AS-mCLB1重组质粒体外抗肿瘤及化疗增敏作用

研究反义全长小鼠细胞周期蛋白B1重组质粒(pAS-mCLB1)体外抗肿瘤及化疗增敏作用。扩增后少量抽提并纯化pAS-mCLB1,通过脂质体将其转染入小鼠露易丝肺癌(LL/2)细胞,RT-PCR和Western印迹测定细胞内细胞周期蛋白B1的表达,观察转染后细胞形态变化,MTT法检测细胞增殖活性,流式细胞仪检测细胞周期及凋亡。细胞转染48h后,用化疗药物健择(gemcitabine;0.2μmol/L)处理24h,MTT法测定健择对细胞的杀伤作用。研究提示pAS-mCLB1转染后LL/2细胞形态明显异常,细胞内细胞周期蛋白B1表达显著下调,细胞周期阻滞于G1期,增殖受抑,凋亡增加;健择对pAS-mCLB1转染后LL/2细胞的杀伤作用显著增强。重组质粒pAS-mCLB1体外具有明显的抗肿瘤作用,并能增强肿瘤细胞对化疗药物的敏感性,估计上述作用与其下调肿瘤细胞内细胞周期蛋白B1表达,从而诱导细胞周期阻滞及凋亡等作用相关。     

干扰PGI调控HIF-1α的表达以及促进乳腺癌细胞凋亡

本实验探究乳腺癌MCF-7细胞中干扰PGI的表达对HIF-1α的调控以及对细胞凋亡的影响。首先,使用慢病毒质粒转染构建干扰PGI(PGI-si RNA)的稳转细胞株,然后使用Hoechst33258染色和流式细胞术(FCM)检测细胞的凋亡,随后,采用RT-PCR检测PGI、HIF-1α和LDHA基因的表达,Western blotting检测Bcl-2、Bax、PGI、LDHA、HIF-1α和ERK蛋白的表达。成功构建PGI-si RNA的MCF-7稳转细胞株后,Hoechst33258染色实验结果显示,PGI-si RNA稳转细胞株中出现核固缩、染色质凝集等形态学改变,FCM结果亦表明PGI-si RNA稳转细胞株发生细胞凋亡。RT-PCR结果显示,PGI-si RNA稳转细胞株中PGI、LDHA和HIF-1α基因表达下调;Western blotting结果显示,PGI-si RNA稳转细胞株中LDHA、HIF-1α、ERK和Bcl-2蛋白表达下调,Bax蛋白表达上调。以上实验结果表明:在乳腺癌MCF-7细胞中,干扰PGI可抑制HIF-1α的表达,同时促进MCF-7细胞凋亡。     

二氢杨梅素通过下调JNK信号拮抗高糖诱导的PC12细胞凋亡

本文探讨二氢杨梅素(dihydromyricetin,DHM)是否通过下调JNK信号拮抗高糖诱导的PC12细胞凋亡.使用四甲基偶氮唑盐法(MTT)检测PC12细胞活力;流式细胞仪检测PC12细胞早、晚期凋亡及死亡细胞率;Hoechst 33258染色观察凋亡细胞核变化;蛋白质印迹法(Western blotting)检测PC12细胞凋亡相关蛋白(Bax、Bcl-2、cleaved-Caspase-3)和p-JNK蛋白的表达.结果发现,不同浓度的葡萄糖(4.5、9.0、13.5、18.0 g/L)分别处理PC12细胞24、48、72、96 h后,发现浓度为13.5 g/L的高糖处理PC12细胞72 h可明显改变细胞形态、降低细胞活力、增加细胞凋亡率,同时促凋亡蛋白(Bax、Caspase-3)表达增加、抗凋亡蛋白Bcl-2表达降低,提示:长时间高糖处理可诱导PC12细胞凋亡.DHM(15μmol/L)预处理能明显改善高糖诱导的PC12细胞凋亡,降低高糖诱导的PC12细胞中JNK和p-JNK蛋白的表达;进一步用JNK激动剂(茴香霉素)处理能取消DHM对高糖诱导PC12细胞凋亡的保护作用.综上,得出结论:DHM通过下调JNK信号拮抗高糖诱导的PC12细胞凋亡.     

过表达Nogo-C对PC12细胞存活及增殖的影响

以PC12细胞为神经元细胞模型,研究Nogo-C对神经元细胞存活及增殖的作用。在PC12细胞中转染过表达Nogo-C,使用G418药物筛选以获得稳定表达的细胞克隆,利用Hoechst33342染色、细胞计数、MTT以及流式细胞仪等技术检测Nogo-C对细胞增殖以及细胞周期的影响。结果表明:(1)Hoechst33342染色未观察到表达Nogo-C的细胞发生明显凋亡;(2)细胞计数及MTT实验观察到转染Nogo-C后的PC12细胞生长增殖活性明显降低;(3)流式细胞仪检测细胞生长周期,正常PC12细胞G1期的百分数为(37.8±7.9)%,S期为(50.4±8.5)%,而转染Nogo-C的PC12细胞G1期为(76.8±4.1)%,S期为(14.7±1.7)%,提示转染Nogo-C的PC12细胞的细胞周期被阻滞在G1期;(4)没有获得稳定表达Nogo-C的PC12细胞模型。实验证明,过表达Nogo-C通过使PC12细胞周期被阻滞在G1期而明显抑制细胞的增殖,但是并不引起细胞的凋亡。     

