雌激素处理的hBMSC对高糖诱导血管内皮细胞损伤的保护作用*

目的: 探讨雌激素处理人骨髓间充质干细胞(hBMSC)对高糖诱导的人脐静脉血管内皮细胞(HUVEC)损伤的保护作用及机制。方法: 采用30 mmol/L葡萄糖刺激hBMSC细胞建立高糖模型并分组:以无刺激者为高糖对照组(HG组)、以20 μmol/L雌激素处理者为高糖雌激素组(HG+E2组)、以5 μmo/L蛋白激酶B(PKB/Akt)抑制剂Triciribine预处理45 min后,再以20 μmol/L雌激素处理者为高糖Akt抑制剂组(HG+E2+Triciribine组)和正常条件培养的hBMSC为正常对照组(NG组)。分别于处理12 h后,采用CCK8法检测各组hBMSC的细胞活力,硝酸还原酶法和ELISA法检测各组培养基上清中NO、VEGF和IL-8的含量(n=6),48 h后采用Western blot检测内皮型一氧化氮合酶(eNOS)和磷酸化eNOS(p-eNOS)蛋白表达水平(n=3)。此外,提取各组hBMSC的培养基上清作为条件培养基(CM)培养人脐静脉血管内皮细胞(HUVEC)并分组为:HG-CM组(HG组条件培养基处理)、HG+E2-CM组(HG+E2组条件培养液处理)、HG+E2+Triciribine-CM组(HG+E2+Triciribine组条件培养基处理)和HG-H组(高糖对照组,30 mmol/L葡萄糖终浓度处理),分别于12 h后,采用CCK8法检测各组HUVEC的细胞活力(n=6),24 h后采用流式细胞术检测各组HUVEC细胞的凋亡率(n=3);48 h后采用划痕实验观察各组HUVEC细胞的迁移率(n=3)。结果: 与NG组相比,HG组中hBMSC细胞活力和细胞内eNOS蛋白磷酸化水平降低(P<0.05),细胞培养液上清中NO、VEGF和IL-8含量减少(P<0.05);与HG组相比,HG+E2组中hBMSC的细胞活力和细胞中eNOS蛋白磷酸化水平显著增加(P<0.05),细胞培养基上清中NO、VEGF和IL-8含量增加(P<0.05),而当hBMSC细胞中Akt蛋白活性被抑制后,HG+E2+Triciribine组中上述结果指标呈反向变化(P<0.05)。此外,与HG-CM组相比,HG+E2-CM组中HUVECs的细胞活力和迁移能力显著增加(P<0.05),细胞凋亡比例降低(P<0.05),而与HG+E2-CM组相比,HG+E2+Triciribine-CM组中HUVECs的细胞活力和迁移能力降低(P<0.05),细胞凋亡比例增加(P< 0.05)。结论: 雌激素可能通过激活hBMSC细胞Akt/eNOS信号通路,促进NO、VEGF和IL-8的分泌,进而增加HUVECs的细胞活力和迁移能力,并抑制细胞凋亡的发生,对高糖诱导的HUVECs细胞损伤发挥保护作用。     

姜黄素类似物EF24诱导A549细胞自噬及凋亡关系的研究

从细胞自噬及凋亡关系角度探讨姜黄素类似物EF24对人肺腺癌细胞（A549）的杀伤机理。选用不同浓度的EF24对体外培养的A549处理,采用MTT方法检查细胞存活率,吖啶橙染色观察细胞形态,蛋白质免疫印迹（Western blot）方法检测与细胞自噬及凋亡相关蛋白的表达及对AMPK-mTOR-S6K信号通路的影响。结果显示,EF24作用24 h的IC50为8.5μmol/L,对A549细胞生长抑制作用优于姜黄素,而接近顺铂。自噬及凋亡蛋白检测显示,在4μmol/L、8μmol/L时A549细胞以自噬为主,在16μmol/L时以凋亡为主;加入100 nmol/L自噬抑制剂渥曼青霉素（wortmannin）后,细胞存活率同比升高。同时还发现,随着EF24浓度的增加,细胞内AMPK-Thr172磷酸化水平上升,mTOR-Ser2481、S6K-Thr389磷酸化水平的下调。由此可见,EF24可通过AMPK的激活下调mTOR-S6K途径抑制细胞生长,在EF24浓度4~8μmol/L范围内,自噬对凋亡起到促进作用。     

