PCR-RFLP在细粒棘球绦虫种株鉴定中的应用

目的根据细粒棘球绦虫线粒体DNA rrnL区段基因特征,建立一种新的细粒棘球绦虫种株（基因型）鉴定技术。方法应用PCR-RFLP方法,对新疆不同地区44个囊型包虫病（CE）病人分离株标本提取DNA,用特异性引物对mtDNA rrnL基因片段进行PCR扩增,将扩增产物用限制性内切酶SspI和Bg1Ⅱ消化,再用琼脂糖凝胶电泳分析片段大小,进行基因型鉴别。结果CE病人分离株PCR扩增产物均不能被限制性内切酶SspI酶切,为细粒棘球绦虫;其中43个病人分离株的PCR扩增产物不能被限制性内切酶Bg1Ⅱ酶切,为细粒棘球绦虫G1基因型;1个病人分离株的PCR扩增产物被Bg1Ⅱ切成158 bp和403 bp 2条DNA片段,为细粒棘球绦虫G6基因型。结论根据细粒棘球绦虫线粒体DNA rrnL区段基因特征建立的PCR-RFLP技术可用于细粒棘球绦虫的基因型鉴别。     

解毒化瘀方对内毒素血症大鼠肝细胞线粒体能量代谢的影响

目的：观察解毒化瘀方对内毒素血症（endotoxemia，ETM）大鼠肝细胞线粒体能量代谢的影响。方法：将56只大鼠随机分为正常对照组及脂多糖（LPS）注射后6、12、24小时组和解毒化瘀方6、12、24小时组。取肝组织分离线粒体，测定其Na^＋-K^＋-ATP酶、Ca^2＋～Mg^2＋-ATP酶活性。同时行高压液相色谱检测线粒体内腺苷酸含量及能荷（EC）水平。结果：在注射LPS 6小时后，线粒体Na^＋-K^＋-ATP酶、Ca^2＋-Mg^2＋-ATP酶活性降低，ATP、ADP含量及EC值减少；24小时后，线粒体Na^＋-K^＋-ATP酶、Ca^2＋-Mg^2＋-ATP酶活性明显降低，ATP、ADP、AMP含量及EC值明显减少。与对应的LPS组比较，经解毒化瘀方处理后，肝细胞线粒体Na^＋-K^＋-ATP酶、Ca^2＋-Mg^2＋-ATP酶活性增加，ATP、ADP、AMP含量及EC值均升高。结论：解毒化瘀方能增加ETM大鼠肝线粒体Ma^＋-K^＋-ATP酶、Ca^2＋-Mg^2＋-ATP酶活性，提高线粒体氧化磷酸化水平，增加线粒体腺苷酸EC值，改善能量代谢，具有保护肝细胞的作用。     

PCR-RFLP技术用于鉴别赫坎按蚊复合体近缘种按蚊的研究

目的区别赫坎按蚊种团内近缘种.方法应用聚合酶链式反应连接的限制性片段长度多态性方法(PCR-RFLP)技术,对辽宁省现场捕获的按蚊用特异性ITS2引物进行PCR扩增,限制性内切酶RsaⅠ和HinfⅠ消化,琼脂糖凝胶电泳分析.结果中华按蚊的PCR扩增产物能被限制性内切酶RsaⅠ酶切成350 bp和200 bp两条酶切DNA条带;嗜人按蚊核糖体DNA(rDNA)的PCR扩增产物能被限制性内切酶HinfⅠ酶切成410 bp的酶切DNA条带; 雷氏按蚊的ITS2基因PCR扩增产物能分别被限制性内切酶RsaⅠ和HinfⅠ酶切,分别显示350 bp和400 bp的酶切DNA条带;八代按蚊的PCR扩增产物没有显示明显的限制性内切酶RsaⅠ或HinfⅠ酶切条带.结论依据rDNA的ITS2区段基因特征建立的PCR-RFLP技术可用于鉴别赫坎按蚊种团的中华按蚊、嗜人按蚊、雷氏按蚊和八代按蚊4个近缘种按蚊.     

