miR-34a调控高糖环境中HK-2细胞增殖作用的初步研究

目的探讨miR-34a对高糖环境中HK-2细胞增殖作用的影响。方法以miR-34a模拟物和miR-34a抑制剂分别转染HK-2细胞,定量PCR检测miR-34a的表达变化,MTT检测细胞增殖活性;Western印迹检测周期相关蛋白CyclinD1和CyclinE的表达变化。生物信息学分析miR-34a的靶基因,对其中的增殖相关转录因子E2F3进行双荧光素酶验证。结果 miR-34a模拟物组miR-34a表达显著上调（P〈0.05）,miR-34a抑制剂组miR-34a显著下调（P〈0.05）。与对照组相比,miR-34a模拟物显著抑制高糖环境中HK-2细胞的增殖作用（P〈0.05）,CyclinD1和CyclinE表达下调（P〈0.05）,miR-34a抑制剂组显著上调高糖环境中HK-2细胞的增殖作用,CyclinD1和CyclinE表达上调（P〈0.05）。TargetScan在线分析软件显示,增殖相关转录因子E2F3可能是miR-34a潜在的靶基因,双荧光素酶分析结果显示E2F3是miR-34a的直接靶基因。结论 miR-34a可以调控高糖环境中HK-2的细胞增殖,其机制可能是通过直接抑制E2F3基因的表达而实现。     

miR-34a调控HEK293细胞自噬作用的初步研究

目的探讨miR-34a对HEK293细胞自噬作用的影响。方法采用miR-34a模拟物(模拟物组)和miR-34a抑制剂(抑制剂组)分别转染HEK293细胞,并设空白对照(对照组)。采用定量PCR检测miR-34a、p53基因表达的变化,Western blotting检测自噬相关蛋白LC3Ⅱ及p53表达的变化,并采用生物信息学方法分析miR-34a对自噬相关基因(Atg6/beclin1、sestrin2、FoxO3等)的可能作用。结果定量PCR结果显示,模拟物组miR-34a表达显著上调(P<0.05),抑制剂组miR-34a表达显著下调(P<0.05)。与对照组相比,miR-34a模拟物可显著抑制HEK293细胞LC3Ⅱ蛋白的表达(P<0.05),而miR-34a抑制剂则显著上调HEK293细胞LC3Ⅱ蛋白的表达(P<0.05)。定量PCR和Western blotting结果显示,各组p53mRNA及蛋白表达无统计学差异。生物信息学分析显示,Atg6/beclin1、sestrin2、FoxO3等自噬相关基因是miR-34a潜在的靶基因。结论miR-34a能抑制HEK293的自噬作用,可能是通过直接抑制...     

miR-141靶向FZD4调节Wnt/β-catenin信号通路促进糖尿病肾病的进展

目的探讨miR-141在糖尿病肾病进展中的作用及其潜在机制。方法通过脂质体转染法分别将 miR-141、miR-141 mimic、miR-141 sponge转入人肾小球系膜细胞中, 高糖培养构建体外模拟糖尿病肾病模型。利用流式分析和ELISA法检测miR-141对细胞凋亡和炎症反应的影响。TargetScan数据库预测 miR-141的下游靶基因, 通过双荧光素酶报告基因实验验证两者的结合作用。过表达靶基因FZD4, 检测FZD4对miR-141在高糖培养的肾小球系膜细胞中发挥的具体作用。利用Western印迹法检测下游Wnt/β-catenin信号通路及下游靶基因的变化。结果 miR-141的上调促进了细胞凋亡以及肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)的表达(P<0.05);miR-141的下调抑制了细胞凋亡以及TNF-α、IL-6的产生(P<0.05)。miR-141负调控FZD4的表达, FZD4的过表达逆转了miR-141对高糖培养肾小球系膜细胞凋亡和炎症因子分泌的促进作用(P<0.05)。敲低miR-141显著促进了Wnt/β-cat...     

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.     

