L-型钙通道对大鼠胚胎海马神经干细胞增殖和分化的调控

为鉴定大鼠胚胎海马神经干细胞(NSCs)是否表达功能性的L-型钙通道,L-型钙通道是否参与了对大鼠胚胎NSCs增殖和分化调控.分离孕15天Wistar大鼠胚胎海马组织,制成单细胞悬液,利用无血清培养技术,在添加bFGF、EGF、N-2和B27 supplement的DMEM/F12培养液中进行培养.采用细胞免疫荧光法对原代至第5代细胞进行鉴定,均有巢蛋白(nestin)的表达,第3代nestin阳性细胞比例达97%.把培养的细胞诱导分化5天后,这些细胞表现为神经元和星形胶质细胞的形态,且分别呈Ⅲ型β-微管蛋白(Tuj1)阳性和胶质纤维酸性蛋白(GFAP)阳性;细胞免疫印迹结果显示,NSCs表达L-型钙通道的Cav1.2α1C亚单位,而无Cav1.3α1D亚单位的表达;利用全细胞膜片钳技术在NSCs上记录到了L-型钙电流,证明了NSCs所表达的L-型钙通道具有功能.进一步对细胞进行药理学干预,发现L-型钙通道的激活不仅可以促进胚胎NSCs的增殖,而且使增殖的NSCs向神经元分化的比例显著增加.以上结果表明,Wistar大鼠胚胎海马NSCs表达功能性的L-型钙通道;L-型钙通道参与了胚胎NSCs增殖和分化的调控.     

星形胶质细胞存在L-型钙通道的新证据

以纯化培养的小鼠星形胶质细胞(Astrocyte,AS)为实验材料,采用激光共聚焦钙成像和荧光分光光度计技术,探讨钙激动剂Bay k8644和钙拮抗剂nimodipine对星形胶质细胞胞内钙离子浓度的影响。结果显示,Bay k8644在0.0001、0.001和0.01mmol/L浓度下均可显著增加星形胶质细胞的细胞内钙水平,而nimodipine在0.001、0.01和0.1mmol/L浓度下均可显著降低星形胶质细胞胞内钙水平,并阻止KCl引起的细胞内钙升高。上述结果表明星形胶质细胞对L-型钙通道激动剂和拮抗剂的反应与神经元的反应相似,提示星形胶质细胞胞膜上也存在L-型钙通道。     

星形胶质细胞诱导成年神经干细胞的神经发生

在中枢神经系统 ,成年后新神经元发生主要见于两个脑区 ,即室管下区 (ｓｕｂｖｅｎｔｒｉｃｕｌａｒｚｏｎｅ)与海马的颗粒下区 (ｓｕｂｇｒａｎｕｌａｒｚｏｎｅ)。正常情况下 ,除上述脑区外的其它脑区能够产生神经胶质细胞 ,但是不能产生神经元。为了研究神经元和 /或神经胶质细胞对来源于成年的神经干细胞分化的影响 ,Ｓｏｎｇ等分离了成年大鼠海马的神经元和星形胶质细胞 ,将其分别或联合与来自成年的、依赖ＦＧＦ 2的神经干细胞共培养 ,意外地发现神经元促进神经干细胞分化为少突胶质细胞 ,而星形胶质细胞则促进神经干细胞分化为神经…     

从小鼠大脑皮层制备高纯度胶质限制前体细胞

目的:探索一种简单易行的分离、制备高纯度胶质限制前体细胞(Glial restricted precursor,GRP)的方法.方法:利用胶质限制前体细胞与星形胶质细胞分层生长的特点,采用摇培的方法将吸附在星形胶质细胞上的GRP分离下来,再利用单克隆形成实验获得单细胞来源的细胞克隆,接着细胞免疫荧光和定向分化实验鉴定细胞种类.结果:经鉴定所形成的单克隆90％为胶质限制前体细胞,它们能够分化成星形胶质细胞和少突胶质细胞,而没有分化成神经元的潜能.结论:利用细胞分层生长的特点和单克隆形成可以简便的制备高纯度的胶质限制前体细胞.     