维生素C对D-半乳糖致亚急性衰老小鼠模型的影响

目的从mtDNA缺失改变，线粒体呼吸酶活力和氧化应激水平的角度研究维生素C（Vc）对D-半乳糖诱导亚急性衰老小鼠模型的影响。方法建立衰老小鼠模型，PCR法检测实验小鼠mtDNA固定片段的缺失情况，氧电极法检测肝线粒体呼吸酶琥珀酸脱氢酶（SUCOX）和NADH氧化酶（NADHOX）活力，荧光法检测肝和脑MDA含量和GPX活性，化学法检测SOD的活性。结果模型组和Vc保护组小鼠都出现mtDNA固定片段缺失，但Vc保护组荧光强度明显低于模型组。与模型组相比，对照组线粒体呼吸酶琥珀酸脱氢酶和NADH氧化酶活性显著降低，Vc保护组线粒体呼吸酶琥珀酸脱氢酶和NADH氧化酶活性显著升高；对照组和Vc保护组小鼠肝脑MDA含量和SOD活性降低。结论Vc可以减轻D-半乳糖诱导亚急性衰老动物模型的氧化损伤，保护mtDNA和增强线粒体氧化呼吸酶活性。     

肺炎支原体P1蛋白结构基因片段的制备及C末端基因片段克隆的研究

目的 制备肺炎支原体P1蛋白结构基因,并在大肠杆菌中克隆肺炎支原体P1蛋白C末端基因片段,为P1蛋白基因片段的扩增、表达及探讨C末端基因片段功能打基础。方法 PCR扩增方法获取P1结构基因。扩增产物用P1蛋白基因特异引物扩增鉴定。用Sal Ⅰ和EcoR Ⅰ双酶酶切消化方法制备P1蛋白C末端基因片段,并与pUC19DNA连接,转入大肠杆菌JM109菌株。用X－gal平板及质粒图谱分析方法筛选重组克隆株,再用限制性核酸内切酶酶切图谱分析鉴定。结果 经PCR扩增MP DNA获得一条5．0kbDNA片段,用P1基因区特异引物扩增鉴定获得阳性结果。重组质粒限制性内切酶批纹图谱显示出两条带,一条为pUC19载体DNA带,另一条是1kb的输入片段。结论 获得肺炎原体P1蛋白结构基因及含P1蛋白C末端DNA片段的重组克隆株。     

编码日本血吸虫线粒体大亚基核糖体基因的亚克隆、测序及同源性分析

目的筛选日本血吸虫成虫 cDNA 文库,得到基因克隆并测序。方法体外将以阳性克隆为模板的 PCR 产物和 pGEM-T 载体连接,转染大肠杆菌 XL1-blue,经抗生素及生色底物 X-gal 初筛,再用限制性内切酶酶切法进一步鉴定为重组质粒后,DNA 自动测序仪测序。序列送blast 基因服务站进行同源性分析。结果构建三个含日本血吸虫 cDNA 基因片段的重组子,其中一个阳性克隆序列为编码日本血吸虫线粒体大亚基核糖体的基因序列。结论获得编码日本血吸虫线粒体大亚基核糖基因片段,为分析其作为候选重组疫苗分子的潜能打下基础。     

不同宿主肝片吸虫DNA限制性片段长度多态性分析

本文首次对分离自黄牛、水牛和牦牛的肝片吸虫的基因组DNA，以BamHⅠ、BglⅡ、EcoRⅠ、HindⅢ和PStⅠ等6种限制性内切酶消化后进行琼脂糖电泳，分析比较它们基因组DNA的限制性片段长度多态性（RestrictionFragmentLengthPolymorphism，RFLP）；同时用地高车标记的牦牛肝片吸虫基因组DNA为探针对不同宿主肝片吸虫DNA进行Southern印迹杂交，限制性内切酸酶切结果显示只有两种限制性内切酶（BamHⅠ和EcoRⅠ）在寄生于牦牛的肝片吸虫中检测到多态性，而水牛肝片吸虫与黄牛肝片吸虫基因组DNA之间没有检测到多态性，根据此结果计算了牦牛肝片吸虫与黄牛肝片吸虫和水牛肝片吸虫基因型间的遗传距离，Southern杂交结果显示牦牛肝片吸虫基因DNA的杂交谱带与黄牛肝片吸虫、水牛肝片吸虫基因组DNA的杂交港带存在一定的差异，本文的实验结果表明牦牛肝片吸虫与黄牛肝片吸虫、水牛肝片吸虫的亲缘关系相对较远，而水牛肝片吸虫与黄牛肝片吸虫的亲缘关系较近。     

