大鼠脑微血管内皮细胞的分离与原代培养

为了建立大鼠脑微血管内皮细胞体外培养模型,探索纯度较高的大鼠脑微血管内皮细胞分离和原代培养的方法并进行形态学观察。采用2～3周龄的SD大鼠,解剖得到大脑皮质,两次酶消化及牛血清白蛋白或葡聚糖和Percoll梯度离心获得较纯的脑微血管段后,接种于涂布基质的培养皿进行原代培养;培养的细胞采用相差显微镜形态学观察、透射电镜观察及Ⅷ因子相关抗原免疫组化检测鉴定。结果发现,培养12h即可见细胞从贴壁的脑微血管段周围长出,细胞呈短梭形,区域性单层生长,5～7天内皮细胞融合,内皮细胞纯度达90％以上;内皮细胞的贴壁和生长有赖于所涂布的基质,纤连蛋白／Ⅳ型胶原优于鼠尾胶和明胶;Ⅷ因子相关抗原免疫组化检测内皮细胞表达阳性,透射电镜观察可见相邻内皮细胞间存在紧密连接结构。提示该方法能成功进行纯度较高的大鼠脑微血管内皮细胞原代培养,可用于脑微血管内皮的生理、生化及药理学研究,亦可用于构建大鼠血脑屏障模型。     

猪骨髓间充质干细胞的分离培养与鉴定

目的:建立猪骨髓间充质干细胞(pMSCs)体外分离培养、纯化和鉴定的方法,为下一步实验研究奠定基础.方法:采用密度梯度离心法获得骨髓单核细胞,接种后形成单层贴壁的成纤维样细胞.免疫荧光及PCR检测细胞表面标志及多能性基因的表达,并鉴定分离细胞的多向诱导分化潜能.结果:体外培养的原代细胞10天达到融合,传代后仍具有成纤维样的形态;免疫荧光结果见波形蛋白(Vimention)和Oct4标记阳性,CD45阴性;PCR分子检测见多能性基因OCT-4,nanog的表达;细胞具有分化为成骨细胞和成脂细胞的能力.结论:采用密度梯度离心法获得的pMSCs体外增殖能力强,纯度高,具有间充质干细胞的特性,pMSCs分离培养体系的成功建立为下一步实验研究奠定基础.     

星形胶质细胞可以诱导内皮细胞系紧密连接的形成

为探索星形胶质细胞在血脑屏障内皮细胞紧密连接形成中的重要意义,通过内皮细胞系ECV304与星形胶质细胞体外接触共培养的方法,采用电镜及内皮细胞紧密连接的银染观察星形胶质细胞对内皮细胞系紧密连接的诱导作用。运用Millipore-ERS系统检测紧密连接的功能状况。结果发现,星形胶质细胞可以诱导内皮细胞系形成广泛而连续的紧密连接并产生较高的跨内皮阻抗(transendothelial electrical resistance,TER),于第10d可达321．3Ωcm^2。提示,星形胶质细胞可以诱导ECV304细胞产生紧密连接。同时,ECV304细胞与星形胶质细胞的体外共培养可以作为研究血脑屏障紧密连接结构与功能的一种可靠而简便的体外实验方法。     

树鼩视网膜微血管内皮细胞永生化细胞株的建立及鉴定

目的 建立树鼩视网膜来源的微血管内皮细胞的体外分离培养技术以及永生化细胞株，为体外利用树鼩视网膜微血管内皮细胞开展相关研究提供新的实验材料。方法 利用Ⅱ型胶原酶、分散酶和DNaseⅠ酶消化法分离培养出原代视网膜微血管内皮细胞，利用差速消化法纯化内皮细胞，然后利用携带SV40T基因的慢病毒转染细胞，再挑取单克隆后进行传代培养。对传至50代以上的细胞进行形态学观察、免疫荧光鉴定以及核型鉴定。结果 采用混合酶消化法能够分离获得微血管内皮细胞，纯化后的细胞呈不规则多角形和梭形。经慢病毒转染后的细胞传代培养后细胞形态一致，到第50代时细胞形态结构仍较好。细胞免疫荧光结果显示标志性蛋白VWF、CD34、Claudin1、ZO-1和永生化SV40T表达阳性。生长曲线结果显示：细胞生长旺盛，第2～4天时处于对数生长期，第4天进入平台期。细胞核型结果显示：染色体数与树鼩染色体相同，即所获取的细胞为永生化的树鼩视网膜微血管内皮细胞。结论 成功建立的树鼩视网膜微血管内皮细胞永生化细胞株具有较好的形态结构与功能，为视网膜病变及眼科相关疾病的研究提供了新的实验材料。     

小鼠小脑颗粒神经元的原代培养与鉴定

本研究通过体外分离培养及鉴定原代小鼠小脑颗粒神经元(cerebellar granule neurons,CGNs),为神经科学研究提供原代神经元细胞模型。取生后第7天的ICR乳鼠小脑,在解剖体视镜下剥离脑膜及血管,经刀片切碎、0.25%胰酶消化、移液器吹打、70μm尼龙滤网制备单细胞悬液,差速贴壁后接种于新鲜配置的培养基。倒置相差显微镜观察不同时间CGNs的形态变化。采用神经元的标记蛋白微管相关蛋白-2(microtubule associated protein 2,MAP2),星形胶质细胞的标记蛋白胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP),小胶质细胞的标记蛋白(type 3 complement receptor,CR3/CD11b)进行免疫荧光检测培养的原代CGNs纯度。原代培养CGNs接种20 min后即贴壁良好,培养24 h后细胞伸出突起,培养第7天神经元成熟,形成丰富的轴突,树突和胞间突触连接。经免疫荧光鉴定,CGNs纯度可达95%以上。成功体外分离培养出高纯度的原代CGNs,可应用于CGNs的体外研究。     

