发生与未发生抗结核药物诱导肝损伤患者血浆中差异性表达的微RNAs分子筛查研究

目的 探讨发生与未发生抗结核药物的肝毒性(ATDH)患者血浆中差异性表达的微RNAs(miRNAs)分子.方法 采集3例ATDH和3例抗结核治疗无肝损伤(非ATDH)患者血浆样本,进行miRNAs芯片检测.对存在差异性表达的miRNAs分子,采用实时定量PCR进行验证.结果 与非ATDH患者相比,ATDH患者血浆中共筛选出12个差异性表达的miRNAs分子,其中表达上调的miRNAs有9个,表达下调的miRNAs有3个.实时定量PCR验证显示,ATDH患者血浆中显著上调的miRNAs有5个,包括miR-4655-3p、miR-1249、miR-1226-5p、miR-3620和miR-1224-5p,明显下调的miRNAs共3个,包括miR-7-5p、miR-424-5p和miR-584-5p.结论 ATDH与非ATDH患者之间在血浆内存在差异性表达的miRNAs分子.     

miR-148-5p靶向H型血管调控因子Slit3的实验研究

目的 观察绝经后骨质疏松症中H型血管调控因子Slit3表达差异,进一步探讨其上游靶向调控miRNAs。方法 雌性SD大鼠随机分假手术组和模型组,Real-time PCR和免疫组化检测骨组织Slit3 mRNA和蛋白表达水平;生物信息学预测可能与Slit3-3’UTR互作的miRNAs,构建野生型和突变型Slit3-3’UTR重组荧光素酶报告载体,和miRNAs表达载体共转染293T细胞,双荧光素酶报告基因测定荧光素酶活性验证miRNA对Slit3-3’UTR的靶向作用。结果 与假手术组相比,模型组Slit3 mRNA表达水平略上调和平均光密度显著上调,生物信息学预测Slit3-3’UTR与miR-148a-5p、miR-148b-5p、miR-410-3p、miR-129-5p和miR-374-5p存在碱基结合位点,野生型Slit3-3’UTR共转染miR-148b-5p荧光活性下调至空白对照组的68.91%,突变型Slit3-3’UTR共转染miR-148b-5p荧光活性未见下调。结论 Slit3可能是绝经后骨质疏松症H型血管的调控基因,miR-148b-5p与Slit3-3’UTR的靶向结合直接调控Slit3的表达水平。     

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.     

LncRNA PCAT1对结直肠癌Colo320细胞的影响及作用机制研究

目的：探讨长链非编码RNA(LncRNA)前列腺癌相关转录本1(PCAT1)对Colo320细胞增殖、侵袭和化疗敏感性的影响及其作用机制。方法：实时荧光定量PCR检测LncRNA PCAT1、miR-145-5p和肌动蛋白结合蛋白1(FSCN1)在结直肠癌组织、Colo320和5-Fu耐药细胞株中的表达;分析LncRNA PCAT1和miR-145-5p、miR-145-5p和FSCN1之间的作用靶点。检测下调PCAT1、miR-145-5p、FSCN1对Colo320细胞增殖和侵袭的影响;检测5-Fu耐药细胞株细胞活力和凋亡率的变化;检测上调LncRNA PCAT1对Colo320细胞增殖、侵袭和化疗敏感性的影响。结果：结直肠癌组织和Colo320细胞中LncRNA PCAT1和FSCN1表达上调,miR-145-5p表达下调;5-Fu耐药细胞株中LncRNA PCAT1和FSCN1表达下调,miR-145-5p表达上调。LncRNA PCAT1靶向miR-145-5p;miR-145-5p靶向FSCN1。上调LncRNA PCAT1通过miR-145-5p促进Colo320细胞增殖...     