线粒体DNA13731点突变与脊髓小脑性共济失调相关性研究

目的 探索线粒体DNA(mtDNA)突变位点与脊髓小脑性共济失调(SCA)的关系.方法 采用聚合酶链反应(PCR)对基因确诊的四个SCA家系10例患者及其亲属共34例与40例健康对照的线粒体ND5基因片段进行扩增,扩增产物进行单链构象多态性分析(SSCP),对SSCP出现异常的样本进行相应mtDNA片段测序.结果 在一家系的1名确诊患者及1名症状前患者检测到mtDNA13731(T>C)点突变.结论 脊髓小脑性共济失调的发生、发展可能与mtDNA突变有关.     

肝细胞癌的临床特征和线粒体D－环DNA突变之间的相关性

据医学空间网12月6日报道（原载J Gastroenterol ．2004Nov；39（11）：1063—8．），在多种人类癌症中发现有线粒体DNA（mtDNA）突变存在。日本的一项研究评价了肝脏中mtDNA突变与人肝细胞癌（HCC）之间的相关性。     

脾气虚大鼠肝细胞超微结构动态变化的实验研究

用过劳及饮食失节法塑造大鼠气虚模型，结果表明，随着造模时间的延长，脾气虚大鼠肝细胞线粒体的肿胀、基质变浅、空泡变淡、嵴断裂及排列紊乱等逐中重，同时空肠细胞线粒体在似变化，提示脾气虚大鼠肝细胞线粒体的变化是一个由轻到重的渐进过程，其病变程度与空脾性细胞线粒体的病变程度密切相关；随着营养物质吸收障碍的逐渐加重，肝细胞线粒体的病变程度也渐趋严重，肝脏合成血浆蛋白的能力逐渐下降，是脾气虚证低蛋白血症逐渐加     

Role of pentoxifylline in non-alcoholic fatty liver disease in high-fat diet-induced obesity in mice

AIM:To study pentoxifylline effects in liver and adipose tissue inflammation in obese mice induced by high-fat diet(HFD).METHODS: Male swiss mice(6-wk old) were fed a highfat diet(HFD; 60% kcal from fat) or AIN-93(control diet; 15% kcal from fat) for 12 wk and received pentoxifylline intraperitoneally(100 mg/kg per day) for the last 14 d. Glucose homeostasis was evaluated by measurements of basal glucose blood levels and insulin tolerance test two days before the end of the protocol. Final body weight was assessed. Epididymal adipose tissue was collected and weighted for adiposity evaluation. Liver and adipose tissue biopsies were homogenized in solubilization buffer and cytokines were measured in supernatant by enzyme immunoassay or multiplex kit, respectively. Hepatic histopathologic analyses were performed in sections of paraformaldehyde-fixed, paraffin-embedded liver specimens stained with hematoxylin-eosin by an independent pathologist. Steatosis(macrovesicular and microvesicular), ballooning degeneration and inflammation were histopathologically determined. Triglycerides measurements were performed after lipid extraction in liver tissue. RESULTS: Pentoxifylline treatment reduced microsteatosis and tumor necrosis factor(TNF)-α in liver(156.3 ± 17.2 and 62.6 ± 7.6 pg/mL of TNF-α for non-treated and treated obese mice, respectively; P 0.05). Serum aspartate aminotransferase levels were also reduced(23.2 ± 6.9 and 12.1 ± 1.6 U/L for nontreated and treated obese mice, respectively; P 0.05) but had no effect on glucose homeostasis. In obese adipose tissue, pentoxifylline reduced TNF-α(106.1 ± 17.6 and 51.1 ± 9.6 pg/mL for non-treated and treated obese mice, respectively; P 0.05) and interleukin-6(340.8 ± 51.3 and 166.6 ± 22.5 pg/mL for non-treated and treated obese mice, respectively; P 0.05) levels; however, leptin(8.1 ± 0.7 and 23.1 ± 2.9 ng/mL for non-treated and treated lean mice, respectively; P 0.05) and plasminogen activator inhibitor-1(600.2 ± 32.3 and 1508.6 ± 210.4 pg/mL for non-treated and treated lean mice, respectively; P 0.05) levels increased in lean adipose tissue. TNF-α level in the liver of lean mice also increased(29.6 ± 6.6 and 75.4 ± 12.6 pg/mL for non-treated and treated lean mice, respectively; P 0.05) while triglycerides presented a tendency to reduction.CONCLUSION: Pentoxifylline was beneficial in obese mice improving liver and adipose tissue inflammation. Unexpectedly, pentoxifylline increased pro-inflammatory markers in the liver and adipose tissue of lean mice.     

