高同型半胱氨酸血症导致大鼠海马基因表达谱变化

目的:观察高同型半胱氨酸(HCY)对大鼠海马基因表达谱的影响。方法:10只雄性SD大鼠随机分为对照组和高HCY组。高HCY组大鼠皮下注射L-HCY(60μg/g),2次/日,连续注射14 d;对照组注射等量生理盐水。造模完成后取血浆分析HCY水平;提取海马组织RNA采用BGISEQ-500技术进行测序。测序数据通过主成分分析进行样本间的比较,找出并剔除离群样品,DEGseq方法分析组间差异表达基因。根据KEGG注释结果以及官方分类,将差异基因进行生物通路分类和富集分析。结果:高HCY组大鼠血HCY明显高于对照组(P0.01)。大鼠海马RNA测序数据使用DEGseq总共筛选到34个有显著性差异表达的mRNA,生物通路分类和富集分析找到最主要的通路是神经活动配体-受体相互作用通路。结论:高HCY血症导致大鼠海马的基因表达谱改变,影响神经活动配体-受体相互作用等生物通路。     

全脑缺血再灌注后海马EphA受体基因表达的变化特点

目的:观察EphA受体在海马全脑缺血再灌注后基因表达的改变。方法:SD大鼠50只随机分为假手术组10只及全脑缺血再灌注组40只,Pulsinelli四血管阻断法建立全脑缺血再灌注模型,采用半定量RT-PCR观察假手术组及缺血组在缺血后不同时间点(6 h、1 d、3 d、7d)EphA受体m RNA含量变化的情况,免疫荧光双标法检测EphA4受体在海马的细胞定位。结果:EphA1-A8及EphA10 RNA在正常海马组织均有表达,EphA4受体含量多。在缺血状态下,EphA1、EphA2、EphA3、EphA6、EphA7及EphA8的mRNA表达水平一过性上调,EphA4、EphA5和EphA10的m RNA表达水平逐步上调;EphA4受体亦是缺血后变化最显著的EphA受体。免疫荧光双标显示EphA4主要分布于海马CA1-CA3区及DG区Neu N阳性锥体神经元。结论:在海马不同的EphA受体对缺血呈现出不同的应答模式,EphA4是海马正常条件下表达最为丰富的EphA受体并且在缺血条件下出现最为显著地表达变化。EphA4受体主要分布于海马CA1-CA3区以及DG区NeuN阳性锥体神经元。     

Dystrophin Delivery to Muscles of mdx Mice Using Lentiviral Vectors Leads to Myogenic Progenitor Targeting and Stable Gene Expression

To explore whether stable transduction of myogenic stem cells using lentiviral vectors could be of benefit for treating dystrophic muscles, we generated vectors expressing a functional microdystrophin/enhanced green fluorescence protein fusion (µDys/eGFP) gene. Lentiviral vector injection into neonatal mdx^4cv muscles resulted in widespread and stable expression of dystrophin for at least 2 years. This expression resulted in a significant amelioration of muscle pathophysiology as assessed by a variety of histological and functional assays. To assess whether this long-term expression was accompanied by stable transduction of satellite cells, we harvested muscle mononuclear cells 1 year after vector injection. Up to 20% of the cultured myoblast colonies expressed the µDys/eGFP transgene following myotube formation. Furthermore, transplantation of the muscle mononuclear cells into secondary mdx^4cv recipients showed their ability to regenerate dystrophin-expressing myofibers in vivo. The ability to isolate myogenic cells able to form dystrophin-positive myotubes or myofibers in vitro and in vivo >1 year postinjection indicates that the vectors stably transduced muscle satellite cells, or a progenitor of such cells, in neonatal mdx^4cv muscles. These studies suggest that integrating lentiviral vectors have potential utility for gene therapy of muscular dystrophy.     

