应用转基因小鼠研究心力衰竭的肾上腺素能机制

目前已有20多种与交感神经肾上腺素能信息系统有关的小鼠品系问世(表1)[1～17],这些品系已形成系统,为心力衰竭(heart failure,HF)的研究提供了有力的工具.多数小鼠品系采用a-肌凝蛋白重链(a-myosin heavy chain, a-MHC)的启动基因来实现转入基因在心脏中的超表达,从而提高肾上腺素能信息系统中某种成分的含量.其他小鼠品系包括非特异性基因删除(disruption)和心脏特异性表达某种抑制肽.目前应用这一类小鼠品系的研究已经积累了大量的信息,许多发现已使我们对HF中肾上腺素能机制有了全新的认识.现阶段采用的研究手段主要有三种:(1)探索小鼠品系的心脏表现型;(2)品系间杂交(crossbreed)来实现基因互补(genetic complementation) ;(3)在小鼠品系上复制实验性 HF.

骨骼肌肌球蛋白分子重链异形体的运动适应

肌球蛋白是一个六聚体的生物大分子，由两条分子量约为19.4-21.2万道尔顿的重链(Myosin Heavy Chain， MHC)和两对分子量约2.0万道尔顿左右的轻链(Myosin Light Chain， MLC)组成。重链由大约1800个氨基酸组成并含有特殊的3-甲基组氨酸肽链；轻链有3种类型：LC1、LC2 和LC3，其中LC1和LC2在碱性条件下容易从肌球蛋白分子上解离下来，故称碱性轻链(AIKaLi Light Chain)，而LC2易受巯基试剂作用而解离，故称巯解轻链(TDNB Light Chain)。

肌球蛋白调节性轻链在慢性心力衰竭大鼠心室肌的表达变化

肌球蛋白是心肌粗肌丝的主要成分,由两条重链(Myosin Heavy Chain,MHC)和两对轻链(Myosin Light Chain,MLC)构成.MLC包括两条基本轻链和两条调节性轻链( Myosin Regulatory Light Chain,MRLC,MYL2),既往多认为MHC异常是导致心肌肥厚、心衰的重要原因,而MYL2仅是MHC的辅助装置,在心肌肥厚的发生过程中起次要作用.然而,近的研究显示,MYL2也可能参与慢性心衰(Chronic Heart Failure,CHF)的发生、发展[1,2].本研究通过制作腹主动脉缩窄大鼠慢性心衰模型,观察MYL2在心室肌中的表达,探讨其在CHF发生中的作用.

人类个体发育中的免疫球蛋白重链可变区基因谱型

免疫球蛋白(immunoglobulin,Ig)介导的体液免疫反应在人体对抗外来抗原侵袭过程中起着重要作用.全部编码免疫球蛋白的基因被称为免疫球蛋白基因谱型(repertoire).人类胎儿和新生儿存在着相对免疫缺陷,针对病原微生物的侵袭,他们仅能产生多反应性、非特异性的低亲和力抗体.这类抗体的产生反映了人类生长发育早期Ig基因谱型的有限性.本文着重进行人类个体发生过程中免疫球蛋白重链(immunoglobulin heavy chain ,IgH)可变区基因谱型研究的综述.

Neuro－2a细胞株用于A型肉毒毒素重链体外实验的可行性研究

目的：探讨Neuro－2a细胞株用于A型肉毒毒素重链（ botulinum neurotoxin serotype A heavy chain， BoNT／A HC）体外细胞模型的可行性。方法：体外培养Neuro－2a细胞检测HC相关受体，确定是否可用于HC作用的体外模型；后于Neuro－2a细胞培养液内加入一定浓度的HC，于不同时点在相差显微镜下摄取图片观察HC对细胞突起生长状况的影响，包括有突起细胞的百分比、细胞突起的长度以及突起总数。结果：（1）培养一定时间的Neuro－2a细胞表达HC的高亲和力（synaptic vesicle 2，SV2）和低亲和力（trisialoganglioside 1b，GT1b）受体。（2）培养液内加入一定剂量的HC可促进Neuro－2a细胞突起再生及增长。在HC影响下，有突起的Neuro－2a细胞百分比、神经突起的总数量以及平均突起的长度皆较对照组明显增加，且差异具有统计学意义（ P＜0．05）。结论：Neuro－2a细胞可用于HC体外研究的细胞模型；Neuro－2a对HC比较敏感；一定剂量的HC能够促进神经突起的生长。