圣草酚调控Akt/mTOR通路对胶质瘤细胞自噬的影响CSCD

为研究圣草酚对胶质瘤细胞自噬的影响,探讨其通过调控Akt/mTOR通路在胶质瘤自噬过程中的作用。本研究采用CCK8和细胞克隆形成实验检测圣草酚对胶质瘤细胞增殖的影响。构建GFP-mRFP-LC3B稳定表达的细胞株,圣草酚(100μmol/L)处理48 h,观察自噬小体的形成情况;然后分别用圣草酚(100μmol/L)、雷帕霉素(200 nmol/L)以及圣草酚(100μmol/L)+雷帕霉素(200 nmol/L)处理48 h后观察自噬流的情况。利用蛋白免疫印迹法检测圣草酚对自噬及Akt/mTOR通路相关蛋白表达水平的影响。建立裸鼠皮下移植瘤模型,观察圣草酚在体内对胶质瘤自噬过程的影响。实验结果显示,圣草酚能够显著抑制胶质瘤细胞的增殖,且呈时间—剂量依赖性(P<0.05)。圣草酚使胶质瘤细胞中绿色自噬小体明显增多,LC3Ⅱ、Beclin 1、ATG5蛋白表达上调。圣草酚处理GFP-mRFP-LC3B稳转细胞株48 h后呈亮黄色,且减弱了雷帕霉素对自噬流的促进作用,并上调P62蛋白的表达。此外,圣草酚可抑制p-Akt、p-mTOR的表达,但该作用被Akt激动剂740 Y-P逆转。体内实验结果同体外一致。说明圣草酚能够抑制胶质瘤细胞的增殖和自噬流,其影响自噬过程的作用机制可能与下调Akt/mTOR通路有关。     

曲古抑菌素A对慢性髓细胞白血病细胞增殖与凋亡的作用及机制研究

该文研究了去乙酰化酶抑制剂曲古抑菌素A(trichostatin A,TSA)对慢性髓细胞白血病(chronic myeloid leukemia,CML)细胞株K562及K562/G01增殖和凋亡的影响及机制。采用不同浓度TSA处理K562及K562/G01细胞,CCK8和克隆形成实验检测细胞增殖能力;流式细胞术、蛋白印迹法和DAPI染色检测细胞凋亡;蛋白印迹法检测细胞自噬相关蛋白及BCR-ABL/STAT5/c-Myc信号轴蛋白水平。结果显示,TSA显著抑制K562及K562/G01细胞的增殖;明显促进K562及K562/G01细胞的凋亡;TSA与伊马替尼(imatinib,IM)联用可更有效地杀伤CML细胞。TSA可抑制BCR-ABL/STAT5/c-Myc信号轴,降低c-Myc蛋白水平;增强CML细胞自噬,自噬抑制剂氯喹(chloroquine,CQ)可部分回复TSA引起的凋亡。综上,TSA通过抑制STAT5信号通路,降低c-Myc蛋白水平,抑制CML细胞增殖;增强CML细胞自噬,促进其凋亡。     

mTOR信号通路诱导的自噬在心肌细胞缺氧损伤中的作用研究

目的:探讨自噬在心肌细胞缺氧损伤中的作用及分子机制。方法:体外分离培养乳鼠心肌细胞,体外建立缺氧/去血清(H/SD)模型以模拟在体的缺血环境。分别给予自噬抑制剂3-甲基腺嘌呤(3MA,5 mM)和mTOR抑制剂雷帕霉素(1.0μg/L)调节心肌细胞自噬水平。分别采用TUNEL染色检测心肌细胞凋亡,Western blot方法检测心肌细胞蛋白表达水平。结果:H/SD损伤可以显著诱导心肌细胞自噬水平(P0.05),并且细胞自噬水平可以被3-MA及雷帕霉素调节。同时,H/SD可以显著增加心肌细胞凋亡(P0.05),而给予3-MA抑制自噬水平可以减少细胞凋亡(P0.05)。相反,雷帕霉素增加自噬同样可以加重缺氧导致的心肌细胞凋亡(P0.05)。H/SD损伤过程中,心肌细胞mTOR信号通路被激活,而自噬抑制剂3-MA可以显著提高缺氧条件下心肌细胞中p-mTOR(Ser2448)的表达水平(P0.05),并增加mTOR下游分子p-p70S6k(P0.05)和p-S6(P0.05)的表达。结论:mTOR信号通路诱导的细胞自噬可能参与了缺氧损伤诱导的心肌细胞凋亡。     

