT-PCR和免疫印迹(Western blot)检测各组DNR处理的THP-1细胞中miR-29b和FZD6表达水平。生物信息学和双荧光素酶报告基因预测并检测miR-29b和FZD6靶向关系。CCK-8、Edu法和流式细胞术分别检测DNR处理的各组THP-1细胞增...     

靶向FHL1的microRNA初步筛选研究

目的 构建含有FHL1基因3'-UTR区的荧光素酶报告基因载体,用以检测与其相互作用的microRNA(miRNA).方法 PCR扩增出FHL1基因3'-UTR区片段,插入到经过改造的荧光素酶报告基因载体pcDNA 3.0中;利用Target Scan5.1软件预测可能与FHL1基因3'UTR区相互作用的miRNA,将miRNA与荧光素酶报告重组子共转染至293T细胞中,检测其荧光活性;构建针对荧光活性结果变化明显的miRNA的种子区缺失突变体,检测荧光活性.结果 测序结果表明,含有FHL1基因3'-UTR区的荧光素酶报告基因载体构建正确;荧光素酶活性实验表明,与对照组相比,miR-200c、miR-146a、miR-146b-5p可使荧光素酶报告重组子的荧光素酶活性降低40％左右,并构建上述miRNA缺失突变体,miR-200c使荧光素酶活性回复.结论 成功构建了FHL1基因3'UTR区的荧光素酶报告基因载体,而miR-200c、miR-146a、miR-146b-5p可以抑制其荧光素酶活性,其中miR-200c可能直接作用于FHL1基因3'-UTR区.     

miRNA-33a抑制人结肠癌细胞HCT-116增殖的机制研究

目的 探讨miRNA-33a抑制人结肠癌细胞HCT-116增殖的机制.方法 化学法合成miRNA-33a mimics、miRNA-33a inhibitor及阴性对照(NC)序列,然后转染至HCT-116细胞.转染后48h,采用Real-time PCR检测细胞中miRNA-33a含量的变化,Western blotting检测细胞中Twist蛋白含量的变化;转染后24、48、72h,采用CCK-8的检测肿瘤细胞增殖活性的变化.采用信息学软件预测miRNA-33a与Twist的结合位点,并通过荧光素酶报告基因法证实预测的结合位点.结果 miRNA-33a mimics及miRNA-33a inhibitor转染后可明显上调或下调HCT-116细胞内miRNA-33a的相对含量(P＜0.05),而NC转染组细胞miRNA-33a含量与对照组比较差异无统计学意义(P＞0.05);miRNA-33a mimics及miRNA-33a inhibitor转染后可明显下调或上调HCT-116细胞内Twist蛋白的表达,与对照组比较差异有统计学意义(p＜0.05),而NC转染组细胞内Twist含量与对照组比较差异无统计学意义(P＞0.05);miRNA-33a mimics及miRNA-33ainhibitor转染后可明显下调或上调HCT-116细胞对数生长期的增殖活性,转染后48h和72h与对照组比较差异均有统计学意义(p＜0.05),而NC转染组细胞增殖活性与对照组比较差异无统计学意义(P＞0.05).荧光素酶实验显示,miRNA-33amimics和miRNA-33a inhibitor转染可以抑制或促进野生型报告基因荧光素酶活性(P＜0.05),而对突变型报告基因荧光素酶活性无明显影响(P＞0.05).结论 miRNA-33a可以通过抑制其靶基因Twist的表达抑制HCT-116细胞的增殖活性.     