戊四氮点燃过程中大鼠海马苔藓纤维发芽变化的研究

目的 :探讨戊四氮 (PTZ)诱导点燃过程中大鼠海马细胞形态的可塑性与慢性癫痫发病机制的关系。方法 :450只大鼠随机分为对照组、药物组 (PTZ 3 5mg/kg腹腔注射 ,每天 1次 )和干预组 (苯巴比妥 3 0mg/kg及PTZ 3 5mg/kg腹腔注射 ,每天 1次 ) ,诱导大鼠点燃过程中采用Timm银染组织化学方法观察海马区苔藓纤维出芽。并分析 3组间苔藓纤维出芽的差异。结果 :药物组大鼠注射PTZ后 ,行为学未出现惊厥 ,脑电图未出现癫痫性放电的点燃前潜伏期内 ,在海马齿状回内分子层和CA3区透明层出现异常苔藓纤维出芽 ,与对照组比较 ,差异有显著性 (P <0 .0 5) ,与干预组对应时间点比较 ,差异亦有显著性 (P <0 .0 5)。结论 :苔藓纤维出芽 海马细胞形态的可塑性是PTZ点燃大鼠慢性癫痫形成的重要机制 ,苯巴比妥可能通过抑制苔藓纤维出芽 ,预防癫痫发生。     

四逆散对运动性疲劳大鼠海马突触素的调节作用

目的 观察四逆散对运动性疲劳大鼠海马内突触素表达的影响及其对学习记忆能力的影响.方法 将大鼠分成正常组,模型组和治疗组,运用力竭游泳方法制造运动性疲劳大鼠模型,造模同时给予治疗组四逆散灌胃10日后,用被动回避实验观测大鼠学习记忆能力的改变,采用免疫组化技术观测大鼠海马CA1区突触素的表达变化.结果 ①各组大鼠的避暗潜伏期/避暗错误次数:正常对照组为(144.25±57.14)s/(0.50±0.80),模型组为(73.00±61.96)s/(1.67±1.15),四逆散组为(166.17±47.92)s/(0.38±0.49);②各组大鼠海马突触素平均光密度值:正常对照组为(0.2636±0.10654),四逆散组为(0.2555±0.16338),高于模型组为(0.0474±0.1837)(P<0.05);③突触素免疫组化染色:正常对照组海马神经细胞周围突触素阳性颗粒密集,染色深,高于四逆散组和模型组.正常对照组的海马神经细胞排列整齐、紧密;模型组大鼠海马细胞排列松散,不紧密;四逆散组海马排列介于正常对照组和模型组之间.结论 四逆散具有改善运动性疲劳大鼠学习记忆的能力,具有增加运动性疲劳大鼠海马突触素表达的作用.     

Lower Resting State Heart Rate Variability Relates to High Pain Catastrophizing in Patients with Chronic Whiplash‐Associated Disorders and Healthy Controls

Vagally mediated heart rate variability (vmHRV) is widely respected as a psychophysiological measure of emotion regulation capacity and serves as a readily available index of executive brain areas that exert an inhibitory influence on subcortical structures. Pain catastrophizing (PC) is conceptualized as the tendency to misinterpret and exaggerate pain‐related situations that may be threatening. Chronic pain patients show lower vmHRV and higher PC. Previously, no study has investigated the association of PC and vmHRV. We examined the association of PC and vmHRV in a sample of patients with chronic whiplash‐associated disorders (WAD, n = 30) and healthy controls (n = 31). Patients with WAD showed lower vmHRV, indexed by high‐frequency HRV (effect size, Cohen's d = 0.442), and greater PC (d = 0.815). Zero‐order and partial correlations controlling for age and sex revealed that vmHRV and PC are inversely related. The results provide evidence for a psychophysiological mechanism underlying PC, in particular in chronic pain patients.     

The Role of Sexual Health Professionals in Developing a Shared Concept of Risky Sexual Behavior as it Relates to HIV Transmission

“Risky sexual behavior” accounts for the majority of new HIV infections regardless of gender, age, geographic location, or ethnicity. The phrase, however, refers to a relatively nebulous concept that hampers development of effective sexual health communication strategies. The purpose of this paper was to propose development of a shared conceptual understanding of “risky sexual behavior.” We reviewed multidisciplinary HIV/AIDS literature to identify definitions of risky sexual behavior. Both the linguistic components and the social mechanisms that contribute to the concept of risky sexual behaviors were noted. Risky sexual behavior was often defined in a subjective manner in the literature, even in the scientific research. We urge a paradigm shift to focus on explicit behaviors and the social context of those behaviors in determining HIV risk. We also propose a new definition that reduces individual biases and promotes a broader discussion of the degree of sexual risk across a diversity of behavioral contexts. Sexual health professionals can strengthen practice and research initiatives by operating from a concise working definition of risky sexual behavior that is broadly transferable and expands beyond a traditional focus on identity‐based groups.     