U0126通过下调p-ERK1/2和GM130的表达对人胃癌细胞MKN-45的影响

探讨ERK1/2通路抑制剂U0126对人胃癌MKN-45细胞增殖、凋亡、侵袭和迁移能力的影响及机制。首先用CCK-8法测定U0126对MKN-45细胞增殖的影响并筛选药物作用的最适浓度和时间用于后续实验,并将实验分为药物处理组,阴性对照组和空白对照组。然后通过流式细胞术、Transwell迁移实验和Matrigel侵袭实验分别检测细胞凋亡、迁移和侵袭能力,RT-q PCR检测各组GM130的m RNA水平的变化,Western blotting检测ERK1/2、p-ERK1/2、MMP-9、MMP-2、GM-130的蛋白水平的变化。实验发现10μmol/L、20μmol/L、30μmol/L、40μmol/L浓度的U0126均能抑制MKN-45细胞的增殖,其中20μmol/L浓度作用48 h效果最强(p0.05),且该药物作用条件下MKN-45细胞凋亡增加,迁移和侵袭作用减弱(p0.05)。Western blotting结果显示,药物处理组ERK1/2蛋白水平不变,而p-ERK1/2、MMP-9、MMP-2、GM-130的蛋白水平均明显低于阴性对照组和空白对照组(p0.05)。RT-q PCR结果显示,药物处理组GM130的m RNA水平明显低于阴性对照组和空白对照组(p0.05)。以上研究结果均表明U0126可促进MKN-45细胞凋亡,降低细胞体外增殖、迁移和侵袭的能力,其机制可能与p-ERK1/2和GM130的表达被抑制有关。     

岩大戟内酯A和岩大戟内酯B通过Akt信号通路影响MCF-7细胞的增殖和凋亡

观察岩大戟内酯A(Jolkinolide A,JA)和岩大戟内酯B(Jolkinolide B,JB)二萜类单体对MSC-7乳腺癌细胞的增殖、凋亡影响,并分析JA或JB的作用机制。若分别用40、60、80μg/m L浓度的JA(或JB)刺激MCF-7细胞,则为不同浓度的JA刺激组或JB刺激组,正常培养的MCF-7为对照组。分别用CCK-8细胞增殖实验检测JA刺激组或JB刺激组的细胞增殖OD值,Western Blot实验检测JA刺激组或JB刺激组MCF-7细胞中Caspase 9蛋白表达,Annexin V-FITC/PI双染检测MCF-7细胞凋亡,酶联免疫吸附实验(ELISA)检测JA刺激组或JB刺激组细胞中Akt、STAT3蛋白的含量。结果发现,与40、60、80μg/m L的JA刺激组MCF-7细胞分别相比,相应浓度JB刺激组的MCF-7细胞增殖OD值均明显降低,但是JB刺激组MCF-7细胞的Caspase 9蛋白相对吸光度、凋亡率显著升高;相对于40、60、80μg/m L JA刺激组,相应浓度的JB-刺激组MCF-7细胞上清液中的VEGF含量分别均降低,相应浓度JB刺激组MCF-7细胞的Akt、STAT3蛋白表达均降低。实验结果表明:岩大戟内酯B明显地通过抑制Akt-STAT3信号通路而抑制MCF-7细胞增殖,促进MCF-7凋亡。     

自噬抑制剂氯奎对低氧诱导肺动脉平滑肌细胞增殖的影响

目的：探讨自噬抑制刺氯喹（cQ）在低氧（hypoxia）调节肺动脉平滑肌细胞（PASMCs）增殖中的作用。方法：将体外培养的大鼠PASMCs分为4组：正常对照组、1％低氧组、50μmol／L氯喹＋1％低氧组、50ttmol／L氯喹组。MTF方法检测各组的PASMCs增殖率;MDC染色检测细胞自噬空泡的变化;Westernblot方法检测微管相关蛋白轻链3（LC3）蛋白的表达变化;划痕法检测细胞迁移的变化。结果：与对照组比较,氯喹组的PASMCs细胞增殖率无明显变化。与对照组比较,1％低氧组PASMCs增殖率明显增加,细胞内出现大量自噬空泡,细胞迁移速度明显增加。细胞LC3-Ⅱ蛋白表达增强。与1％低氧组比较,氯喹与低氧联合作用时细胞自噬空泡的积聚以及rE3．II蛋白表达增强,但细胞增殖率和迁移明显降低。结论：低氧激活自噬过程并促进了PASMCs增殖和迁移,而自噬抑制剂氯喹在一定程度上通过抑制自噬进程,达到抑制肺动脉平滑肌细胞增殖和迁移的作用。     

体外缺血缺氧条件下大鼠骨髓间充质干细胞凋亡发生的机制研究

目的:研究体外大鼠骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cells,BMSCs)在缺血缺氧条件下发生凋亡的作用机制。方法:采取大鼠骨髓,以密度梯度离心分离出单个核细胞(MNCs),于体外培养并由牛垂体提取物(PEX)诱导扩增传代培养出骨髓间充质干细胞(MSCs)。经形态学和流式细胞仪检测MSCs表面标志物鉴定后,骨髓间充质干细胞(BMSCs)在缺血缺氧条件下培养,通过Annexin V/PI双染细胞凋亡检测比较不同组别细胞的凋亡率和蛋白印迹法(western blot)来观察细胞中蛋白的变化。结果:①经形态学观察和流式细胞仪检测MSCs表面标志物鉴定,提示骨髓间充质干细胞培养成功。②对照组(无缺血缺氧)与缺血缺氧组比较,缺血缺氧组的凋亡率显著性增加,而通过磷酸化Akt的表达量显著性增加提示PI3K(Phosphoinosi-tide-3kinase)/Akt(ProteinkinaseB,PKB)信号通路被激活(P<0.05);同时缺血缺氧组与缺血缺氧+PI3K/Akt抑制剂(LY294002)组比较,缺血缺氧+PI3K/Akt抑制剂(LY294002)组的凋亡率显著降低,而通过磷酸化Akt的表达量显著减少提示PI3K/Akt信号通路被抑制(P<0.05)。结论:PI3K/Akt信号通路对体外缺血缺氧条件下培养的骨髓间充质干细胞凋亡发生有关键性作用。     