miR-29a抑制胃癌细胞内靶基因VEGF-A的表达及意义

目的 验证胃癌细胞株SGC-7901内miR-29a对靶基因VEGF-A的直接调控作用.方法 采用负载miR-29a的腺病毒载体（Ad-miR29a）感染人胃癌细胞株SGC-7901,上调SGC-7901细胞内miR-29a的表达丰度后,采用RT-qPCR及Western blot法分别检测SGC-7901细胞内VEGF-A在mRNA及蛋白水平的表达;进一步采用双荧光素酶实验及突变实验,验证miR-29a是否通过结合在VEGF-A 3＇UTR而直接抑制靶基因表达.结果 与感染阴性对照Ad-LacZ的SGC-7901细胞相比,感染Ad-miR29a的SGC-7901细胞内VEGF-A蛋白表达下降（0.42±0.02 vs.0.91±0.03,P＜0.01）,且VEGF-A mRNA水平亦下调（0.75±0.21 vs.1.15±0.25,P＜0.05）;荧光素酶实验显示,与阴性对照相比,转染miR-29a mimic后,HEK293细胞萤火虫荧光素酶活性明显下降（0.56±0.13 vs.0.93±0.06,P＜0.05）;而在VEGF-A 3＇UTR与miR-29a的结合位点被突变之后,HEK293细胞萤火虫荧光素酶活性得以恢复.结论 miR-29a通过结合于VEGF-A 3＇UTR相应位点,直接抑制VEGF-A在mRNA及蛋白水平的表达.miR-29a可能成为胃癌基因治疗的有效靶标.     

MicroRNA-34a通过Notch1对膀胱肿瘤细胞株T24增殖的影响

目的 探讨膀胱肿瘤细胞株中microRNA-34a(miR-34a)与Notch1的相互作用关系以及过表达miR-34a对T24细胞增殖的影响.方法 通过生物信息学软件预测miR-34a与Notch1的作用位点,并通过荧光素酶实验验证两者的直接调控关系.在膀胱癌细胞株T24过表达miR-34a,采用实时定量PCR和蛋白印迹检测Notch1表达水平的变化;分别通过新型四唑氮盐(MTS)实验和流式细胞术检测细胞增殖、凋亡以及细胞周期的变化.结果 T24细胞中荧光素酶报告基因实验显示,miR-34a在膀胱癌细胞中能与报告基因结合,使萤火虫荧光强度减弱(P=0.006).过表达miR-34a后,T24细胞内源性Notch1的mRNA水平和蛋白水平均明显下调;T24细胞生长明显受抑(P＜0.001),并呈现一定时间依赖性;凋亡率增加(P=0.003),G0-G1期细胞显著增多(P=0.002).结论 过表达miR-34a能通过降低靶基因Notch1的表达,抑制膀胱肿瘤细胞的增殖.     

miR-193b在HepG2细胞中下调K-Ras蛋白表达

目的探讨microRNA-193b（miR-193b）在HepG2细胞中对K-Ras蛋白表达的调控作用。方法 Tar-getScan软件预测获得miR-193b调控候选靶基因K-ras;构建包含预测miR-193b作用靶位点的K-rasmRNA3′UTR野生型和突变型荧光素酶报告基因载体pGL-KRAS和pGL-Mu-KRAS,检测转染HepG2细胞的荧光素酶活性;合成miR-193b的dsRNA,转染HepG2细胞,实时定量PCR和Western印迹检测对K-rasmRNA和蛋白表达的影响。结果与结论 miR-193b可以与K-rasmRNA3′UTR结合。在HepG2中,miR-193b在mRNA和蛋白水平下调K-ras基因表达。     

miR-761通过靶向作用CRKL抑制卵巢癌SKOV3细胞增殖研究

目的探讨miR-761对卵巢癌SKOV3细胞增殖的影响以及相关机制。方法采用Lipofectamine 2000将miR-761过表达和沉默质粒载体分别转染卵巢癌SKOV3细胞后,采用Real-Time PCR验证其转染效率;采用CCK8法检测miR-761对卵巢癌SKOV3细胞增殖的影响;采用Real-Time PCR检测卵巢癌SKOV3细胞中CRKL mRNA表达变化。采用双荧光素酶报告基因验证miR-761和CRKL的存在靶向结合并明确其结合位点。将CRKL(+)和CRKL(-)慢病毒载体分别转染至卵巢癌SKOV3细胞;采用CCK8法检测CRKL对卵巢癌SKOV3细胞增殖的影响。将CRKL(+)和CRKL(-)质粒分别转染至已经建立的miR-761过表达和沉默细胞,采用CCK8检测卵巢癌SKOV3细胞增殖变化。结果与对照组比较,miR-761过表达显著抑制了卵巢癌SKOV3细胞的增殖,极大降低了CRKL在SKOV3细胞中的mRNA和蛋白水平;miR-761和CRKL存在靶向结合,并明确了其结合位点;CRKL过表达显著增强了卵巢癌SKOV3细胞增殖能力;CRKL过表达有效阻断了miR-761抑制SKOV3细胞增殖的作用。结论 miR-761能够通过靶向下调CRKL,抑制卵巢癌SKOV3细胞增殖。     

