毛细管电泳技术在疾病基因诊断方面的应用

近年来分子生物学和遗传学的迅速发展,使人们对基因结构与功能的认识日益加深。科学工作者们发现基因的某些突变可导致疾病的发生或易感性增加。因此可以通过对疾病相关基因的分析进行疾病诊断或预告;对于那些尚未确定致病基因的遗传病也可利用DNA标记进行遗传连锁分析?..     

Hijacking Dorsal Raphe to Improve Moods and Metabolism via BDNF Gene Transfer in Mice

Background: Mood disorders such as depression and metabolic diseases like obesity and diabetes have significant comorbidities; furthermore, each disease increases the risk of the other one. Continuing use of antidepressant medication is associated with an elevated risk of type 2 diabetes and weight gain. On the other hand, antiobesity drugs are found to have varying neuropsychiatric adverse event profiles. Therefore, there is an urgent need to develop more effective and safer therapies for mood disorders and metabolic diseases with minimal side effects. Both brain derived neurotrophic factor (BDNF) and serotonin (5-HT) can regulate moods in the limbic system and metabolism in the hypothalamus, respectively. The dorsal raphe nucleus (DR) in the midbrain contains the largest number of serotonin-expressing neurons as well as other neurons expressing various neurotransmitters. Both serotonergic and nonserotonergic DR neurons send projections widely to the forebrain including the limbic system and the hypothalamus. This study aimed to investigate whether BDNF gene transfer in DR neurons can improve moods and metabolism. Methods: Recombinant adeno-associated virus (AAV) was microinjected into DR to achieve BDNF gene transfer. Chronic unpredictable mild stress (CUMS) model of depression in C57BL/6 mice was established to investigate the role of DR BDNF gene transfer in depression. Forced swimming test (FST) and sucrose consumption test (SCT) were used to examine the depressive-like behaviors. Open field test (OPT) and elevated zero maze (EZM) were used to examine the anxiety-like behaviors. Diet-induced obesity (DIO) and leptin receptor mutant db/db mice were employed to investigate the role of DR BDNF gene transfer in regulating metabolism. Metabolic profiling included body weight, food intake, fat mass, serum factors, energy expenditure, glucose tolerance and insulin sensitivity. Quantitative RT-PCR was performed to determine expression changes of several key genes in DR, hypothalamus, liver and fat tissues. Tph2 conditional knockout mice were used to examine whether 5-HT is essential for BDNF in improving metabolism. In addition, chemogenetic activation of DR neurons via AAV-mediated hM3Dq expression was also performed to examine whether there is a similar effect as BDNF. Results: FST and SCT showed significant anti-depressant effects of DR BDNF gene transfer in the CUMS model, meanwhile, OPT and EZM showed significant anxiolytic effects. BDNF gene transfer in DR also markedly ameliorated obesity and hyperglycemia in DIO and db/db mice, which was revealed by lightened body weight, decreased food intake, increased energy expenditure, improved glucose tolerance and insulin sensitivity, reduced levels of serum factors like insulin and leptin. The mRNA expression of genes related to 5-HT signaling such as tph2 and vmat2 was significantly increased in DR after BDNF gene transfer. Expression levels of multiple genes regulating energy balance in the hypothalamus, liver, and fat tissues also changed. Conditional knockout of tph2, encoding the rate-limiting enzyme of 5-HT synthesis, did not block the effects of DR BDNF gene transfer in improving metabolism. Chemogenetic activation of DR neurons also had the effects of improving metabolism in DIO mice. Conclusions: BDNF gene transfer in DR of mice improves moods and metabolism. This study surmounts the “hypothalamocentric” paradigm dominating in metabolism research by connecting metabolism and moods via dorsal raphe.     

生物电阻抗法测定身体脂肪率在社区2型糖尿病肥胖患者筛查中的应用

【目的】评价生物电阻抗法测定身体脂肪率(body fat ratio,BFR)在筛查社区糖尿病肥胖患者与BMI判定的一致性。【方法】采用生物电阻抗法测定472名社区糖尿病患者BFR,同时测定其身高和体重,计算BMI,采用ROC曲线判定BFR的准确性和有效性。【结果】社区472名糖尿病患者肥胖率为14.8%,超重率为41.5%;男性与女性BMI差异无统计学意义(P>0.05)。全人群BFR判定肥胖与BMI的一致性Kappa=0.206,女性BFR判定肥胖与BMI一致性Kappa=0.469。利用ROC曲线对BFR进行评价后发现,在全人群中,BFR判定糖尿病患者肥胖、超重、正常及低体重一致性效果一般;对女性进行ROC曲线评价后,BFR判定女性糖尿病患者肥胖、超重、正常及低体重一致性较好。【结论】社区糖尿病患者肥胖患病率及体脂率均较高,体脂率判定女性糖尿病患者肥胖与BMI一致性较好。     

