MFN1介导的线粒体融合在心肌细胞凋亡中的作用研究

目的:探讨线粒体融合关键蛋白MFN1介导的线粒体融合在调控心肌细胞凋亡中的作用。方法:通过si RNA降低体外培养H9C2心肌细胞中MFN1的表达后,采用Western blot检测线粒体细胞色素c(Cyto c)释放及其下游凋亡效应分子Caspase9与Caspase3活性,流式细胞术检测细胞内活性氧(ROS)的产生情况,流式细胞术检测细胞凋亡的情况。结果:干扰MFN1可显著促进H9C2心肌细胞内细胞色素c由线粒体释放至胞浆,促进Caspase9与Caspase3的激活,增加细胞内活性氧ROS产生并提高细胞凋亡率(均P0.05)。结论:MFN1介导的线粒体融合可保护心肌细胞凋亡,其机制可能与抑制ROS产生与细胞色素C释放有关。     

羟基乙酰化姜黄素诱导的声动力治疗对巨噬细胞凋亡与坏死的影响

目的:探讨羟基乙酰化姜黄素(HAC)介导的声动力治疗对人急性单核细胞白血病细胞核(THP-1)源巨噬细胞凋亡的影响。方法:体外诱导THP-1源巨噬细胞,通过单纯药物和单纯超声对细胞存活率的影响优化出声动力治疗的最适条件。将细胞分为4组(1×105cells/ml):对照组、单纯超声组、单纯HAC组和声动力治疗组。应用声动力体外治疗巨噬细胞,药物HAC终浓度为5μg/ml,超声强度为0.5 W/cm2,超声时间为5 min。不同治疗方法后用无血清培养液培养6 h,用CCK-8法测定细胞存活率的变化,用Annexin V/PI法测定细胞凋亡、坏死变化。结果:CCK-8法测得声动力治疗后,细胞存活率明显下降(P0.01),Annexin V/PI法测得声动力治疗后,细胞凋亡率和坏死率均升高,凋亡/坏死比升高(P0.01)。结论:羟基乙酰化姜黄素介导的声动力治疗对THP-1巨噬细胞的凋亡有明显的诱导效应。     

华蟾素对MDA - MB -231细胞线粒体膜电位及活性氧的影响

目的:研究华蟾素诱导乳腺癌MDA - MB - 231细胞凋亡过程中,细胞内活性氧(ROS)及线粒体膜电位(△Ψm)的变化,探讨华蟾素对乳腺癌细胞的作用机制.方法:用不同浓度的华蟾素作用于MDA - MB - 231细胞24h后,分别用荧光探针罗丹明123和荧光探针DCFH-DA进行荧光染色,用流式细胞仪检测细胞内线粒体膜电位和活性氧的变化.结果:不同浓度的华蟾素作用于MDA - MB - 231细胞后,随着药物浓度的增加(0、12.5、25、37.5、50μg/ml),细胞内的ROS水平显著升高,荧光强度从3 609±24上升为6 263±35;同时,线粒体膜电位(△Ψm)显著下降,荧光强度从242±6降低到173±4.结论:华蟾素作用细胞后,使得细胞内活性氧水平显著升高,同时,线粒体膜电位显著下降,推测华蟾素对MDA - MB - 231细胞通过线粒体途径诱发细胞凋亡.     

姜黄素对H_2O_2诱导的HT29细胞氧化损伤的保护作用及机制研究

研究姜黄素对H_2O_2诱导HT29细胞氧化应激的保护作用及其可能的分子机制。分别采用低、中、高浓度姜黄素处理H_2O_2诱导的HT29细胞氧化损伤模型,并设置H_2O_2模型组和正常对照组;MTT法确定H_2O_2最佳损伤浓度和时间及姜黄素(2.5、5、10μmol/L)对H_2O_2诱导的HT29细胞活性的影响;Annexin V/PI双标记流式细胞术检测细胞凋亡情况;PI染色流式细胞术检测细胞周期变化;DCFH-DA荧光探针检测细胞内活性氧(ROS);JC-1染色检测细胞线粒体膜电位;比色法测定乳酸脱氢酶(LDH)释放量,以及丙二醛(MDA)、超氧化物歧化酶(SOD)、caspase-3和caspase-9的水平。结果显示姜黄素组(2.5、5、10μmol/L)明显提高H_2O_2诱导的HT29细胞的存活率(P0.05,P0.01);与H_2O_2模型组相比,姜黄素组细胞凋亡率降低(P0.01),增殖指数增高(P0.05,P0.01),LDH释放量和细胞内ROS降低(P0.05,P0.01),线粒体膜电位上升(P0.01),SOD活力增加(P0.01),MDA降低(P0.01),caspase-3和caspase-9活性增强(P0.01),并呈剂量-效应关系。结果表明姜黄素在一定剂量范围内对H_2O_2诱导的HT29细胞氧化损伤具有较好的保护作用,该作用可能与清除ROS,减轻DNA氧化损伤,抑制线粒体通路介导的细胞凋亡有关。     

