植物rRNA基因组织模式的荧光原位杂交比较分析

采用荧光原位杂交技术,对分属5个科的10种植物的分生细胞的18S-25S rRNA基因(45S rDNA)的组织模式进行了比较分析.45S rDNA探针在所有供试植物的间期核都产生了两种杂交信号:荧光强、位于核仁周边的纽和荧光较弱分布于核仁内的点,表明不同植物间期核的rDNA染色质的组织模式相似.在每种植物的部分间期细胞都观察到点与纽相连或从纽发出的情况,而且点的数目越多纽就变得越小,点的有无和数目的多少与细胞的活性呈正相关.这些事实表明,纽代表了处于凝缩状态的非活性的rDNA染色质,点是由纽解凝缩而来,rDNA异染色质解凝缩形成点是植物rRNA基因活跃转录的细胞学表现,在同一物种中点的多少代表了间期核rDNA转录活性的强弱.我们的结果支持点是核仁内活性rRNA基因组织的结构单位及rRNA合成发生地点的推论.我们的结果还显示,不同植物间期核的rDNA染色质的组织也存在一些差异,其中核仁内点的最大数目有较大的不同.在所有供试植物的有丝分裂前中期细胞,45S rDNA探针在rDNA位点都产生了松散的信号块和许多点,表明植物的rDNA位点在有丝分裂前中期还有较活跃的转录.     

Upregulation of P53 promotes nucleus pulposus cell apoptosis in intervertebral disc degeneration through upregulating NDRG2

P53 is an apoptosis marker which is involved in determining nucleus pulposus (NP) cell fate. Little is known about P53 interaction with N-Myc downstream-regulated gene 2 (NDRG2) in intervertebral disc degeneration (IVDD). Here, we studied the role of the P53-NDRG2 axis in IVDD. We found that NDRG2 was expressed in NP tissue obtained from patients with IVDD. The level of NDRG2 was positively related to the severity of IVDD, as determined by Pfirrmann grading. Subsequently, we overexpressed NDRG2 in human NP cells by adenoviral transfection and studied the effects of increased levels of NDRG2 on the viability and apoptosis of these cells. NDRG2 overexpression induced NP cell apoptosis and reduced viability in NP cells obtained from patient with IVDD. We also found that the level of P53 was elevated in NP cells from patients with IVDD and treatment with exogenous P53 upregulated NDRG2 in NP cells. Last, IVDD model was established in P53 knockout mice and the pathological changes in the intervertebral discs and NDRG2 expression were examined. P53 knockout can reduce the damage of NP tissues after IVDD surgery to some extent. Restoration of NDRG2 antagonized the effect of P53 knockout on IVDD. Collectively, this study suggests that elevated P53 in NP cells stimulates apoptosis of the cells by upregulating NDRG2 expression, thereby exacerbating IVDD.     

KF核及B(o)tzinger复合体内GABA能神经元向膈神经核的投射

实验在６只成年猫上进行。将ＷＧＡ－ＨＲＰ微量注入Ｃ５膈神经核内,通过逆行追踪及ＧＡＢＡ免疫组织化学ＦＩＴＣ荧光双重标记方法,研究了脑干内ＧＡＢＡ能神经元向膈神经核的投射。结果在脑桥ＫＦ核和面神经后核周围区（即Ｂｏｔｚｉｎｇｅｒ复合体）观察到ＧＡＢＡ－ＨＲＰ双标神经元。另外,在中缝大核、旁巨细胞外侧核及前庭神经核也观察到双标神经元。本实验结果表明：发自上述脑干神经核团,特别是ＫＦ核及Ｂｏｔｚｉｎｇｅ     

谷氨酸及NMDA受体拮抗剂MK-801对大鼠伏核痛兴奋神经元电活动的影响

本文研究了谷氨酸(glutamic acid,Glu)及其NMDA受体拮抗剂5-甲基二氢丙环庚烯亚胺马来酸(MK-801)对人鼠伏核(nucleus accumbens,NAc)痛兴奋神经元(pain-excitation neurons,PEN)痛诱发反应的影响。电刺激坐骨神经作为伤害性刺激,用玻璃微电极记录NAc的PEN放电,观察脑室内注射Glu和NAc内注射MK-801对大鼠NAc中PEN伤害性诱发活动的影响。结果显示,伤害性刺激可使NAc的PEN电活动增强;脑室内注射Glu(10nmol／10μl)可使NAc的PEN伤害性诱发放电频率增加;NAc内注射MK-801(1．0nmol／0．5μl)可阻断这种作用;MK-801本身也可部分抑制PEN伤害性诱发反应。上述结果表明,Glu对PEN伤害性反应的易化作用是通过NMDA受体介导的：Glu和NMDA受体参与NAc伤害性信息传递的调制。     

