前列腺基质细胞的原代培养方法

目的:建立一种操作简单、成功率高、重复性好的前列腺增生组织原代基质细胞(PSC)培养方法。方法:采用胶原酶消化法、组织块贴壁法和胰酶消化组织块贴壁法,从70岁及以上男性的良性前列腺增生组织中分离培养PSC,通过显微镜观察比较PSC的数量、形态、培养周期,用免疫荧光染色法鉴定PSC的纯度。结果:胶原酶消化法得到的贴壁细胞少,细胞体积较小且形态无法铺展,增殖能力较弱;组织块贴壁法培养72h后细胞会从组织边缘缓慢爬出,生长周期长;胰酶消化组织块贴壁法,细胞培养7d后基本融合,折光性强,细胞多呈长梭形,通过免疫荧光染色鉴定,基质细胞纯度在95%以上。结论:利用胰酶消化组织块贴壁法建立了一种易行、高效且重复性好的前列腺增生组织基质细胞培养方法。     

雪貂脂肪间充质干细胞的分离、分化与鉴定

目的分离、多向诱导分化并进行鉴定雪貂脂肪间充质干细胞。方法无菌采取雪貂腹部皮下脂肪,机械剪碎为0.5 mm~2,采用含0.075%的胶原酶I消化,应用高糖-DMEM培养基(添加4 ng/mLβ-FGF)培养,观察细胞形态特征,流式细胞术分析细胞表面抗原标志的表达,并检测其体外成脂、成骨、成软骨分化能力,还进一步尝试了诱导神经分化。结果运用组织块贴壁法可以从新鲜脂肪组织中分离到贴壁生长的类似成纤维细胞样细胞,流式分析结果显示高表达CD29(53.1%),CD90(99.6%),CD105(93.7%),低表达CD11b(36.3%),CD45(28.3%)。这些细胞在体外经诱导可以分化成为脂肪、骨、软骨和神经元。结论从雪貂脂肪组织中可以分离得到脂肪间充质干细胞,这些细胞具有多向分化能力。     

小鼠血管壁CD34+干细胞的分离、培养及鉴定

血管壁干细胞(vascular wall-resident stem cells, VW-SCs)在维持血管正常功能以及损伤血管的修复过程中发挥着关键性的作用,对其功能特性的研究将有助于干细胞的调控及应用。本文旨在建立稳定的VW-SCs分离、培养及分选鉴定技术,为进一步深入研究VW-SCs在生理和疾病状态下的增殖、迁移和分化等机制提供丰富、可靠的细胞来源。首先利用组织块贴壁法获取小鼠主动脉外膜以及肠系膜动脉血管壁细胞,传代培养到细胞数量至少达到1×10⁷后经磁珠分选和流式细胞术鉴定CD34⁺的VW-SCs;其次采用细胞免疫荧光染色检测干细胞标志物(CD34、Flk-1、c-kit、Sca-1)以及平滑肌标志物(SM22、SM MHC)、内皮标志物(CD31)和细胞核内分裂增殖相关蛋白(Ki-67);血管壁CD34+干细胞的定向分化采用EBM-2内皮分化诱导培养基和FM-2成纤维细胞培养基分别培养7天和3天;利用细胞免疫荧光染色和q-PCR检测内皮细胞标志物(CD31, VWF)和成纤维细胞标志物(Vimentin, PDGFRα)的表达。此外...     

鼻黏膜间充质干细胞的培养、鉴定及诱导分化

目的建立Sprague Dawley(SD)大鼠鼻黏膜间充质干细胞(NM-MSCs)的获取、分离、培养方法,初步了解其生物学特性。方法通过SD大鼠鼻黏膜贴壁法体外分离、培养NM-MSCs,光镜下细胞形态学观察,用免疫荧光技术检测间充质干细胞和神经干细胞标记物,用流式细胞术检测细胞表面标记物,再诱导其向骨组织及脂肪组织方向分化,最后对其进行细胞周期检测及分析。结果分离培养的NM-MSCs光镜下以梭形与多角形细胞为主,呈放射状排列,且生长旺盛;免疫荧光染色显示NM-MSCs同时表达STRO-1和Nestin;第4代NM-MSCs不表达CD19、CD31、CD34、CD45及HLADR细胞表面标记物,但表达CD90、CD105基质细胞标记物;NM-MSCs经成骨、成脂诱导后,茜素红染色和油红O染色均呈阳性;细胞周期分析显示NM-MSCs符合干细胞生长的特性。结论 SD大鼠NM-MSCs组织块贴壁法获取容易,操作简单,且可大量扩增用于细胞实验研究,经体外诱导后具有多向分化潜能,具有间充质干细胞的一般生物学特性。SD大鼠鼻黏膜组织块贴壁法为组织细胞工程研究提供了充足的种子细胞来源。     