评价血液ST13和BCL11B基因表达水平在帕金森病早期诊断中的潜在价值

目的:研究外周血ST13和BCL11B基因表达水平作为帕金森病(PD)早期分子诊断标志物的可能性。方法:利用实时定量PCR,比较外周血ST13和BCL11B基因的表达水平在各阶段PD患者中的变化,并计算与健康对照组的统计学差异。结果:PD患者外周血ST13和BCL11B的表达水平明显降低。BCL11B的表达在包括Hoehn-YahrⅠ级患者在内的所有PD患者中均显著下降,而ST13的表达只有在Hoehn-YahrⅡ级以上患者中才出现明显变化。结论:ST13和BCL11B基因表达水平都有作为潜在PD分子诊断标志物的可能,但就筛查早期PD患者而言,BCL11B可能更具优势。     

脓毒症对大鼠海马区caspase-3表达的影响

目的 探讨脓毒症对大鼠海马区神经元的凋亡蛋白酶caspase-3表达的影响。 方法 80只30日龄健康雄性Wistar大鼠,随机分为盲肠结扎穿孔术（CLP） 组（n=50）和对照组（n=30）。CLP组采用CLP建立脓毒症模型,对照组行假手术不造成脓毒症模型。于手术后6、12、24 h,CLP组和对照组分别取10只大鼠,5只做神经行为学评分,另外5只处死取脑,采用蛋白免疫印迹法观察caspase-3的表达,术后24 h,两组各取3只大鼠,采用免疫荧光检测caspase-3的表达。 结果 对照组大鼠大脑海马区仅微量表达caspase-3,神经行为学评分较高。CLP组大鼠caspase-3的表达量于CLP后6 h就开始升高,24 h达高峰,均明显高于对照组（P＜0.05）,大鼠的神经行为学评分在CLP后6 h开始降低,并随着时间逐渐下降,均明显低于对照组（P＜0.05）。 结论 脓毒症脑损伤时大鼠的神经行为学评分降低,海马区神经元caspase-3表达上调,并随时间变化而波动。     

Stable Human FIX Expression After 0.9G Intrauterine Gene Transfer of Self-complementary Adeno-associated Viral Vector 5 and 8 in Macaques

Intrauterine gene transfer (IUGT) offers ontological advantages including immune naiveté mediating tolerance to the vector and transgenic products, and effecting a cure before development of irreversible pathology. Despite proof-of-principle in rodent models, expression efficacy with a therapeutic transgene has yet to be demonstrated in a preclinical nonhuman primate (NHP) model. We aimed to determine the efficacy of human Factor IX (hFIX) expression after adeno-associated-viral (AAV)-mediated IUGT in NHP. We injected 1.0–1.95 × 10¹³ vector genomes (vg)/kg of self-complementary (sc) AAV5 and 8 with a LP1-driven hFIX transgene intravenously in 0.9G late gestation NHP fetuses, leading to widespread transduction with liver tropism. Liver-specific hFIX expression was stably maintained between 8 and 112% of normal activity in injected offspring followed up for 2–22 months. AAV8 induced higher hFIX expression (P = 0.005) and milder immune response than AAV5. Random hepatocellular integration was found with no hotspots. Transplacental spread led to low-level maternal tissue transduction, without evidence of immunotoxicity or germline transduction in maternal oocytes. A single intravenous injection of scAAV-LP1-hFIXco to NHP fetuses in late-gestation produced sustained clinically-relevant levels of hFIX with liver-specific expression and a non-neutralizing immune response. These data are encouraging for conditions where gene transfer has the potential to avert perinatal death and long-term irreversible sequelae.     

谷氨酸转运体-1在脑出血早期痫性发作大鼠海马组织的表达

目的:观察脑出血早期痫性发作大鼠海马组织谷氨酸转运体(GLT-1)的表达。方法:应用立体定向技术制备大鼠脑出血模型,同时监测脑电活动,35只大鼠根据是否造成脑出血和是否发生痫性放电分为假手术组(n=7)、脑出血组(n=21)及痫性发作组(n=7)。采用HE染色观察大鼠海马组织形态学变化,采用免疫组织化学法检测海马组织GLT-1阳性表达。结果:HE染色结果显示脑出血组、痫性发作组海马组织出现神经细胞变性、坏死或凋亡。假手术组、脑出血组及痫性发作组GLT-1阳性表达面积分别为(583.2±23.6)μm2、(419.6±29.3)μm2、(358.2±19.4)μm2,脑出血组、痫性发作组GLT-1阳性表达低于假手术组(P<0.05),痫性发作组GLT-1免疫阳性表达低于脑出血组(P<0.05)。结论:大鼠脑出血早期痫性发作与海马区GLT-1表达下调有关。     