膀胱癌患者血清miR-21、miR-119a表达量与肿瘤恶性增殖及侵袭的相关性分析

目的 探讨血清miR-21和miR-119a是否可以预测膀胱肿瘤并分析其与肿瘤恶性增殖及侵袭的关系。方法 膀胱癌患者及健康体检者各48例,分为膀胱癌组和正常对照组。荧光定量PCR法检测血清和组织中miR-21和miR-119a的表达水平。蛋白质印迹法检测PTEN,PI3K,AKT,CyclinD1,TGF-β,β-Catenin,N-cadherin和E-cadherin的表达。流式细胞术检测细胞凋亡率和周期分布。Perason相关分析血清miR-21和miR-119a表达与侵袭性基因的相关性。结果 与正常对照组比较,膀胱癌组血清miR-21和miR-119a相对表达量均升高（均P<0.05）;血清miR-21和miR-119a对膀胱癌的分型具有良好的鉴别能力,其AUC分别为0.995和0.996。与癌旁正常组织比较,膀胱癌组织miR-21和miR-119a相对表达量均升高（均P<0.05）,PTEN和E-cadheri相对表达量降低（均P<0.05）,PI3K、AKT、CyclinD1、TGF-β、β-catenin和N-cadherin相对表达量均升高（均P<0.05）。与膀胱癌正常组织比较,膀胱癌组织凋亡率较低,膀胱癌组织G1期的比例低于膀胱癌正常组织,S、G2期的比例则高于膀胱癌正常组织（均P<0.05）;Pearson相关分析显示,血清miR-21和miR-119a表达水平与PI3K、AKT、CyclinD1、TGF-β、β-catenin及N-cadherin表达呈正相关（均P<0.05）,与PTEN和E-cadherin表达呈负相关（均P<0.05）。结论 血清中miR-21和miR-119a的表达水平对膀胱癌的预测具有较高的价值,膀胱癌患者血清和癌组织中miR-21、miR-119a表达水平均升高,且血清miR-21、miR-119a与增殖和侵袭相关基因相关,提示血清miR-21、miR-119a可能是膀胱癌的潜在生物标志物。     

白藜芦醇抑制膀胱癌细胞生长增殖及影响miR-34a表达的研究

目的观察白藜芦醇(resveratrol,Res)对人膀胱癌细胞T24生长增殖的影响,初步探讨miR-34a在Res影响T24细胞中的作用。方法不同浓度Res分别作用T24细胞24 h,采用甲基噻唑基四唑(methyl thiazolyl tetrazolium,MTT)法检测细胞增殖,流式细胞术检测细胞凋亡,Western blotting检测p53蛋白水平,实时定量PCR(Real-time PCR)检测T24细胞miR-34a的基因表达。结果 Res剂量依赖性地抑制T24细胞增殖,诱导细胞凋亡,明显增加p53的蛋白表达。此外,Res处理后T24细胞中miR-34a的基因表达亦显著升高。结论 Res能够有效抑制膀胱癌细胞生长增殖,可能与诱导细胞凋亡、上调miR-34a及p53表达有关。     

miR-200a靶向胰岛素样生长因子2抑制肾母细胞瘤细胞G401的迁移与侵袭

目的 探究miR-200a靶向调控胰岛素样生长因子2(insulin-like growth factor 2,IGF2)对肾母细胞瘤细胞G401迁移与侵袭的影响。方法 收集30例肾母细胞瘤患者的癌组织及癌旁组织,RT-PCR检测miR-200a和IGF2的表达水平。双荧光素酶实验检测miR-200a对IGF2的靶向作用。G401细胞过表达miR-200a模拟物后,RT-PCR和蛋白印迹法分别检测IGF2mRNA和蛋白表达水平。转染细胞进行分组：miR-NC组、miR-200a模拟物组、pIGF2组、pIGF2+miR-200a模拟物组,划痕实验检测各组细胞迁移能力,Transwell检测各组细胞侵袭能力。结果 与癌旁组织比较,癌组织中miR-200a在癌组织中表达量显著下降（t=6.76,P<0.01）;而IGF2的mRNA以及蛋白水平显著升高（t=12.41、t=8.75,P<0.01）。与IGF2 WT+miR-NC比较,IGF2 WT+miR-200a模拟物荧光素酶活性显著降低（t=6.33,P<0.01）。与miR-NC组比较,miR-200a模拟物组IGF...     

MiR-92a及其靶抑癌基因BCL11b在辐射诱导小鼠胸腺淋巴瘤中的作用

目的 采用电离辐射诱导的小鼠胸腺淋巴瘤动物模型,检测抑癌基因BCL11b和miR-92a的表达变化,探讨miR-92a对BCL11b基因的调控作用.方法 BALB/c小鼠采用γ射线全身照射,单次照射剂量和剂量率分别为1.75 Gy和0.382 Gy/min,每周1次,连续照射4周,计算电离辐射诱导小鼠胸腺淋巴瘤发生率.采用实时定量PCR法检测miR-92a表达变化情况,Westernblot方法检测BCL11b蛋白表达变化情况;将miR-92a序列插入载体pcDNA-DEST-47中构建真核表达质粒;构建BCL11b基因3'UTR-荧光素酶报告质粒,与pcDNA-DEST-miR-92a质粒共转染至EL-4细胞,分析miR-92a对BCL11b表达的调控作用.结果 辐射诱导的BALB/c小鼠胸腺淋巴瘤的发生率为58.51％.胸腺淋巴瘤细胞中BCL11b表达下调,而miR-92a表达上调,两者之间存在显著的负相关(r=-0.827,P＜0.05).psiCHECK 2-BCL11b和pcDNA-DEST-miR-92a两种质粒共转染组的荧光素酶表达明显低于单独转染的空载体和靶基因BCL11b组(t=3.42,P＜0.05).结论 电离辐射诱导的小鼠胸腺淋巴瘤中miR-92a与BCL11b表达的负相关,提示miR-92a可能参与了辐射诱发肿瘤中BCL11b蛋白表达的调控作用.     