牦牛输卵管上皮细胞分离培养和纯化鉴定

为建立牦牛输卵管上皮细胞原代培养及纯化方法,通过选取牦牛输卵管,运用机械刮取法和0.25%胰蛋白酶消化两种方法分离上皮细胞进行体外培养。对不同分离方法的培养效果比较,培养细胞进行形态学观察与传代培养、MTT比色检测细胞活力并制定生长曲线,原代及传代上皮细胞的免疫组织化学鉴定,冷冻解冻后经台盼蓝排斥试验检测活细胞数。结果表明该试验分离出的原代细胞,纯化后传代培养,经鉴定为牦牛输卵管上皮细胞,培养的细胞生长状况良好,建立了一套牦牛输卵管上皮细胞分离培养及纯化鉴定的方法。     

大鼠主动脉内皮细胞的分离培养和鉴定

目的:探索大鼠主动脉原代内皮细胞体外培养方法,为体外研究提供细胞模型。方法:分离大鼠主动脉,直接贴壁于培养皿中,荧光倒置显微镜观察细胞形态,免疫组化Ⅷ因子相关抗原染色鉴定细胞。结果:约24小时组织块边缘有游离的新生细胞长出,7天即融合成片。消化传代后细胞呈短梭形或三角形,单层生长,铺路石状,Ⅷ因子表达阳性,呈指数增殖。冻存后复苏细胞活性均超过90%。结论:用贴壁法成功建立了大鼠血管内皮细胞体外培养方法,冻存细胞存活率高,为体外研究提供了稳定的模型。     

微流控芯片对乳腺癌细胞MDA-MB-231的捕获及再培养研究

目的:利用实验室构建的微流控芯片对乳腺癌细胞(MDA-MB-231)进行捕获,提高捕获率并保证细胞活性,实现再培养。抗肿瘤药物阿霉素处理正常培养和再培养的细胞,分析细胞内的基因表达变化。方法:对微流控芯片进行基底修饰,利用MUC1抗原与抗体特异性结合捕获肿瘤细胞,优化捕获条件提高捕获率。对微流控芯片捕获的细胞进行分离、收集和再培养。用1μmol/L阿霉素对正常培养和再培养的细胞分别孵育24h,然后提取RNA并逆转录合成c DNA。选择乳腺癌细胞中高表达及与肿瘤转移相关的基因FN1、ITGA6和LAMB3设计引物,以c DNA为模板分别进行RT-PCR扩增,对琼脂糖凝胶电泳结果进行灰度分析。结果:经MUC1抗体修饰的微流控芯片能有效地捕获肿瘤细胞,捕获率达80%±3%,释放率约98%,细胞释放后存活率高实现再培养。阿霉素对正常培养和再培养的乳腺癌细胞中FN1、ITGA6和LAMB3的基因表达均有抑制作用。结论:MUC1抗体修饰的微流控芯片能有效捕获乳腺癌细胞并实现再培养,捕获前后细胞内基因表达无显著差异,均能产生药物敏感性。     

人轮状病毒G1P[8]型感染树鼩原代小肠上皮细胞模型的建立

目的探究树鼩原代小肠上皮细胞的增殖特性,建立人轮状G1P[8]型病毒体外感染树鼩原代小肠上皮的细胞模型。方法采用胶原酶XI和中性蛋白酶I联合消化法获取树鼩原代小肠上皮细胞,经纯化和鉴定,用人轮状G1P[8]型病毒感染细胞,测定培养上清的病毒滴度和载量,并用Western blot和间接免疫荧光检测法检测人轮状病毒G1P[8]型VP6蛋白的表达情况,评价人轮状G1P[8]型病毒体外对树鼩原代小肠上皮细胞的感染性。结果分离的树鼩原代小肠上皮细胞经传代培养纯化,获得纯度达90%树鼩原代小肠上皮细胞。对树鼩原代小肠上皮细胞、原代肾细胞、HCT116细胞和MA104细胞进行轮状病毒易感性比较,确定树鼩原代小肠上皮细胞可以被人轮状病毒G1P[8]感染,培养72 h时病毒滴度可达到2.0×105TCID_(50)/m L。经Western blot和间接免疫荧光发现在人轮状G1P[8]型病毒感染树鼩原代小肠上皮细胞1~5 d均能检测到人轮状病毒VP6蛋白的表达和分布。结论确立了树鼩原代小肠上皮细胞的分离、纯化与培养方法,并建立了人轮状G1P[8]型病毒感染树鼩原代小肠上皮细胞的体外模型。     

树鼩原代皮肤成纤维细胞的分离培养技术

建立稳定的树鼩(Tupaiabelangeri)皮肤成纤维细胞的体外培养体系,可为有关此类细胞的实验和疾病树鼩细胞模型提供技术支持。取树鼩大腿内侧皮肤用组织块贴壁法和胶原酶Ⅰ消化法分离皮肤细胞,胰蛋白酶差别消化法纯化细胞;用MEM(10%FBS)完全培养基和含低血清生长添加物(LSGS)的培养基培养细胞;免疫荧光和蛋白印迹法鉴定细胞,并测定细胞的生长、冻存和复苏特性。经树鼩皮肤细胞分离效果比较,胶原酶消化法比组织块贴壁法更适合用于树鼩原代皮肤细胞分离;对分离及冻存复苏后细胞生长状况观察比较发现,添加了LSGS的MEM培养基更利于细胞存活、生长;细胞形态观察、免疫荧光和蛋白印迹检测鉴定所分离的细胞为树鼩皮肤成纤维细胞。成功建立了树鼩原代皮肤细胞的分离、纯化方法,并优化了该细胞的培养条件。     

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.