岳阳市城区小学生血吸虫病防治健康教育现状调查

目的 调查湖南省岳阳市城区小学生血吸虫病防治知识、行为现状及健康教育需求情况,为制定科学有效的小学生血吸虫病健康教育方案提供参考依据。方法 采用分层整群抽样方法,抽取岳阳市城区洞庭湖湖畔学校和中心城区学校各2所,每所再从五、六年级分别抽取2个班的学生,通过调查问卷了解小学生血吸虫病相关知识、预防行为及健康教育需求,并进行统计分析。结果 共调查湖畔小学353人、中心城区小学363人,两组学校小学生年龄、性别、年级构成差异无统计学意义（t=-0.494,χ²_性别=1.615,χ²_年级=2.152;P均>0.05）。学生血吸虫病防治知识总知晓率为42.60%（305/716）,其中,湖畔学校小学生血防知识知晓率（52.97%）高于中心城区（32.51%）,差异有统计学意义（χ²=30.661,P<0.05）;学生行为正确率为76.68%（549/716）,血防知识知晓组行为正确率（81.31%）高于不知晓组（71.24%）,差异有统计学意义（χ²=6.384,P<0.05）。学生血防知识主要来源于老师（47.49%）;91.90%的学生愿意了解更多的血防知识,最喜欢的血防知识学习方式是参加课外活动（50.42%）。结论 岳阳市城区小学生的血防知识知晓率及行为正确率偏低,对血吸虫病防治健康教育需求较高。建议针对学生开展形式多样的血防健康教育,以增强学生的血吸虫病防护意识,提高自我防护能力。     

RNA-Seq在老年神经退行性疾病研究中的应用

阿尔茨海默病及帕金森病是老年人最常见的两种神经退行性疾病,但其发病机制及治疗是研究的热点。随着高通量测序技术的进步及成本的下降,RNA-Seq也成为神经退行性疾病机制研究及生物标志物发现的有力手段。RNA-Seq相对于microarray具有高灵敏度、高准确性、高重复性以及噪声低等优势,在阿尔茨海默病及帕金森病研究中有较为广泛的应用,包括检测差异表达基因,可变剪接、新长链非编码RNA预测分析和miRNAs调控等,但是容易受病理复杂性及样本等因素影响。目前阿尔茨海默病及帕金森病转录组研究相比于癌症等还不够深入,在临床诊断及治疗应用还面临较大挑战。但是随着新技术及新方法的发展,RNA-Seq将进一步推动神经退行性相关疾病的研究和临床转化。     

Rising incidence of oesophageal adenocarcinoma in men in Australia

Adenocarcinomas of the oesophagus and of the gastric cardia have been reported to be increasing in incidence in many countries, while the incidence of squamous cell carcinoma of the oesophagus is stable and non-cardia gastric cancers are decreasing in incidence. Age-standardized incidence rates for the years 1982–93 for oesophageal adenocarcinoma and non-adenocarcinoma, and gastric cardia and non-cardia cancers were calculated based on state cancer registry incidence data. Time trends in the age-standardized rates were assessed using linear regression. A consistent increasing trend in the incidence of oesophageal adenocarcinoma in men was seen in all states of Australia and was statistically significant in all states except South Australia. There were no consistent nationwide trends in the incidence of oesophageal adenocarcinoma in women, although a trend towards an increase in the incidence of this cancer reached statistical significance ( P < 0.05) in three states (New South Wales, Victoria, Queensland). There were no important trends in the incidence of oesophageal non-adenocarcinoma in either men or women. There were no consistent nationwide changes in the incidence of gastric cardia cancer in either men or women, although this cancer was significantly increasing in Tasmania in both men and women. The incidence of cancer of the stomach not arising at the gastric cardia was significantly decreasing in men in all states and was also decreasing in women in all states, although in women this decrease was statistically significant only in New South Wales, Victoria and Western Australia. There has been a dramatic increase in the incidence of oesophageal adenocarcinoma in men in Australia. The incidence of this cancer in men is now approximately equal with that of non-adenocarcinoma of the oesophagus. The incidence of non-cardia stomach cancer continues to fall.     