miR-182调控RECK信号通路在非小细胞肺癌进展中的作用

目的探讨miR-182在非小细胞肺癌(NSCLC)发展中的作用及其相关机制。方法采用实时定量PCR检测NSCLC细胞系以及30组配对的NSCLC患者癌组织和癌旁组织样本中miR-182的表达水平。在NSCLC细胞株A549和H1299中过表达miR-182后,测定细胞增殖活性及侵袭转移能力。利用荧光素酶活性实验鉴定miR-182的预测靶基因(RECK)。采用qRT-PCR和Western blotting分别检测转染miR-182类似物和miR-182抑制物后RECK mRNA和蛋白的表达水平。结果miR-182在NSCLC细胞系和癌组织中的表达水平明显高于癌旁组织,在伴淋巴结转移的肺癌患者癌组织中的表达水平明显高于不伴淋巴结转移者,差异有统计学意义(P<0.01)。过表达miR-182可增强NSCLC细胞株A549和H1299的增殖、侵袭和迁移能力。双荧光素酶实验结果表明,RECK的翻译水平受miR-182直接调控。qRT-PCR和Western blotting 检测结果表明,转染miR-182类似物后,RECK表达水平降低,转染miR-182抑制物后,RECK表达水平升高。结论miR-182可调控RECK的表达,促进NSCLC的进展。     

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.     

A Little to a Lot or a Lot to a Little?

PURPOSE: To determine whether a little dose to a large normal lung volume or a high dose to a small lung volume is more critical for induction of clinical pneumonitis. The second question is if dose-volume histogram (DVH) parameters are more reliable, if the lungs are analyzed as separate organs or as a whole organ. PATIENTS AND METHODS: We analyzed the clinical and DVH data from 49 patients treated for a thoracic malignancy using 3-D conformal treatment plans. 18 patients had developed a clinical pneumonitis (CTC II or III). The majority of patients (n = 48) received radiochemotherapy for non-small-cell lung cancer (NSCLC) with a combination of paclitaxel and carboplatin. Patients were generally treated 5 fx/week, single dose 2 Gy, using a two-series approach (shrinking field) up to a total dose of 60-70 Gy. For every individual patient, the overall dose distribution was recalculated in the Helax-TMS by means of adding dose plans according to the total dose applied in each series. The lungs were defined both as separate organs and as a whole organ. Low-dose volume (< or = 10 Gy, Vlow), moderate-dose volume (> 10-40 Gy, Vmod) and high-dose volume (> 40 Gy, Vhigh), as well as V10-V40 and mean lung dose (MLD) were defined from the cumulative DVH. Dose-effect relationships were fitted with a logistic regression model. RESULTS: Manifestation of clinical pneumonitis was within 3 months from termination of irradiation in all cases. For the ipsilateral lung, the incidence of pneumonitis was closely correlated to Vhigh. The pneumonitis rate increased from 13% up to 60%. By contrast, with increasing Vlow the pneumonitis rate dropped to < 10%. A similar but less pronounced effect was seen for the total lung. The lung volumes Vlow, Vmod and Vhigh of the ipsilateral, contralateral and whole lung were significantly correlated to the corresponding MLD. The incidence of pneumonitis increased with increasing MLD for the ipsilateral lung with a D50 of 32 Gy and a gamma 50 of 0.98. For the whole lung, the observed increase was less steep. MLD showed a close correlation to NTCP calculated by the Kutcher model. However, NTCP calculation overestimated the pneumonitis risk for the ipsilateral lung and underestimated the risk for the whole lung due to the steeper gradient. The logistic regression curve for the DVH parameters V10-V40 showed an increase of steepness toward higher doses. From the logistic regression curves, a DVH template indicating critical borders of V10-V40 was generated for the ipsilateral as well as for the total lung. CONCLUSION: Our data indicate that it is reasonable to disperse the dose outside the target volume over large areas in order to reduce the volumes of lung receiving > 40 Gy. Reducing the high-dose volume reduces the pneumonitis rate more than a corresponding reduction in the low-dose regions of the DVH. Landmarks for DVH optimization as defined in this analysis may serve as a basis for DVH contrains in IMRT planning. Separate organ analysis produced more reliable results and should be preferred to whole-organ analysis, if techniques mainly involving one side of the lung are applied. Further validation of these constraints is necessary prior to general recommendation.     