微小核糖核酸- 29b通过靶向髓样细胞白血病- 1影响脑缺血/再灌注损伤时神经细胞凋亡

【摘要】 目的 确定微小核糖核酸（microRNA,miRNA,miR）在脑缺血/再灌注（I/R）损伤中对神经细胞凋亡的影响,并探讨其潜在机制。方法 培养小鼠成神经细胞瘤（N2a）细胞,构建氧糖剥夺（OGD）/复氧（R）模型,模拟体外脑I/R状态。N2a细胞随机分为空白对照组（control）、OGD/R组（OGD/R）、OGD/R＋转染miR-29b模拟物组（mimics）、OGD/R＋转染miR-29b抑制剂组（inhibitor）、OGD/R＋转染miR-29b模拟物阴性对照组（mimics NC）、OGD/R＋转染miR-29b抑制剂阴性对照组（inhibitor NC）。实时定量聚合酶链反应（PCR）检测各组miR-29表达变化。细胞计数试剂盒（CCK）-8和流式细胞术分别检测miR-29b和miR-29b抑制剂对N2a细胞活力和凋亡的影响。免疫印迹法检测抗凋亡蛋白髓样细胞白血病（MCL）-1、B淋巴细胞瘤（BCL）-2及半胱氨酸天冬氨酸特异性蛋白酶（caspase）-3表达。双荧光素酶分析验证miR-29b与MCL-1间相互作用。结果 与control组相比,OGD/R组miR-29b表达明显升高。与OGD/R组相比,mimics组N2a细胞凋亡增加,存活率降低,MCL-1、BCL-2表达下调,caspase-3表达上调,而这种效应在inhibitor组受到逆转。双荧光素酶分析显示miR-29b可通过与MCL-1的3’非翻译区（UTR）端结合下调MCL-1表达。结论 miR-29b在脑I/R损伤过程中通过靶向MCL-1促进神经细胞凋亡。这为缺血性脑卒中治疗提供了一潜在的新靶点。     

程序性细胞死亡4基因增强胰腺癌细胞放射敏感性的研究

目的 探讨程序性细胞死亡4（PDCD4）对胰腺癌细胞放疗敏感性的影响及机制。方法 收集胰腺癌组织及对应的癌旁组织,采用RT-PCR和Western blot检测PDCD4表达水平。以人胰腺癌细胞Sw1990为研究对象,细胞转染PDCD4过表达载体（pIRES2-PDCD4组）、空载体（pIRES2组）,同时以只加入转染试剂的细胞为未转染组,RT-PCR和Western blot检测PDCD4表达水平。细胞经放射处理后,流式细胞术检测细胞凋亡情况,细胞克隆实验检测细胞放射敏感性,Western blot检测细胞中β-连环蛋白、Wnt信号通路下游靶基因c-myc、活化的含半胱氨酸的天冬氨酸蛋白水解酶3（Cleaved Caspase-3）表达水平。结果 胰腺癌组织中PDCD4 mRNA和蛋白表达水平均明显低于癌旁组织（t=4.869、9.208,P<0.05）。pIRES2-PDCD4组细胞中PDCD4 mRNA和蛋白表达水平均明显高于未转染组（t=9.074、18.927,P<0.05）。照射处理后,pIRES2-PDCD4组细胞凋亡率及细胞中Cleaved Caspase-3水平均明显高于未转染组（t=3.670、4.086,P<0.05）,而细胞中β-连环蛋白、c-myc表达水平均明显低于未转染组（t=9.242、17.644,P<0.05）。pIRES2-PDCD4组放射敏感性高于未转染组,增敏比为1.843。结论 PDCD4能够增加胰腺癌细胞放射敏感性,促进胰腺癌细胞凋亡,作用机制可能与Wnt信号通路有关。     