致凋亡的声动力疗法诱导巨噬细胞线粒体钙升高的机制研究

目的:研究致凋亡的声动力疗法诱导巨噬细胞线粒体钙升高的机制。方法:应用佛波酯(PMA)诱导THP-1单核细胞分化为巨噬细胞进行实验研究。选用5-氨基酮戊酸(ALA)作为声敏剂,进行声动力治疗(SDT)。应用流式细胞术证实SDT显著促进了细胞凋亡;应用Rhod 2/AM实时监测线粒体Ca~(2+)水平;通过蛋白质免疫印迹对全细胞蛋白中的Bax、Cleaved-caspase3、电压依赖性阴离子通道1(VDAC1)和三磷酸肌醇Ⅲ型受体(IP3R-Ⅲ)进行检测;应用VDAC1抗体进行免疫共沉淀,检测VDAC1和IP3R-Ⅲ之间的相互作用;实时监测线粒体Ca~(2+)水平,检测VDAC抑制剂DIDS和IP3Rs抑制剂2-ABP对SDT效果的影响。结果:与对照组相比,仅SDT组出现了显著的细胞凋亡(P0.001)。与对照组相比,ALA对线粒体Ca~(2+)水平无明显影响,超声诱导了线粒体Ca~(2+)水平的明显升高,SDT诱导了线粒体Ca~(2+)水平快速且大幅度的升高,且去除超声后仍维持在较高水平。与对照组相比,超声对Bax、Cleaved-caspase3、VDAC1和IP3R-Ⅲ的表达无明显影响,ALA诱导了VDAC1(P0.01)和IP3R-Ⅲ表达量的增加(P0.05),SDT诱导了Bax(P0.001)、Cleaved-caspase3(P0.001)、VDAC1(P0.01)和IP3R-Ⅲ(P0.05)表达量的增加,VDAC1和IP3R-Ⅲ的增加幅度与ALA组接近;ALA和SDT均诱导了VDAC1和IP3R-Ⅲ之间相互作用的显著增强(P0.05)。DIDS和2-ABP均明显抑制了SDT诱导的线粒体Ca~(2+)增加。结论:在致凋亡的SDT作用于THP-1巨噬细胞的过程中,ALA诱导了线粒体外膜Ca~(2+)转运通道VDAC1和内质网重要Ca~(2+)转运通道IP3R-Ⅲ的表达量增加与二者间相互作用的增强,在内质网和线粒体之间建立了大量的Ca~(2+)转运通道,超声的作用则在于触发这些Ca~(2+)转运通道的开放,进而引发线粒体钙的迅速增加。这是后续线粒体凋亡通路启动的重要机制之一。     

线粒体移植增强人神经胶质瘤U87细胞的辐射敏感性

线粒体移植( mitochondrial transplantation)是从患者正常组织分离线粒体然后注入线粒体损伤或缺失的部位,使损伤细胞获得救治、器官功能得以恢复的全新干预技术。本研究重点探讨线粒体移植对人神经胶质瘤细胞(U87)辐射敏感性的影响。提取人星形胶质细胞(human astrocytes, HA)的线粒体,用荧光探针Mito-Tracker Red进行标记后再与U87细胞共培养,激光共聚焦显微镜观察发现,Mito-Tracker Red的红色荧光大量出现在U87细胞内;随后对进入细胞内的游离线粒体荧光强度进行定量分析,游离线粒体和U87细胞共培养12 h时,单细胞内荧光强度由本底值0.08上升至1.83,表明游离线粒体可以通过共培养方式进入U87细胞内。随后给予U87细胞X射线辐照,Western印迹结果显示,联合组与单独辐照组相比,Cyto-C和Bax的表达量分别由179.5%和198.5%升高至251.72%和256.10%,Bcl-2的表达量由57.17%降低至22.23%;使用Annexin V/PI双染法检测到联合组U87细胞凋亡量相比单独辐照组的3.1%和23.5%上升至19.2%和43.8%;RT-CES检测结果表明,96 h内联合组U87细胞生长曲线被抑制;联合组克隆形成率相对单独辐照组的53%下降至29.3%。鬼笔环肽染色发现,线粒体移植组细胞表面丝状伪足相比对照组明显减少。本研究提示,线粒体移植能够促进辐射诱导的肿瘤细胞凋亡,对U87细胞具有辐射增敏作用,其作用机制与重新激活线粒体凋亡通路、降低肿瘤恶性程度有关。     