Neuroglobin基因体内转染对水杨酸钠给药后小鼠下丘核神经元听反应的影响

实验以48只成年健康昆明小鼠为实验对象,研究GeneJamer转染试剂介导的neuroglobin(NGB)基因体内转染对水杨酸钠给药后小鼠下丘核区听反应的影响。实验分4组,每组12只。A1组:对照组1(阴性对照,将GeneJamer转染试剂6μl和pEGFP-C12μg混合后注入下丘核脑区);A2组:对照组2[阳性对照,将GeneJamer转染试剂(6μl)和pEGFP-NGB(质粒载体pEGFP-C1与NGB基因全编码序列构建的重组子2μg)混合后注入下丘核脑区];B组:水杨酸钠给药组(450mg/kg·d-1)+pEGFP-C1;C组:水杨酸钠(450mg/kg.d-1)+pEGFP-NGB组。以直接注射法将GeneJamer转染试剂和重组质粒pEGFP-NGB混合后注入小鼠下丘核区。采用RT-PCR和Westernblot技术检测小鼠下丘核区NGBmRNA和蛋白的表达;采用细胞外记录技术,研究小鼠下丘核区神经元在水杨酸钠给药后转染重组质粒pEGFP-NGB对强度-发放率函数(刺激声强与实验鼠下丘核区神经元在接受声刺激所产生的电发放的关系曲线)、强度-潜伏期函数(刺激声强与实验鼠下丘核区神经元在接受声刺激至产生电发放潜伏期之间的关系曲线)和频率调谐曲线(实验鼠下丘核区神经元在各个频率纯音刺激下起反应的阈值绘制的曲线)的影响。实验观察到:(1)经GeneJamer转染试剂介导NGB基因可有效地转染小鼠下丘核区脑组织并得到表达。(2)水杨酸钠给药后神经元的强度-发放率函数曲线升高。对照组A1、A2各项指标进行比较均无统计学意义。对照组A1、A2和水杨酸钠+pEGFP-NGB组神经元的强度-发放率函数以非单调型(随刺激强度增加时,发放率表现为先降后升呈“V”形或“U”形)为主,分别占74.6%、72.2%和59.3%,水杨酸钠给药组以不规则型强度-发放率函数为主,占47%,与对照组A1、A2和水杨酸钠+pEGFP-NGB组比较,有显著性差异(P<0.01、P<0.01、P<0.05)。(3)水杨酸钠给药后神经元的强度-潜伏期函数曲线降低。对照组A1、A2各项指标进行比较均无统计学意义。水杨酸钠给药组以非单调型强度-潜伏期率函数为主,与对照组A1、A2和水杨酸钠+pEGFP-NGB组比较,有显著性差异(P<0.01、P<0.05)。(4)A1和A2对照组听反应神经元的调谐曲线,Q-10dB值均大于5.00,其调谐曲线为狭窄型。记录水杨酸钠给药组72个听神经元的调谐曲线,有53个神经元的Q-10dB值小于5.00,Q-10dB值最小为2.12,其调谐曲线为宽阔型;其余19个神经元的Q-10dB值大于5.00,属于狭窄型调谐曲线。水杨酸钠+pEGFP-NGB组67个听神经元的调谐曲线,有12个神经元的Q-10dB值小于5.00,Q-10dB值最小为2.87,其调谐曲线为宽阔型,其它的神经元的值大于5.00。它们的调谐曲线均属狭窄型。以上结果提示外源性NGB基因在水杨酸钠给药后小鼠下丘核区局部高表达,提示GeneJamer转染试剂介导NGB体内转基因治疗水杨酸钠引起的下丘核区的损伤的方法是可行的。实验小鼠转染NGB基因后可逆转因水杨酸钠给药引起的强度-发放率函数曲线升高以及强度-潜伏期函数曲线降低,并可逆转水杨酸钠引起的部分听神经元对声刺激强度的编码类型。     

L-type voltage-dependent calcium channels facilitate acetylation of histone H3 through PKCγ phosphorylation in mice with methamphetamine-induced place preference

The present study investigated regulation of histone acetylation by L-type voltage-dependent calcium channels (VDCCs), one of the machineries to provide Ca(2+) signals. Acetylation of histone through the phosphorylation of protein kinase Cγ (PKCγ) in the development of methamphetamine (METH)-induced place preference was demonstrated in the limbic forebrain predominantly but also in the nucleus accumbens of α1C subunit knockout mice. Chronic administration of METH produced a significant place preference in mice, which was dose-dependently inhibited by both chelerythrine (a PKC inhibitor) and nifedipine (an L-type VDCC blocker). Protein levels of acetylated histone H3 and p-PKCγ significantly increased in the limbic forebrain of mice showing METH-induced place preference, and it was also significantly attenuated by pre-treatment with chelerythrine or nifedipine. METH-induced place preference was also significantly attenuated by deletion of half the α1C gene, which is one of the subunits forming Ca(2+) channels. Furthermore, increased acetylation of histone H3 was found in specific gene-promoter regions related to synaptic plasticity, such as Nrxn, Syp, Dlg4, Gria1, Grin2a, Grin2b, Camk2a, Creb, and cyclin-dependent kinase 5, in wild-type mice showing METH-induced place preference, while such enhancement of multiple synaptic plasticity genes was significantly attenuated by a deletion of half the α1C gene. These findings suggest that L-type VDCCs play an important role in the development of METH-induced place preference by facilitating acetylation of histone H3 in association with enhanced expression of synaptic plasticity genes via PKCγ phosphorylation following an increase in the intracellular Ca(2+) concentration.     

Arginine vasopressin in the caudate nucleus plays an antinociceptive role in the rat

Our previous work has shown that arginine vasopressin (AVP) regulates antinociception through brain nuclei rather than the spinal cord and peripheral organs. The present study investigated the nociceptive effect of AVP in the caudate nucleus (CdN) of the rat. Microinjection of AVP into the CdN increased pain threshold in a dose-dependent manner, while local administration of AVP-receptor antagonist-d(CH(2))(5)Tyr(Et)DAVP decreased pain threshold. Pain stimulation elevated AVP concentration in CdN perfuse liquid. CdN pretreatment with AVP-receptor antagonist completely reversed AVP's effect on pain threshold in the CdN. The data suggest that AVP in the CdN is involved in antinociception.