microRNA-196a2单核苷酸多态性对靶基因IL-2表达的影响

目的观察基因单核苷酸多态性（SNP）对成熟microRNA（miRNA）-196a2在人角质形成细胞HaCaT细胞中表达水平的影响,及对预测的靶信使RNA（mRNA）IL-2 3＇非翻译区（UTR）结合能力的影响,探讨miRNA-196a2 SNP是否与皮肤病等自身免疫性疾病关联。方法在HaCaT细胞中转染构建miR-196a2-T（野生型）和miR-196a2-C（突变型）的表达质粒,RT-PCR检测表达效果;将两种基因型的表达质粒与预测的靶mRNA IL-2 3＇UTR的报告基因质粒共转染细胞,双荧光素酶报告基因实验分别检测结合能力,并使用SPSS17.0软件统计分析。结果成功构建miR-196a2-T/C表达质粒和IL-23＇UTR报告基因质粒;miRNA-196a2野生型和突变型的表达质粒在HaCaT细胞中的表达水平无显著差异（P〉0.05）;IL-2是miR-196a2的靶基因,SNP影响miR-196a2与IL-2 3＇UTR的结合（P〈0.05）。结论 MiR-196a2可与IL-2结合调节其转录后水平,从而参与免疫反应,可能影响多种皮肤病等自身免疫性疾病的进程。     

成批煤气爆燃烧伤的救治体会

报道治疗因煤气泄漏爆炸烧伤病员7例，经应用MEBT技术积极救治，其中6例于伤后17天痊愈出院，另一例特重伤员也于伤后30天康复出院。作者认为：应严格按照MEBT／MEBO要求进行规范治疗，正确处理生命体征、休克、感染、脏器功能和创面的辨证关系。同时，不能忽视外科营养在过程中的重要作用，它是创面修复的物质保证。     

Glutamine supplementation in vitro and in vivo,in exercise and in immunodepression

In situations of stress, such as clinical trauma, starvation or prolonged, strenuous exercise, the concentration of glutamine in the blood is decreased, often substantially. In endurance athletes this decrease occurs concomitantly with relatively transient immunodepression. Glutamine is used as a fuel by some cells of the immune system. Provision of glutamine or a glutamine precursor, such as branched chain amino acids, has been seen to have a beneficial effect on gut function, on morbidity and mortality, and on some aspects of immune cell function in clinical studies. It has also been seen to decrease the self-reported incidence of illness in endurance athletes. So far, there is no firm evidence as to precisely which aspect of the immune system is affected by glutamine feeding during the transient immunodepression that occurs after prolonged, strenuous exercise. However, there is increasing evidence that neutrophils may be implicated. Other aspects of glutamine and glutamine supplementation are also addressed.     

Sex differences in neuromuscular fatigability in response to load carriage in the field in British Army recruits

Objectives

Women are resistant to neuromuscular fatigue compared to men in response to a range of exercise tasks. The sex differences in the neuromuscular responses to load carriage have yet to be investigated.

新疆石河子地区奶牛隐性乳房炎的调查与分析

采用LMT法对新疆石河子地区三个规模化奶牛场泌乳牛群进行了隐性乳房炎检测,共检测994头泌乳牛3976个乳区.结果表明:奶牛隐性乳房炎阳性率为81.9%,乳区阳性率为49.1%.数据分析表明,不同年龄、胎次以及有无卧床奶牛的隐性乳房炎阳性率差异显著(P<0.05),后乳区的隐性乳房炎感染率显著高于前乳区感染率(P<0.05),隐性乳房炎乳区发生数也明显影响当日产奶量(P<0.05).     

Decrease in eccentric hamstring strength in runners in the Tirol Speed Marathon

Background

The local muscular endurance of knee flexors, during eccentric work in particular, is important in preventing or delaying kinematic changes associated with fatigue during treadmill running. This result, however, may not be transferable to overground running.

Objective

To test the hypothesis that overground running is associated with eccentric hamstring fatigue.

Methods

Thirteen runners (12 male and one female) performed an isokinetic muscle test three to four days before and 18 hours after a marathon. Both legs were tested. The testing protocol consisted of concentric and eccentric quadriceps and hamstring contractions.

Results

There were no significant differences between peak torque before and after the race, except that eccentric peak hamstring torque (both thighs) was reduced.

Conclusion

Overground running (running a marathon) is associated with eccentric hamstring fatigue. Eccentric hamstring fatigue may be a potential risk factor for knee and soft tissue injuries during running. Eccentric hamstring training should therefore be introduced as an integral part of the training programme of runners.     