微小RNA在结肠慢传输型便秘病变组织中的差异表达

目的 观察微小RNA(miRNA)在结肠慢传输型便秘(STC)病变结肠组织和正常结肠组织中的差异表达.方法 收集6例STC病变结肠组织和正常结肠组织标本,应用miRNA芯片检测miRNA在STC病变结肠组织和正常结肠组织中的表达.结果 5种miRNA在STC病变结肠组织正常结肠组织中的表达差异有统计学意义(P＜0.05).与正常结肠组织比较,miR-20b、miR-27b、miR-30b、miR-128及miR-129-3p在STC结肠组织中表达下调,统计学组间差异倍数均在1.2倍以上,其中miR-128及miR-129-3p下调明显,统计学组间差异倍数大于2倍.结论 部分miRNA在STC结肠组织和正常结肠组织中存在差异表达,可能参与了STC发生的分子机制.     

miR-30b/Snail调控糖尿病肾病肾小管上皮细胞EMT

目的:探讨miR-30b/Snail调控高糖诱导人源肾小管上皮细胞(HK2)-间充质转化(EMT)的可能机制,为糖尿病肾病(DN)间质纤维化的防治提供新的实验依据及思路。方法:通过生物信息学预测调控Snail的候选miRNAs;在db/db小鼠肾组织中验证所有候选miRNAs的表达并分别与Snail做pearson相关性分析,筛选出与Snail负相关最显著的miRNA;以高糖诱导的HK2细胞为载体,转染该miRNA mimics 72 h后,采用real-time PCR、western blot方法,观察该miRNA、Snail以及EMT相关蛋白的表达改变。结果:调控Snail的候选miRNAs共6个,分别是miR-30b-5p、miR-25-3p、miR-199a-5p、miR-128-3p、miR-22-3p、miR-27-3p,其中miR-30b与Snail存在明显负相关,r2=0.775 49;高糖诱导HK2细胞miR-30b表达下调,肾小管上皮细胞标志蛋白E-cadherin表达减少;过表达miR-30b-5p可抑制HK2细胞Snail表达,同时上调E-cadherin表达,而Vimentin、α-SMA表达下调。结论:miR-30b/Snail参与调控高糖引起的肾小管上皮细胞EMT。     

MiR-506-3p与自噬相关因子在烧伤后皮肤成纤维细胞中的表达研究

目的：分析烧伤组织中miR-506-3p的表达及其对自噬水平的调控。方法：提取正常皮肤组织、烧伤皮肤组织和热处理成纤维细胞细胞的总RNA,采用荧光定量RT-qPCR、Western Blot分别检测各细胞miR-506-3p和自噬相关因子(LC3-Ⅱ/Ⅰ比值、Atg5、p62及CollagenⅠ)表达水平;采用同样方法检测miR-506-3p模拟物或miR-506-3p抑制剂转染到真皮成纤维细胞中对上述自噬相关因子表达水平的影响,并利用CCK-8方法检测转染细胞的增殖效率。结果：与正常组织相比,烧伤组织和热处理成纤维细胞中,miR-506-3p的表达下调,自噬相关因子LC3-Ⅱ/Ⅰ比值、Atg5及Collagen Ⅰ的表达显著上调(P<0.01),而p62表达下调(P<0.05);miR-506-3p抑制剂转染到皮肤成纤维细胞中,miR-506-3p表达显著降低,促进了细胞增殖和自噬发生,而miR-506-3p的过表达则显示出相反的作用(P<0.05)。结论：miR-506-3p在烧伤皮肤组织恢复中发挥着积极作用,对调节成纤维细胞的自噬水平具有重要作用,有可能成为烧伤...     

miR-338—3p在肝癌组织中的表达及意义

目的 检测miRNA 在肝癌中的表达谱,探讨miR-338-3p 在肝癌组织中的表达及与肝癌临床病理参数的关系和意义.方法 采用液相芯片技术分析20 对肝癌组织与其癌旁组织中114 种miRNA 表达谱的差异.Real-time RT-PCR 验证另36 对肝癌组织中miR-338-3p 下调表达及其与肝癌临床参数的相关性.结果 31 种miRNA 在肝癌组织与非肝癌组织间呈现差异表达,其中12 种miRNA 在癌组织的表达较癌旁组织上调,19 种miRNA 在癌组织中表达下调.miR-338-3p下调表达程度与肝癌恶性程度、肿瘤分化程度、肝内和肝外转移及门静脉癌栓有关,与患者性别、AFP、HBsAg、是否合并肝硬变等不相关（P 〈 0.01）.miR-338-3p 的表达量肝癌组织低于非肿瘤组织,转移性肝癌低于非转移性癌组织,miR-338-3p 的表达量随着肿瘤级别的增加而逐近降低（P 〈0.01）.结论 肝癌组织与癌旁组织之间存在miRNA 差异表达,其中miR-338-3p 下调表达程度与肝恶性行为相关.     