成年小鼠原代皮肤成纤维细胞的分离培养及其生物学特性

目的:建立一种周期短、成本低的成年小鼠原代皮肤成纤维细胞分离培养方法,并探索其生物学特性。方法:取8~12周龄BALB/c小鼠背部、尾尖、耳部皮肤,配制2种血清的细胞培养液,采用组织块贴壁法、酶消化法、酶消化组织块贴壁法进行原代皮肤成纤维细胞的培养,通过显微镜观察比较原代细胞的数量、形态、培养周期及纯度;通过免疫荧光、CCK-8、UVB辐照、流式细胞术进行生物学特性鉴别。结果:背部皮肤组织块贴壁使用Gibco胎牛血清培养7 d无细胞游出,CLARK特级胎牛血清细胞游出较多。背部皮肤经酶消化法得到细胞贴壁少;经组织块贴壁法细胞生长慢,培养周期长;酶消化组织块贴壁法细胞游出速度快、数量多、呈长梭形。尾尖取材量少,得到细胞少;耳部皮肤取材方便,但细胞纯度低。CCK8增殖曲线呈S型;相较于对照组,UVB辐照后细胞凋亡率增高17%。结论:CLARK特级胎牛血清、背部皮肤取材、酶消化组织块贴壁法是培养成年小鼠原代皮肤成纤维细胞最优的方案,可增加细胞得量、缩短培养周期,降低成本。     

树鼩原代小胶质细胞的分离培养、纯化与鉴定

本文旨在建立树鼩(Tupaia belangeri)小胶质细胞原代培养及纯化的方法,为利用新型实验动物树鼩进行相关研究工作提供实验材料。将新生树鼩大脑皮质机械分离,皮质组织块用胰蛋白酶消化后制成细胞悬液;培养9～10 d后,分别采用直立手拍法、温和胰酶消化法以及恒温振荡法分离纯化树鼩小胶质细胞,通过差速贴壁进一步纯化。荧光显微镜下,利用小胶质细胞的特异性标记物CD11b抗体进行鉴定。结果显示,小胶质细胞分离培养第3天时呈静息状态,表现为梭形、杆状、分支状等不规则形态。细胞免疫荧光CD11b呈阳性。不同纯化方法细胞免疫荧光并计数显示,直立手拍法所获得的细胞产量明显高于恒温振荡法(P 0.05),细胞阳性率( 96%)明显高于温和胰酶消化法( 90%,P 0.05)。直立手拍法可获得产量及纯度高的树鼩原代小胶质细胞。     

基于c-Kit~+显微形态标记及流式分选分离心脏Telocytes

目的:建立基于显微形态标记及流式分选分离大鼠心脏Telocytes(CTs)的方法。方法:采用抗体c-Kit免疫磁珠法获得原代心脏Telocytes,显微注射Di I标记具"Telopode"典型形态的细胞,使用流式分离及回收单个Di I+细胞;使用免疫荧光技术和RT-PCR方法对经回收的单个细胞来源的细胞进行表型鉴定。结果:显微注射Di I能较好地标记具Telocytes典型形态的细胞,结合流式分选及单细胞回收,能有效回收经标记的Di I+细胞,经回收的Di I标记阳性Telocytes的贴壁率为14.9%,增殖率为5.6%,呈克隆样生长率为2.4%,该呈克隆样生长的细胞能通过消化传代。免疫荧光染色证明,该回收Telocytes表达其相对特异性表面标记物c-Kit和CD34,RT-PCR的结果也证明:经回收Telocytes表达其相对特异基因c-Kit、CD34、Vimentin和PDGFR-β。结论:研究所建立的方法能有效分离及单细胞回收高纯度的心脏Telocytes,经回收的心脏Telocytes具有增殖及传代能力,且能维持其特异表型。     