Oxalosis in primary hyperoxaluria in infancy

Primary hyperoxaluria (PH1) is a rare inborn autosomal recessive metabolic disorder due to the deficiency of hepatic alanine-glyoxylate-aminotransferase. This deficiency results in excessive synthesis and urinary excretion of oxalate, inducing renal stone formation and deposition of calcium oxalate in the kidney, bone, myocardium, and vessels (systemic oxalosis, SO) in the most severely affected individuals. We report renal and skeletal changes in a 3-month-old girl with PH1 and SO. Intense cortico-medullary hyperechogenicity and increased homogeneous radiopacity of normal-sized kidneys suggested the diagnosis of SO. Skeletal survey showed osteopenia and characteristic symmetrical metaphyseal transverse bands in long bones, progressively becoming more dense and migrating towards the diaphysis. Multiple pathological and slowly healing fractures of the limbs occurred at the dense band level. A radiopaque rim was then observed in flat bones, epiphyseal nuclei, and vertebral bodies. Inflammatory granulomatous reaction, induced by the presence of oxalate crystals in the marrow spaces, coexisted with progressively evident radiological signs of secondary hyperparathyroidism, with partially overlapping features. The patient was treated by peritoneal dialysis and hemodialysis until combined liver–kidney transplantation. There are no previous reports of infants treated with hemodialysis for more than 2 years.     

Interventional radiology in oncology in private practice

Interventional oncology in private practice requires expert training and can be performed in a stand-alone facility for type 1 procedures in a hospital setting for type 2 and 3 procedures where subspecialized radiologists, state-of-the-art equipment, and postprocedure hospital monitoring are available. A multidisciplinary effort with oncologists, internal medicine physicians and anesthesiologists is necessary. The practice of interventional oncology requires around the clock availability, meticulous and established protocols and procedures and a financial investment. On the other hand, it is professionally gratifying because of constant technical advances and the impact on patients.     

胎龄和日龄对新生儿局部脑血流的影响

目的探讨胎龄及日龄对新生儿局部脑血流(rCBF)变化的影响及规律.方法对早产儿组、足月早期新生儿组、足月晚期新生儿组进行99Tcm-双半胱乙酯(ECD)SPECT脑血流灌注断层显像,并作半定量分析和读片分析.结果早产儿组原始感觉运动区和枕叶rCBF低于足月早期新生儿组(P<0.05),显著低于足月晚期新生儿组(P<0.01),其额叶中份、顶叶rCBF也低于足月晚期新生儿组(P<0.05),其余各叶皮质rCBF随胎龄和日龄的增大有增加趋势.早产儿组脑血流显像图与足月早期新生儿组和晚期新生儿组比较有明显差异.结论早产儿与足月儿rCBF的变化可能与新生儿大脑皮质相应区域发育成熟度和时间顺序有关.     

Radiological strategy in acute stroke in children

The aim of the study was to estimate the preponderance of patterns of pediatric stroke, ischemic or hemorrhagic, their etiologies and the correct diagnostic protocol for acute management.Forty-one consecutive pediatric patients (age range 5-16 years) with an acute stroke observed in acute phase during a 10-year period, were retrospectively evaluated. Twenty-three patients underwent magnetic resonance imaging (MRI), 3 cases were studied by computed tomography (CT) without MRI, and 15 underwent both CT and MRI studies. In 9 cases, intra-arterial digital subtraction angiography (IADSA) was performed after non-invasive preliminary assessment.Seventeen hemorrhagic (41%) and 24 ischemic (59%) strokes were found. Among hemorrhagic forms, 5 cases were due to arteriovenous malformation (AVM), 7 to cavernoma, and 2 to aneurysm. Among ischemic forms, 2 were due to sickle-cell disease, 1 to hyperomocysteinemia, 1 to moyamoya syndrome, 1 to pseudoxantoma elasticum, 3 to prothrombotic state, 1 to Fabry's disease, 1 concomitant with CO intoxication, 5 to venous sinus thrombosis, and 4 to cardio-embolic state. Etiology remains unknown in 8 cases (20.5%).This study shows a moderate prevalence of ischemic over hemorrhagic strokes. Moreover, personal experience suggests that MRI is always more informative than CT and in selected cases should be the first-choice examination in the acute phase.