miRNA在糖尿病肾病中的临床应用进展

微小RNAs（ microRNAs,miRNAs） 是一类内源性非编码单链小分子RNA,通过介导靶mRNA降解或靶基因翻译抑制来调节靶基因的表达.糖尿病肾病是糖尿病的主要并发症,也是终末期肾脏病的首位病因,严重威胁人类健康.目前研究表明,miRNA 在糖尿病肾病的发生、发展过程中发挥重要作用.一些miRNA 的表达异常（如miR-192、miR-200s、miR-29a、miR-29c、miR-21、miR-377、miR-323b-5p、miR-429和miR-93等的上调或下调）与糖尿病肾病的发生、发展密切相关.目前研究还发现在体内调控miRNAs的表达是可行的,为miRNAs应用于临床糖尿病肾病的治疗提供了理论依据.     

微小RNA在膀胱肿瘤组织的表达及其临床意义

目的研究微小RNA（MicroRNAs/miRNAs）microRNAs在人类膀胱肿瘤组织中的表达及其作用。方法取安徽医科大学附属省立医院2009年手术切除的45例膀胱肿瘤组织标本及其肿瘤旁正常组织标本。通过microRNAs微阵点杂交法检测肿瘤组织及其癌旁正常组织的手术标本中microRNAs的表达情况,分析癌组织及其癌旁正常膀胱组织进行定性判断（癌和非癌）。结果人类膀胱肿瘤组织与正常膀胱组织相比,miR-223、miR-26b、miR-221、miR-103-1、miR-185、miR-23b、miR-203、miR-17-5p、miR-23a和miR-205表达显著上调（P〈0.005）。结论 miRNAs与膀胱肿瘤的发生发展有密切的联系,有可能成为一个有效的肿瘤标志物以进行膀胱肿瘤的早期预测和诊断。     

Atrial natriuretic peptide in dehydrated Long-Evans rats and Brattleboro rats

Atrial natriuretic peptide (ANP) was measured by radioimmunoassay in atrial and plasma extracts from normal Long-Evans (LE) rats and Brattleboro-strain diabetes insipidus (DI) rats. LE rats, dehydrated for 72 hours, had an increased plasma osmolality and plasma vasopressin. They also demonstrated a higher atrial immunoreactive ANP (IR-ANP) content than hydrated animals (72 hr dehydration: 178.2 +/- 30.4 micrograms/g wet weight atria, mean +/- SE, control: 60.4 +/- 8.2; P less than 0.001). Plasma IR-ANP in dehydrated LE rats tended to be lower than hydrated LE but this was not statistically significant [72 hr dehydration: 61.9 +/- 5.9 pg/ml, control: 82.4 +/- 8.2]. IR-ANP concentration in atrial extracts from DI rats, without detectable plasma vasopressin levels but with increased plasma osmolality, was not different from that in hydrated LE rats (DI: 100.6 +/- 13.2 micrograms/g). There was also no significant difference between plasma IR-ANP in DI and hydrated LE rats (DI: 100.2 +/- 11.9 pg/ml). The atrial IR-ANP concentration in DI rats was decreased by infusion with either arginine-vasopressin (AVP) or 1-deamino-8-arginine vasopressin (DDAVP), and plasma IR-ANP was increased significantly by both infusions (AVP: 171.3 +/- 18.1 pg/ml, DDAVP: 179.5 +/- 24.6). Thus, changes in atrial and plasma IR-ANP concentration appeared to be associated with changes in water balance but not with plasma AVP levels, indicating that the changes in volume may be a more important factor controlling ANP release in vivo than vasopressin itself.     