富马酸二甲酯对人角质形成细胞HaCat生长和自噬的影响

该文探讨了富马酸二甲酯(dimethyl fumarate,DMF)对人角质形成细胞Ha Cat增殖、细胞周期和细胞自噬的调节作用以及相关机理的影响。应用MTT实验和流式细胞术分别检测细胞增殖和细胞周期的变化。并用免疫荧光染色、Western blot技术检测自噬相关蛋白的水平与分布。结果显示,DMF在25μmol/L时,可较明显地抑制Ha Cat细胞的增殖,当浓度达到150μmol/L时,细胞几乎完全失去增殖能力。低剂量DMF(25~50μmol/L)同时也可显著抑制Ha Cat细胞DNA合成,与对照组相比,S期细胞减少50%(P0.01),而G1期细胞增高近20%(P0.05);高剂量DMF(100μmol/L)可导致G2与M期阻滞(P0.01),而氧自由基抑制剂NAC(N-Acetyl-L-cysteine)预处理则几乎完全逆转这一现象。DMF处理诱导细胞出现明显的自噬特征,表现为LC3-II(microtubule associated protein 1light chain 3-II)免疫荧光染色呈点状分布并与溶酶体颗粒重合,LC3蛋白质脂化为LC3-II以及自噬相关蛋白成熟型Cathepsin D、LAMP1(lysosome-associated membrane glycoprotein 1)、p62水平增加。此外,DMF对ERK(extracellular regulated protein kinases)、AKT(phosphatidylinositol 3 kinase)以及m TOR(mammalian target of rapamycin)信号相关蛋白的磷酸化也有显著的抑制作用。这些结果表明,DMF可显著抑制人角质形成细胞增殖、抑制DNA合成、诱导细胞发生自噬,其机理可能与DMF抑制AKT/m TOR、ERK信号功能有关。     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.     

真菌类遗传学分析的知识结构教学

本文以认知结构理论为指导,讨论了真菌类遗传分析与高等动植物遗传分析的内在联系,认为利用这种内在联系进行教学可收到好的效果并说明了作者的具体教学过程。 Abstract:In the paper, the relationship between genetic analysis of Fungi and genetic analysis of high animal and plant was discussed.A good results were obtained when we adopted this method in the teaching.     

龙胆科药用植物化学成分的研究现状

龙胆科植物在我国的分布范围很广,且多数为药用植物,其多数种属的药用植物,至今其化学成分尚未被系统研究。综述了目前龙胆科药用植物的化学成分的研究现状及一般提取方法,对近年来发现的环烯醚萜及裂环烯醚萜类化合物进行了总结,为本科药用植物的更深入研究提供了参考。     

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Summary Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l⁻¹ glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l⁻¹) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.     

Specificity of action of piperidylcholine derivatives as substrates of cholinesterases of various origin

The review deals with study of enzymologic properties of a novel highly specific acetylcholinesterase substrate, N-(β-acetoxyethyl) piperidinium iodomethylate (“piperidylcholine”), and its 30 derivatives that were tested as effectors of cholinesterases of mammals and various species of Pacific squids. It was proven for the first time that responsible for specificity of action was structure of cyclic ammonium grouping of the alcohol part of molecule of the ester substrate. Analysis of specificity is performed based on enzymatic hydrolysis parameters—activity of catalytic center of cholinesterases and bimolecular constant of the reaction rate that are determined at optimal and low substrate concentrations. Among the specially synthesized group of thioester compounds there is revealed one more highly specific acetylcholinesterase substrate—N-(β-acetoxyethyl) piperidinium.     

Interconnection of parameters of regulation system of lipid peroxidation and of morphophysiological parameters of mouse liver

A complex analysis of seasonal fluctuations of the mean group parameters of the system of regulation of lipid peroxidation has been performed in liver of Balb/c mice. Association of lipid characteristics and morphophysiological parameters is studied in the Balb/c mouse liver. An inter-connection is revealed between the liver index and the amount of lysoforms of phospholipids, the scale and character of the interconnection differing essentially depending on proportion of phos-phatidylcholine in mouse liver phospholipids.