A型肉毒素重链对Neuro－2 a细胞的促神经突起再生作用

目的：观察A型肉毒素重链（ botulinum neurotoxin serotype A heavy chain， BoNT／A HC）对Neuro－2的促神经再生作用。方法：（1）在细胞培养液中加入不同浓度的BoNT／A HC，于3个时点收集细胞做免疫荧光检测轴突长度和有突起细胞的百分比。（2）在培养液中加入同一浓度的BoNT／A HC，分别作用不同时间收集细胞，通过免疫荧光检测平均吸光度对磷酸化ERK1／2进行半定量分析。结果：BoNT／A HC作用3个时点后轴突长度及有突起细胞百分比与对照组相比，低浓度效果不显著，其余浓度皆显著增高（ P＜0．05）；BoNT／A Hc组平均吸光度与对照组比较，10 min 差异无统计学意义，其余时点均有意义（ P＜0．05）。结论：一定剂量的BoNT／A HC不仅可以促进神经细胞轴突增长，还可以促进细胞长出突起，并使再生信号蛋白磷酸化ERK1／2的表达增加，表明A型肉毒素重链对Neuro－2细胞的促神经突起再生作用可能与ERK1／2的磷酸化有关。

AIM:Cytochrome P450 epoxygenase 2J2 and epoxyeicosatrienoic acids ( EETs) are known to protect against cardiac hypertrophy and heart failure, which involve activation of 5′-AMP-activated protein kinase ( AMPK) and Akt.Although the functional roles of AMPK and Akt are well established , the significance of crosstalk between them in the development of cardiac hypertrophy and anti -hy-pertrophy of CYP2J2 and EETs remains unclear .Here, we investigated whether CYP 2J2 and its metabolites EETs protected against cardiac hypertrophy by activating AMPKα2 and Akt1.Moreover, we tested whether EETs enhanced crosstalk between AMPKα2 and phosphorylated Akt1 ( p-Akt1), and stimulated the nuclear translocation of p-Akt1, to exert their anti-hypertrophic effects. METHODS:The recombinant rAAV9 vector was coupled to CYP2J2 and the rAAV9-CYP2J2 construct was injected into the caudal vein of AMPKα2-/-and littermate control mice .AMPKα2 -/-and littermate control mice that overexpressed CYP 2J2 in heart were treated with angiotensin II (Ang II) for 2 weeks.Hemodynamic and cardiac functions were also evaluated after 14 days of infusion with Ang II or saline.RESULTS:Interestingly, the overexpression of CYP2J2 suppressed cardiac hypertrophy , including decreased heart size, cross sectional area of cardiomyocytes , markers of cardiac hypertrophy [ brain natriuretic peptide ( BNP) ,β-myosin heavy chain (β-MHC) and skeletal muscle α-actin (ACTA1)] and increased levels of atrial natriuretic peptide (ANP) in the heart tissue and plasma of wild-type mice but not AMPKα2 -/-mice.Measurement of left ventricular ejection fraction and fractional shortening showed that CYP2J2 overexpression prevented Ang II-induced ventricular systolic dysfunction in mice .Moreover, an Ang II-induced reduction in cardiac function, demonstrated by decreased dp/dtmax and dp/dtmin, was prevented by overexpression of CYP2J2.Mechanistically, the CYP2J2 metabolites 11,12-EET activated AMPKα2 to induce the nuclear translocation of p-Akt1, which increased production of ANP and thereby inhibited the development of cardiac hypertrophy .Furthermore , by co-immunoprecipitation analysis , we found that full-length Akt1 and an Akt1 fragment containing amino acids 150-408, which constitute the protein kinase domain , but not other frag-ments of Akt1, bind to the AMPKγ1 subunit.AMPKα2β2γ1 and p-Akt1 interact through the direct binding of the AMPKγ1 subunit to the Akt1 protein kinase domain.This interaction was enhanced by 11,12-EET.CONCLUSION:Our studies reveal a novel mechanism in which CYP2J2 and EETs enhanced Akt1 nuclear translocation through interaction with AMPKα2β2γ1 and protect against cardiac hy-pertrophy and suggest that overexpression of CYP 2J2 might have clinical potential to suppress cardiac hypertrophy and heart failure .