Vasography in rats

Vasovasostomy was performed in 20 rats. Vasography then was used to determine the patency of the vas. The scrotum was entered through a longitudinal incision and the scrotal contents were extruded extravaginally. The vas was divided near the epididymis and a 0.4/0.7-mm cannula was inserted into the abdominal end of the vas and contrast medium was injected under fluoroscopic control. The results were documented by radiography. It is concluded that vasography is a reliable method for assessing the success of vasovasostomy in rats.     

Comparative morphometric analysis of cochlear vessels in Wistar-Kyoto rats, spontaneously hypertensive rats, and aged spontaneously hypertensive rats

Vessel density and the ratio of the tissue area to the vessel surface area were studied by morphometric analysis techniques in normal rats, spontaneously hypertensive rats (SHR), and aged spontaneously hypertensive rats (aged SHR). Horseradish peroxidase was injected intravenously and the animals were killed 10 minutes later. The temporal bones were harvested, fixed in glutaraldehyde, and decalcified in 10% ethylenediamine tetraacetic acid (EDTA). After 7 days of decalcification, the cochleas were dissected and incubated with a diaminobenzidine tetrahydrochloride solution. Sections with stained vessels were projected onto the digitizing plate with the help of the camera lucida. The computer was used to calculate tissue area, vessel length, and vessel surface area. A statistically significant increase (p less than 0.05) in both the tissue area to vessel length ratio and the tissue area to vessel surface area ratio was demonstrated in the SHR and the aged SHR groups when compared to the WKY in the stria vascularis. No statistically significant difference was found between the two SHR groups. These data show a decrease of the vessel density in the capillary beds of the stria vascularis in spontaneously hypertensive rats. No statistically significant difference was found in the diameters of the capillary among the three groups.     

Glucose-6-phosphate dehydrogenase in kidneys of lead-poisoned rats and adrenalectomized rats

Glucose-6-phosphate dehydrogenase (G6PDH) activity, assayed biochemically, was significantly increased in kidney homogenates of lead-poisoned rats when compared with controls. Histochemically, G6PDH activity was greatly increased both in the distal tubules and the macula densa, but showed no significant changes in the proximal tubules. Biochemical assay of G6PDH in kidney homogenates of adrenalectomized rats was three times that in control animals. In this condition also, histochemical staining showed G6PDH activity to be increased in both macula densa and distal tubules. This demonstrates an increase in G6PHD in two completely different experimental conditions and suggests that the distal renal tubule in the rat might operate in functional unity with the macula densa.     

Experimental vaginitis in rats

The authors present an experimental model of vaginal inflammation in female rats, through local instillation of different substances causing inflammation of varying degrees. The inflammatory reaction was studied macroscopically, histologically and biochemically. Using polysiloxane as a protective substance of vaginal mucosa was found to be of therapeutic interest.     

Intramuscular urography in rats

In addition to macroscopic studies, microscopic studies, and biologic tests, urography is a valuable method for investigating renal function in rats. Intravenous urography is complicated, however, by the difficulty of puncturing a tail vein or the femoral vein. Intramuscular urography, on the other hand, is a simple and reliable method for radiographic examination of the kidneys and ureters, and gives information on morphology as well as physiology. In this study in 40 rats, intramuscular urography was performed under general anesthesia, and the contrast medium was injected into the gluteal muscles. Dense nephrograms and optimum films of the calices and the ureters were obtained approximately 40 minutes after injection.     

Morphine-halothane interaction in rats

The effects of morphine, halothane, and their various combinations on the purposeful movement (PM) response and the heart rate (HR) increase caused by noxious stimulation were studied in 250 rat experiments. Doses that block the PM and HR responses for the single agent and for combinations were determined with a probit procedure and compared with an isobolographic analysis. As was evidenced by the PM response, the combined anesthetic effect of morphine and halothane, with some deviations, may be defined as additive. It also was found that the combined administration of morphine and halothane results in an antagonism for suppression of the HR increase to noxious stimulation. Halothane antagonized morphine to a much greater extent than morphine to halothane.