壮通饮对体外培养新生大鼠海马神经干细胞增殖分化的影响

探讨壮通饮对体外培养大鼠大脑海马区内源性神经干细胞(neural stem cells,NSCS)分化作用的影响。利用无血清DMEM/F12(含20 ng/mL bFGF和EGF及2%B27)体外培养技术从新生Wistar大鼠大脑海马区中培养NSCS,在NSCS分化过程中添加不同剂量(20、40和80 mg/L)壮通饮进行干预,以免疫荧光及细胞化学方法对NSCS及其分化后的细胞进行鉴定。从大脑海马区分离培养的NSCS能够表达巢蛋白(Nestin),并具有分化为神经元、星形胶质细胞及少突胶质细胞的能力。在同一接种密度条件下,不同剂量壮通饮干预组的Nestin阳性细胞表达数分别为:20 mg/L组:21.5±2.9;40 mg/L组:18.6±2.4;80 mg/L组:16.7±2.2,与对照组(24.8±3.1)比较明显减少(P0.05)。而壮通饮干预组表达神经元核心抗原(NeuN)、胶原纤维酸性蛋白(GFAP)、髓鞘碱性蛋白(MBP)的阳性细胞数较对照组明显增加(P0.05)。通过本实验证实壮通饮能诱导大脑海马区NSCS分化,具有一定的促进神经再生的作用。     

低氧促进大鼠心脏成纤维细胞DNA合成及Ⅰ、Ⅲ胶原mRNA表达

目的:研究低氧(2%氧)对成年Wistar大鼠心脏成纤维细胞DNA合成及Ⅰ、Ⅲ型胶原前a肽链表达的影响.方法:分离培养成年Wistar大鼠心脏成纤维细胞,采用液体闪烁计数方法检测心脏成纤维细胞的DNA合成速率,采用原位杂交技术检测Ⅰ、Ⅲ型胶原前α肽链mRNA的表达.结果:成年Wistar大鼠心脏成纤维细胞在低氧第6h、12 h时3H-TdR掺入量较常氧组显著增加,分别增加34%(P＜0.05)和36%(P＜0.01);低氧第4 h、8 h、12 h Ⅰ型胶原前α肽链mRNA表达显著高于常氧培养的细胞;低氧第2 h,Ⅲ型胶原前α肽链mRNA表达显著高于常氧培养的细胞.结论:低氧能够直接促进体外培养的成年Wistar大鼠心脏成纤维细胞DNA合成和Ⅰ、Ⅲ型胶原前α肽链表达,提示低氧对心脏成纤维细胞生长和胶原表达的直接调节可能是低氧性心肌纤维化的重要机制.     

大鼠少突胶质前体细胞(OPCs)纯化培养及糖氧剥夺(OGD)模型的建立

目的建立大鼠脑少突胶质前体细胞(oligodendrocyte precursor cells,OPCs)分离纯化培养及糖氧剥夺(oxygen glucose deprivation,OGD)模型。方法出生3d内的SD大鼠乳鼠取脑,经胰蛋白酶消化法培养混合胶质细胞,混合培养10d后,震摇及差速贴壁法分离纯化OPCs,纯化培养3d后鉴定、诱导分化OPCs为少突胶质细胞(oligodendrocyte,OL)及进一步OGD干预。免疫荧光法鉴定OPCs纯度及分化为OL的能力;MTT法检测OGD(37℃,1%O2,5%CO2)干预0.5h、1h、2h及4h时细胞活力改变,Edu染色检测细胞增殖情况。结果免疫荧光显示纯化培养的OPCs 95%以上表达NG2+A2B5,且可分化为MBP阳性的OL。OGD 2h时,MTT显示细胞活力明显下降,Ed U染色阳性率明显降低。结论震摇及差速贴壁法可获得高纯度的OPCs,且细胞具有分化为OL的能力。2h可作为OPCs OGD模型缺血缺氧损伤合适时间。     