心肌干细胞培养和纯化的实验方法研究

目的:探讨心肌干细胞(CSC)体外分离、培养和纯化方法,以建立重复性好、稳定性高的CSC培养的方法.方法:用不同的消化方法来分C57BL小鼠心脏组织中的细胞,将获得的细胞置于自行拟定的心肌干细胞培养液中培养.以c-kit、Sca-l和Sca-l/c-kit作为干细胞标记,用免疫磁珠法纯化细胞.用流式细胞仪、免疫荧光显微镜、激光共聚焦显微镜检测分选后所得细胞表面标记表达,并对比观察分选前后细胞生长情况,进一步了解CSC的生物学特性.结果:①从小鼠心肌组织中分离培养所得的细胞中,含有一种类似于文献报道中所提及CSC形态的细胞.②用免疫磁珠法可以分选出高纯度的CSC,实验中分选出CSC分别具有c-kit+-CD34-lin-、Sea-I+-CD34low-Lin-、Sea-1+c--kit+-CD34low-Lin-的表型.③分选后的干细胞经过1~2d的滞留期后,贴壁生长的细胞形态相对均一;而传代后细胞贴壁迅速,细胞形态多样.结论:从C57BL小鼠心脏中可以分离、培养获得CSC,并可用免疫磁珠法得以纯化.     

毛囊干细胞体外诱导成血管内皮细胞的实验研究

目的建立简单、可靠的毛囊干细胞体外定向分化为血管内皮细胞的方法。方法无菌条件下切取1周龄SD大鼠触须部皮肤,Dispase酶和Ⅳ型胶原酶混合液消化,显微镜下分离毛囊隆突部,改良的组织块贴壁法培养rHFSCs,差速贴壁法纯化,流式细胞仪检测及细胞免疫荧光染色联合鉴定。用含10 ng/ml和20 ng/ml VEGF165作为主要诱导因子将细胞分为两种浓度组别,不传代的情况下分别在1、2、3周内观察诱导后细胞形态;通过流式细胞和细胞免疫荧光染色检测分化效率;选取最佳诱导因子浓度和工作时间,对诱导后的细胞进行体外官腔形成实验,并在透射电镜下观察W-P小体。流式细胞术检测分化效率实验数据的比较采用单因素方差分析及独立t检验。结果分离、培养、纯化的rHFSCs克隆能力好,活力强,生长曲线呈S型。流式细胞及免疫荧光染色联合检测β1(98.9%)、α6整合素(97.9%),CK15(68.1%)、P63(98.5%)呈高表达,阴性表达CD31(13.6%)、VE-cadherin(17.9%)。在诱导液的作用下,两组细胞从1周到3周后,均由扁平铺路石样改变到完全被长梭形样细胞占据。流式细胞及免疫荧光染色检测CD31和VE-cadherin显示,CD31在诱导1周时表达最强,两种诱导因子比较无差异[(65.27±0.57%)vs(66.13±0.60)%,t=1.812,P=0.145]。而VE-cadherin在诱导1周时表达也最强,10ng/ml要优于20ng/ml VEGF165的诱导作用[(95.57±0.85)%vs(78.10±1.25)%,t=19.977,P=0.001]。10 ng/ml VEGF165诱导1周后的细胞体外管腔形成实验阳性,在透射电镜下可以观察到内皮细胞特有结构W-P小体。结论毛囊干细胞体外在10ng/ml VEGF165的诱导下,1周后可成功得到性能良好的血管内皮细胞。可为组织工程血管、细胞移植治疗缺血性疾病等提供理想的种子细胞。     

青年和老年小鼠棕色脂肪来源间充质干细胞生物学特性比较

旨在探讨青年和老年小鼠肩胛骨间棕色脂肪来源间充质干细胞(Brown adipose-derived mesenchymal stem cells,BADSCs)生物学特性的比较。用细胞流式检测第3代BADSCs表面抗原表达,MTT法检测细胞增殖情况,7-ADD-AnnexinV双染色观察细胞凋亡情况和β-半乳糖苷酶染色法检测细胞衰老情况,比较两组细胞的成骨和成脂分化差异。结果显示,第3代BADSCs表面抗原CD29、CD105和CD44的表达均为阳性、CD73为低表达。MTT法显示老年小鼠的BADSCs增殖能力与青年小鼠相比没有显著性差异(P0.05)。7-ADD-AnnexinV双染色显示青年小鼠的BADSCs早凋比例4.63±0.87%,老年的BADSCs早凋比例9.88±0.81%,且有显著性差异(P0.05)。β-半乳糖苷酶染色法显示青年组染成蓝色细胞数为2.33±0.3,老年组染成蓝色细胞数为6.66±1.2,有显著性差异(P0.05)。青年组成骨分化OD值为1.26±0.046,老年组成骨分化OD值为0.88±0.047,有显著性差异(P0.05)。青年组成脂分化OD值为0.9808±0.066,老年组成脂分化OD值为0.769±0.035,有显著性差异(P0.05)。青年组的BADSCs更倾向于成骨分化,老年组的BADSCs更倾向于成脂分化。老年组小鼠凋亡和衰老细胞比例增加;成骨和成脂分化能力下降。     

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.