Identification of a cannabimimetic indole as a designer drug in a herbal product

A cannabimimetic indole has been identified as a new adulterant in a herbal product being sold illegally in Japan for its expected narcotic effect. Liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry analyses indicated that the product contained two major compounds. One was identified as a cannabinoid analog (1RS,3SR)-3-[4-(1,1-dimethyloctyl)-2-hydroxyphenyl]cyclohexan-1-ol (1) by direct comparison with the authentic compound, which we reported previously. The other compound (2) showed a molecular weight of 341 daltons, and accurate mass spectral measurements showed its elemental composition to be C₂₄H₂₃NO. Both mass and nuclear magnetic resonance spectrometric data revealed that 2 was 1-pentyl-3-(1-naphthoyl)indole [or naphthalen-1-yl-(1-pentylindol-3-yl)methanone] being identical to JWH-018, which was synthesized by Wiley and coworkers in 1998. This compound was reported as a potent cannabinoid receptor agonist possessing a pharmacological cannabimimetic activity.     

Suicide by a rifle with a muzzle brake: a particular entrance wound

The authors describe a case in which a 21-year-old male committed suicide using a rifle equipped with a muzzle brake, placed in contact with his head. A muzzle brake for firearms is a device positioned at the mouth of a weapon, which dissipates the gases, that are generated subsequent to the gun being discharged. Generally, when a gun is pressed against an anatomical region, where the skin lies on the bone surface without interposition of other soft parts, the entrance wound consists of many lacerated branches, originating from a central lack of tissue. In this case report, the use of a rifle with a muzzle brake generated an unexpected wound, circular in shape, with a diameter of 0.8 cm. This lesion was surrounded by a bruised area, circular in shape, and an abrasion collar of the height of 0.5 cm. In addition, a muzzle imprint mark consisting of intradermal bruises, composed of a narrow red line concentrically encircling the entrance hole, was found. In order to analyze in detail if this particular entrance wound could be associated with a weapon equipped with the muzzle brake, ballistic tests – with weapons compatible with that used by the victim – were performed.     

Death of a seven-month-old child in a washing machine: a case report

The authors present a case which brings out a unique modality of child homicide by placing the baby in a washing machine and turning it on. The murder was perpetrated by the baby’s mother, who suffered from a serious depressive disorder. A postmortem RX and then a forensic autopsy were performed, followed by histologic examinations and toxicology. On the basis of the results of the autopsy, as well as the histology and the negative toxicological data, the cause of death was identified as acute asphyxia. This diagnosis was rendered in light of the absence of other causes of death, as well as the presence of typical signs of asphyxia, such as epicardial and pleural petechiae and, above all, the microscopic examinations, which pointed out a massive acute pulmonary emphysema. Regarding the cause of the asphyxia, at least two mechanisms can be identified: drowning and smothering. In addition, the histology of the brain revealed some findings that can be regarded as a consequence of the barotrauma due to the centrifugal force applied by the rotating drum of the washing machine. Another remarkable aspect is that we are dealing with a mentally-ill assailant. In fact, the baby’s mother, after a psychiatric examination, was confirmed to be suffering from a mental illness—a severe depressive disorder—and so she was adjudicated not-guilty-by-reason-of-insanity. This case warrants attention because of its uniqueness and complexity and, above all, its usefulness in the understanding of the pathophysiology of this particular manner of death.

     

Hemodynamic during a postexertional asystolia in a healthy athlete: a case study

Hemodynamic events leading to spontaneous postexertional vasovagal syncope are not completely understood because of the lack of beat-to-beat data. We report a case study of a young athlete who undergoes a syncopal episode during the recovery period following a maximal cycle-ergometer test. The episode was monitored by an impedance cardiograph which can gather noninvasively beat-to-beat the flow of heart rate (HR), stroke volume (SV), cardiac output (CO), diastolic filling rate (SV/DT), and myocardial contractility index (PEP/LVET). The most important findings of this report are the dramatic reduction of SV/DT preceding the syncope, the increment of SV together with the reduction of HR preceding and following the syncope, the prompt recovery of CO values after the syncopal episode despite the bradycardia, and the reduction of PEP/LVET after the syncope. This report confirms the importance of active recovery immediately after strenuous exercise and supports the hypothesis that the reduction of SV/DT in the presence of an inotropic stimulation can trigger the vasovagal reaction.     

Atypical manifestation of a fistula between a hydatid cyst of a liver and a gallbladder: A case report

The hydatid cyst of the liver is the most common location of hydatid disease. Complications in this form are dominated by superinfection of the cyst and rupture into the bile ducts or the peritoneal cavity. We report the case of a 54-year-old female patient with a hydatid cyst of the liver complicated by a rupture in the gallbladder revealed by urinary symptoms.