一种手动解离、免疫磁珠分选加TIC培养液改良的小鼠原代海马小胶质细胞培养法

本文旨在研究手动解离、免疫磁珠分选加TIC培养液改良原代小胶质细胞的培养方法,获得高活性、高纯度且与在体状态更接近的小鼠海马原代小胶质细胞,以用于小胶质细胞的相关研究。选取2～4周龄SPF级C57BL/6J小鼠,PBS灌注后取海马组织剪碎,使用成年鼠脑组织解离试剂盒手动解离海马组织,获得单细胞悬液,再经去碎片试剂去除髓鞘等组织碎片。在4°C孵育CD11b免疫磁珠15 min后,细胞悬液经免疫磁珠细胞分选(magnetic activated cell sorting, MACS)两次过柱提高小胶质细胞纯度。将分选后细胞离心重悬,种在多聚赖氨酸包被后的24孔培养板中,用完全培养基或TIC培养液(以TGF-β、IL-34和胆固醇为主要营养成分的无血清培养基)培养4 d后进行其他检测。结果显示:(1)使用成年鼠脑组织解离试剂盒手动解离可得到含有(56.03±2.10)%活细胞的单细胞悬液;(2)与免疫抗体包被筛选法(immunopanning)相比,经过MACS两步分选后,可得到更多且纯度高达(86.20±0.68)%的小胶质细胞;(3)使用TIC培养液培养4 d后,可获得分枝状、形态类似静息状态的原代小胶质细胞;(4) q RT-PCR检测显示,与完全培养基培养相比,TIC培养液培养的小胶质细胞在4 d和7 d后TNF-α、IL-1β及CCL2 m RNA表达水平更接近急性分离后的小胶质细胞。上述结果提示,采用手动解离海马组织,经MACS两步分选及TIC培养后,可以获得高纯度、高活性且接近在体静息状态的小胶质细胞。     

成年大鼠神经元和星形胶质细胞细胞周期蛋白表达的差异研究

目的比较研究成年大鼠细胞周期蛋白在神经元和星形胶质细胞的表达差异。方法应用免疫荧光和激光扫描共聚焦显微镜观察成年大鼠生理状态下大脑皮层或海马CA1区神经元和星形胶质细胞细胞周期素D1、E、A、B1、(CyclinD1、E、A、B1)的表达。结果成年大鼠海马CA1区和大脑皮层的神经元有Cyclin D1、E、A和B1的表达,细胞核和细胞浆均有表达,以胞核为主;星形胶质细胞也有上述细胞周期蛋白的表达但细胞数目较少,并且表达这些指标的星形胶质细胞多聚集在海马CA1区。结论成年大鼠大脑皮层和海马区的神经元和星形胶质细胞均表达细胞周期蛋白,而其在神经元的表达较星形胶质细胞更为普遍。     

神经干细胞向少突胶质前体细胞的定向分化诱导

本研究采用神经胶质瘤细胞株(B104 neuroblatoma cells,B104 cells)培养上清(B104CM)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF),将冷冻复苏的大鼠胚胎脊髓神经干细胞(neural stem cells,NSCs)定向诱导为少突胶质前体细胞(oligodendrocyte precusor cells,OPCs)。形态学和免疫组化的结果显示,诱导后95％以上的细胞具有双极或多极突起的典型OPCs形态,并表达A285和血小板源生长因子受体-α(platelet derived growth factor receptor-α,PDGFR-α等0PCs标志,所有PDGFR-α阳性的OPCs均不表达β-Tublin Ⅲ,其中仅少量细胞表达胶质原纤维酸性蛋白(glia fibrillary acidic protein,GFAP)。在B104CM和bFGF共存的培养条件下,悬浮培养的OPCs可大量增殖形成少突胶质细胞球,该细胞球可通过传代继续扩增,且扩增的OPCs仍能维持其特有的形态和自我增殖的特性。撤去bFGF和B104CM后,OPCs能进一步分化为成熟的少突胶质细胞(oligodendrocytes,OLs)或Ⅱ型星形胶质细胞。实验表明,诱导NSCs产生的OPCs在形态、增殖以及分化格局等方面均与已报道的存在于胚胎脑区的O-2A前体细胞相类似。该培养系统可为实验性细胞移植的研究提供丰富的细胞来源。     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.