利用GCaMP6f蛋白对小鼠伏隔核神经元活动的钙离子成像

目的在小鼠伏隔核表达GCa MP6f蛋白,并在束缚状态下,应用钙成像技术实时监测小鼠伏隔核内活化的神经元。方法首先,包装并纯化aav-Syn1-GCa MP6f-P2A-Tomato病毒,并通过感染原代神经元验证目的蛋白的表达和功能。然后,通过脑立体定位技术注射aav-Syn1-GCa MP6f-P2A-Tomato病毒于小鼠伏隔核脑区,动物恢复21 d后,插入光纤,在其清醒状态下,给予束缚刺激,用钙成像技术实时记录伏隔核神经元的活化。结果表达GCa MP6f的原代神经元在给予谷氨酸刺激后,神经元被活化,Ca2+内流而产生绿色荧光;通过在体实验,给予小鼠束缚刺激,可在视野内检测到因伏隔核神经元活化而发出的绿色荧光信号;鼠脑切片同样可检测到位于胞质的绿色荧光信号与位于细胞核的病毒红色荧光标记共定位于相同细胞。结论 aav-Syn1-GCa MP6f-P2A-Tomato腺相关病毒可成功用于在体活化神经元的实时监测。通过在小鼠伏隔核定点注射该病毒,给予束缚刺激,应用钙成像技术可实时记录到小鼠伏隔核的活化神经元。     

上海市金山区健康人群麻疹抗体监测

麻疹是一种严重危害儿童健康的急性呼吸道传染病。上海市金山地区 1991～ 1999年每年对 8个月～ 2 0岁的健康人群进行麻疹抗体的监测 ,以了解人群麻疹的免疫水平。现将监测结果报告如下。1　材料与方法1.1　对象对金山区 17个乡、镇每年抽取 1个乡、镇 ,对年龄范围在 8月龄到 2 0岁的 ,按 8个月～、3岁～、5岁～、7岁～、9岁～、11岁～、15～ 2 0岁分成 7个组 ,每组抽取约 3 0人的末梢血进行抗体测定。1.2　方法麻疹抗体用血凝抑制试验测定。抗体滴度≥ 1:2的为阳性。用等级相关、等级秩和检验及 χ2 检验的统计学方法进行评价。2　结果2 .1…     

麻疹 流行性腮腺炎 风疹活疫苗(M-M-RⅡ)免疫效果观察及免疫程序探讨

选择在8～9月龄时已完成麻疹减毒活疫苗（MV）初免的11～16月龄儿童为观察对象,对麻疹、流行性腮腺炎（腮腺炎）、风疹活疫苗（M－M－RⅡ）的免疫效果进行了观察,并探讨其免疫程序．M－M－RⅡ免疫后麻疹血凝抑制（HI）抗体阳性率100％,抗体≥4倍增长率为55．42％,几何平均滴度（GMT）1：46．60,较免疫前增长2．27倍;腮腺炎HI抗体阳转率95.23％,GMT1：9．60;风疹HI抗体阳转率100％,GMT1：392．47．作者认为,在上海市M－M－RⅡ初始免疫程序确定为已完成MV初免的12～18月龄儿童是合理的,且能产生理想的免疫效果．     

应用毛细管无胶筛分电泳诊断缺失型杜氏肌营养不良基因

毛细管电泳是一项新兴的分离、分析技术,它以高效、快速、重复性好、自动化及微量等优点,在核酸分离分析的各方面均较以往的方法更具优势。本工作应用毛细管无胶筛分电泳与多重ＰＣＲ技术相结合对缺失型杜氏肌营养不良的病人进行了直接基因诊断。与传统方法相比,本技术在分离速度、分辨率及灵敏度方面均有较大改善。毛细管电泳技术将广泛用于遗传病基因诊断。     

小剂量甲氨蝶呤治疗高甲状腺过氧化物酶抗体血症观察

目的：观察小剂量甲氨蝶呤（MTX）对高甲状腺过氧化物酶抗体（TPOAb）血症的疗效及安全性。方法：140例高TPOAb血症桥本甲状腺炎（HT）患者,分为治疗组110例和对照组30例。两组均给予小剂量、短期的抗甲亢和（或）补充左甲状腺素（L-T4）治疗。治疗组在此基础上加服甲氨蝶呤片（每周7.5～10mg）,观察时间3～24个月。比较两组治疗前后TPOAb水平变化、转阴率、甲减患者服用L-T4剂量的变化,及甲功正常者甲状腺功能的变化。结果：治疗后两组TPOAb水平持续明显下降,24个月后,治疗组TPOAb水平明显低于对照组,下降率则明显高于对照组（P〈0.01）;治疗组转阴率为19.1％,明显高于对照组（P〈0.01）。结论：小剂量MTX联合小剂量短期抗甲亢治疗,能显著降低HT患者TPOAb水平,延缓甚至终止TPOAb对甲状腺的破坏进程,优于常规抗甲亢治疗。     

夜发性额叶癫痫三家系临床和遗传学特征

目的 分析夜发性额叶癫痫3个家系的临床、脑电图和遗传学特征.方法 在3个夜发性额叶癫痫家系患者及部分亲属中收集临床、脑电图及神经影像学等资料,并采用测序方法筛查烟碱型乙酰胆碱受体(nAChR)α4、β2和α2亚单位编码基因(CHRNA4、CHRNB2和CHRNA2).结果 3个家系中有6例患者(其中男5例),平均年龄(20.5±11.5)岁,平均发病年龄(7.3±5.5)岁,临床表现为夜发性额叶发作,具体发作类型包括姿势性发作2例,躯体自动症发作4例,最多每夜发作6次.发作间期、发作期视频脑电图2例患者表现为正常或动作伪差,2例表现为前部导联慢波节律,3例出现前部导联棘波、棘慢波及尖波.神经系统及神经影像检查未见异常.抗癫痫药物治疗反应良好.CHRNA4、CHRNB2和CHRNA2部分序列(包含跨膜区1～3)筛查未见突变.结论 夜发性额叶癫痫是一种遗传异质性癫痫综合征.