差异表达基因Hepsin在前列腺癌中的表达及其意义

目的探讨cDNA芯片筛选出的前列腺癌（PC）差异表达基因Hepsin在PC中的表达及其意义。方法应用RT—PCR检测Hepsin在正常前列腺组织、PC组织及PC细胞系中的表达，并进行半定量分析，同时采用免疫印迹法检测Hepsin的表达，采用免疫组织化学染色法检测40例PC标本、15例前列腺增生（BPH）标本和6例正常前列腺标本的Hepsin表达水平。结果在mRNA水平，Hepsin在PC中的表达水平高于正常前列腺组织（P=0．026），但在PC细胞系中的表达并不增高。在蛋白表达水平，正常前列腺组织未表达Hepsin，而PC及PC细胞系均表达Hepsin。PC和BPH标本免疫组化检测均为阳性，但两者间有非常高度显著性差异（P=0．000），而正常前列腺标本为阴性。结论Hepsin在PC中的表达增高，有可能作为早期诊断的标志以及基因治疗的靶基因。     

抑肽酶在转基因烟草中的表达

目的　在烟草中高效表达抑肽酶(bpti)。方法　克隆抑肽酶基因,以烟草叶片为外植体,通过根癌农杆菌将其导入烟草,经卡那霉素抗性筛选和PCR扩增检测基因的整合;抽提转染植株叶片中的抑肽酶,进行活性测定。结果　抗性植株整合并高效表达了抑肽酶。结论　构建的含抑肽酶基因的植物表达载体pBI1 2 1 bpti具有正确的阅读框,并能在异源植物中表达。     

烫伤增强大鼠TNF-α基因表达对内毒素刺激的敏感性

目的　证实严重烫伤大鼠TNF α基因表达对内毒素 (LPS)刺激敏感性增强 ,阐明创伤机体无明显感染条件下血清TNF α升高的机理。方法　给予同剂量LPS体内注射 ,观察烫伤组 ,烫伤休克组及其正常对照组血清TNF α及肝脏TNF αmRNA的变化。结果　同剂量的LPS刺激后 ,正常对照组血清TNF α及肝脏TNF αmRNA升高不显著 ,而烫伤组 ,烫伤休克组血清TNF α及肝脏TNF αmRNA明显升高。结论　TNF α基因表达对LPS敏感性增强 ,导致创伤患者TNF α水平明显升高 ,提示防治创伤患者轻微感染在临床治疗休克、多功能器官衰竭中非常重要     

一氧化氮合酶对增生性瘢痕的影响

目的:观察一氧化氮合酶(NOS)在增生性瘢痕与正常皮肤组织及细胞中的表达特征及其差别,并通过应用一氧化氮(NO)供体或NOS抑制剂对培养的瘢痕成纤维细胞进行体外药物干扰,探索NO、NOS在增生性瘢痕形成中的作用。方法:取临床增生性瘢痕手术患者的瘢痕组织及周围少许正常皮肤,体外培养获得皮肤和瘢痕成纤维细胞,通过免疫组化及RT-PCR方法比较两种组织及细胞中NOS的表达及分布的差异。分别以100~500μM的NO供体硝普钠(SNP)、NOS抑制剂N-硝基-L-精氨酸甲酯(L-NAME)作用于瘢痕成纤维细胞,观察细胞增殖的变化;并用免疫细胞化学和RT-PCR方法比较作用前后成纤维细胞中I、III型胶原表达的变化。结果:免疫组化检测及RT-PCR方法检测显示增生性瘢痕组织及细胞中NOS表达相对于皮肤组织及成纤维细胞中明显减少(P<0.05)。瘢痕细胞经SNP作用后,细胞增殖显著降低。免疫细胞化学方法和RT-PCR方法检测I、III型胶原结果显示,从SNP200μM组开始I、III型胶原表达显著降低(P<0.05)。经L-NAME200μM作用后,I、III型胶原表达显著增加(P<0.05)。结论:增生性瘢痕组织及细胞中NOS含量较正常皮肤组织及细胞明显减少,NO供体SNP对体外培养的增生性瘢痕成纤维细胞增殖以及胶原表达起到抑制作用,NOS抑制剂L-NAME对其胶原表达则起到促进作用,结果提示NOS表达降低可能与瘢痕增生密切相关,因此改变NO的产生及NOS的活性可望调控瘢痕成纤维细胞的生物学行为。