线粒体分裂抑制剂Mdivi-1改善血管紧张素Ⅱ介导的内皮功能紊乱（英文）

动力相关蛋白(dynamin-related protein 1,Drp1)与线粒体的动态变化有着密切的联系,是介导线粒体分裂的主要功能蛋白。本研究旨在探讨血管紧张素II(angiotensin II,Ang II)对血管内皮细胞线粒体融合和分裂的影响以及Drp1功能抑制剂Mdivi-1对Ang II介导的内皮损伤是否有减轻作用。Ang II或联合Drp1抑制剂Mdivi-1处理人脐静脉内皮细胞(human umbilical vascular endothelial cells,HUVECs)后,用Western blot检测Drp1、e NOS和凋亡相关酶的蛋白表达,用Mito Tracker Red染色观察细胞内线粒体形态,用JC-1探针染色检测线粒体膜电位变化,用DCFH-DA染色检测细胞活性氧簇(reactive oxygen species,ROS)生成,用Transwell实验检测细胞迁移情况,用Annexin V/PI染色检测细胞凋亡情况。结果显示,Ang II处理12 h后,HUVECs的Drp1的表达水平显著升高,内皮细胞迁移、凋亡及ROS的生成显著增加,e NOS表达量显著降低;同时,Ang II处理还诱导了线粒体形态的改变,使网状的线粒体变成了短管状,并伴随着线粒体膜电位的下降。Mdivi-1可以显著逆转Ang II对内皮功能的上述损伤作用,提高内皮细胞线粒体膜电位及e NOS的表达量,降低细胞内ROS水平,抑制内皮细胞凋亡及迁移能力。以上结果提示,Drp1抑制剂Mdivi-1可以减轻Ang II介导的内皮损伤。     

β-拉帕醌通过下调过氧化物酶V诱导SW480结肠癌细胞凋亡

过氧化物酶V(peroxiredoxin V,Prx V)是过氧化物酶(peroxiredoxin)家族中的一员,广泛存在于线粒体、溶酶体及细胞质中,具有有效地清除细胞内一氧化氮(nitric oxide,NO)和活性氧(reactive oxygen species,ROS)的功能。该文阐明β-拉帕醌(β-lapachone)引起SW480结肠癌细胞凋亡过程中Prx V的作用及其机制。该研究利用β-拉帕醌处理SW480细胞系,通过3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT]法、荧光显微镜照相、流式细胞术、蛋白质免疫印迹分析等方法检测细胞存活率、细胞内ROS水平、细胞凋亡情况以及凋亡相关蛋白质水平的变化。结果表明,β-拉帕醌通过抑制Prx V水平,致使细胞内ROS水平升高,激活线粒体依赖性凋亡途径,导致SW480结肠癌细胞凋亡。研究结果初步揭示了β-拉帕醌诱发SW480结肠癌细胞发生凋亡的机制,为结肠癌的治疗提供了新思路。     

丹酚酸B通过抑制线粒体功能增加胶质瘤细胞的放疗敏感性

目的:研究药用植物丹参的根茎提取成分丹酚酸B对人胶质瘤U251细胞的放疗增敏作用,并探讨其可能的分子机制。方法:使用1μM浓度的丹酚酸B处理胶质瘤U251细胞,并用等量PBS建立对照组,使用射线照射建立放射治疗模型。MTT法检测细胞活力;流式细胞术检测细胞凋亡;荧光染色检测活性氧ROS含量及线粒体肿胀程度。结果:丹酚酸B能够显著降低射线照射后U251细胞活力,并增加其凋亡(P0.05)。丹酚酸B能够显著增加射线照射后U251细胞活性氧ROS的产生,并增加线粒体的肿胀程度(P0.05)。结论:丹酚酸B能够通过诱导内源性凋亡增加胶质瘤细胞的放疗敏感性,这种作用可能是通过抑制线粒体功能而实现的。     

MitoQ对高糖诱导的心肌细胞线粒体功能影响

目的:探讨MitoQ对高糖诱导的心肌细胞线粒体功能影响。方法:常规获取与纯化SD大鼠新生仔鼠心肌细胞,分为对照组、高糖组、实验组。对照组用含10%血清的DMEM培养基(5.5 mmol/L葡萄糖)培养;高糖组用含血清的高糖DMEM培养基(33mmol/L葡萄糖)培养;实验组用含血清的高糖DMEM培养基(33 mmol/L葡萄糖)和MitoQ。MTT法检测心肌细胞存活率,氯离子荧光探针检测细胞内氯离子浓度,流式细胞术检测各组心肌细胞凋亡率,超氧化物阴离子荧光染色检测心肌细胞活性氧(reactive oxygen,ROS)含量,利用ATP检测试剂盒检测心肌细胞中的ATP水平,Western blot法检测心肌细胞胱天蛋白酶3(caspase-3)蛋白水平。结果:与对照组相比,高糖组的心肌细胞增凋亡率、ROS产生、氯离子相对浓度均明显增加,ATP显著降低(P0.05),细胞内caspase-3蛋白表达显著上升(P0.05);与高糖组相比,实验组凋亡率降低,ROS产生、细胞内caspase-3蛋白表达均显著降低(P0.05)。结论:高糖会引起心肌细胞线粒体障碍,造成心肌细胞凋亡,MitoQ可降低细胞内ROS和caspase-3水平,抑制心肌细胞凋亡,改善心肌细胞线粒体功能。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

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.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.