Role of microRNA in regulation of myeloma-related angiogenesis and survival

Multiple myeloma (MM) is a malignant disease caused by clonal proliferation of plasma cells that result in monoclonal gammopathy and severe end organ damage. Despite the uniform clinical signs, the disease is very diverse in terms of the nature and sequence of the underlying molecular events. Multiple cellular processes are involved in helping the malignant cells to remain viable and maintain proliferative properties in the hypoxic microenvironment of the bone marrow. Specifically, the process of angiogenesis, triggered by the interactions between the malignant MM cells and the stroma cells around them, was found to be critical for MM progression. In this review we highlight the current understanding about the epigenetic regulation of the proliferation and apoptosis of MM cells and its dependency on angiogenesis in the bone marrow that is carried out by different microRNAs.     

Propranolol-induced hypertension in treatment of cocaine intoxication

The case of a patient with apparent cocaine toxicity and drug-mediated hypertension and tachycardia is presented. IV propranolol was used as the initial treatment for his hyperadrenergic state, resulting in a decrease in heart rate but a paroxsymal increase in blood pressure. The patient required nitroprusside for control of elevated blood pressure. A mechanism of unopposed alpha stimulation as a result of beta-2 receptor blockade is proposed, and a cautious approach to the use of propranolol in these patients is suggested.     

Use of fab fragments of digoxin-specific antibodies in the therapy of massive digoxin poisoning

A case of massive digoxin ingestion with multiple arrhythmias, consisting of high grade A-V block and ventricular ectopy not responsive to lidocaine, is described. The arrhythmias ceased following administration of digoxin-specific Fab fragments. The patient improved and was transferred to the psychiatric unit.     

Comparison of intravenous and oral routes of theophylline loading in acute asthma

In a prospective, randomized clinical trial, 19 patients with an acute exacerbation of asthma were given a loading dose of aminophylline by the IV (n = 10) or oral route (n = 9) of administration following treatment with epinephrine. Plasma concentrations of theophylline were measured prior to giving the loading dose, and one, two, three, and 24 to 48 hours later. Therapeutic effectiveness was evaluated by analyzing spirometric measurements prior to giving the loading dose, and one, three, and 24 to 48 hours later. Side effects also were recorded. In the IV group, the mean peak plasma theophylline concentration was 15.1 micrograms/mL one hour after loading, and in the oral group the mean peak serum theophylline concentration was 14.2 micrograms/mL three hours after loading. There was no correlation between theophylline concentrations and normalized change in spirometric values. There was no significant difference in spirometric values between the IV and oral groups. Nausea was slightly more common in the IV group. We conclude that there is no therapeutic advantage to giving a loading dose of aminophylline by the IV route rather than orally in patients with mild-to-moderate exacerbation of asthma initially treated with epinephrine.     

Histamine antagonists in the treatment of shock hyperglycemia in the rat

The present study was undertaken to determine the effect of exogenous histamine and histamine blockers on blood glucose and hepatic glycogen in the rat. Forty-one nonfasted male Sprague-Dawley rats that had been anesthetized with intraperitoneal injections of urethane were injected intravenously (femoral) with histamine (10 mg/kg) five minutes after pretreatment with Ringer's solution (control), diphenhydramine (1 mg/kg) (H-1 blocker); metiamide (1 mg/kg) (H-2 blocker); or a combination of these blockers. Mean arterial pressure (carotid), blood glucose, and hepatic glycogen were measured. Within 30 minutes, histamine evoked a significant increase in blood glucose, and a decrease in hepatic glycogen, and a reduction in blood pressure. However, rats treated with the H-2 blocker metiamide or with a combination of H-1 and H-2 blockers did not show as significant a hypotensive response as rats treated with the H-1 blocker diphenhydramine alone. The hyperglycemic-glycogenolytic response to histamine was modified by diphenhydramine as well as by a combination of blockers, but not by metiamide alone. These results suggest that a) the hypotension did not initiate the hyperglycemic and glycogenolytic response; b) the H-2 blocker metiamide has little effect on the hyperglycemic response to exogenous histamine; and c) the H-1 blocker diphenhydramine may have antihyperglycemic properties.