Sim2基因对PC12细胞分化的影响

[摘要] 目的 探讨位于Down综合征关键位点的Sim2基因对PC12细胞分化的影响及其机制。 方法 以pcDNA3-mSim2真核表达载体稳定转染PC12细胞,以倒置相差显微镜镜观察PC12细胞神经突起的变化;以RT-PCR方法检测神经元分化相关基因GAP43和Synapsin I mRNA表达水平的变化;流式细胞仪检测GAP43蛋白的表达。 结果 RT-PCR结果显示, pcDNA3-mSim2转染后,mSim2 mRNA表达明显上调;与对照组相比,转染mSim2的PC12细胞突起数量显著减少,长度明显变短;GAP43和Synapsin I mRNA表达水平明显降低（P<0.05）;流式细胞仪检测发现,转染mSim2的PC12细胞GAP43蛋白表达水平显著降低（P<0.05）。 结论 Sim2基因可通过影响神经元的分化参与Down综合征的发生。     

siRNA沉默Survivin基因对鼻咽癌CNE-2细胞凋亡的影响

合成Survivin小干扰RNA(small interfering RNA,siRNA)序列,用于转染人鼻咽癌细胞株CNE2,体外观察Survivin siRNA转染后CNE-2细胞Survivin mRNA、蛋白的表达、细胞增殖和凋亡情况。设计合成3段特异性Survivin siRNA,用siRNA转染鼻咽癌CNE-2细胞株,设置空白对照和阴性对照组,通过Real-time PCR检测CNE-2细胞的Survivin mRNA相对表达量,Western blotting检测Survivin蛋白的表达,流式细胞术及原位细胞凋亡检测转染后细胞凋亡情况。siRNA转染CNE-2细胞后,siRNA 1组和3组mRNA表达抑制率分别为(68.46±7.94)%、(49.44±3.78)%,siRNA 2组结果无显著差异(p0.05)。蛋白相对表达量只有siRNA 1组降低,表达抑制率为(30.61±2.47)%,流式细胞术和原位细胞凋亡检测转染后CNE-2细胞数量明显降低,细胞凋亡比例增高。siRNA干扰沉默Survivin基因能有效抑制CNE-2细胞的增殖和诱导细胞凋亡。本研究为Survivin基因可能作为治疗鼻咽癌的一个靶点,为Survivin基因沉默可能是治疗鼻咽癌高效的途径提供体外实验支持。     

磁性纳米粒介导沉默PI3Kγ基因调控肿瘤相关巨噬细胞抗小鼠肺癌细胞效应的研究

为了探讨超顺磁性Fe3O4纳米粒子(superparamagnetic iron oxide nanoparticles,SPIONs)介导的磷脂酰肌醇3激酶γ(phosphatidylinositol 3 kinaseγ,PI3Kγ)抑制表达调控的肿瘤相关巨噬细胞(tumor-associated macrophages,TAM)对小鼠Lewis肺癌细胞(Lewis lung carcinoma,LLC)增殖和凋亡的影响,该研究构建了能启动巨噬细胞(macrophage,MФ)特异性表达PI3Kγ催化亚基p100 siRNA的pSilencer-EGFP-SP-p110质粒,通过SPIONs负载成磁性纳米质粒复合物(SPIONs-DNA),在强磁作用下转染MФ,通过普鲁士蓝染色法检测SPIONs-DNA在细胞内的分布,Real-time PCR和Western blot检测细胞PI3Kγp110亚基的表达水平。建立M1、M2型MФ模型,将SPIONs-DNA在强磁作用下转染M2型MФ,通过Real-time PCR和Western blot鉴定细胞表型,明确M2型MФ转化为M1型的强度。采用Transwell系统建立SPIONs-DNA转染的M2型MФ与小鼠LLC细胞的共培养模型,通过锥虫蓝染色法检测LLC细胞的活细胞数并绘制细胞生长曲线,CCK-8法检测LLC细胞增殖情况,硝酸还原酶法检测共培养液上清中NO含量,流式细胞术检测LLC细胞凋亡情况。结果显示,制备的SPIONs-DNA在强磁作用下成功转染MФ并大量分布在细胞胞核周围,SPIONs-DNA转染组细胞PI3Kγp110 mRNA和蛋白表达水平显著低于空白细胞对照组(P<0.05)。建立的M1型MФ高表达iNOS(P<0.001),M2型MФ高表达ARG-1(P<0.001)。M2型MФ转染SPIONs-DNA后细胞iNOS mRNA和蛋白的表达显著增加(P<0.001),ARG-1 mRNA和蛋白的表达显著降低(P<0.01)。在共培养组中,SPIONs-DNA转染的M2型MФ组能大量分泌NO,LLC细胞生长和增殖能力显著降低(P<0.05),凋亡率显著增高(P<0.01)。结果表明,磁性纳米粒负载pSilencer-EGFP-SP-p110重组质粒能够特异性靶向抑制巨噬细胞PI3Kγp110的表达,诱导M2型MФ转化为M1型;其转染的M2型MФ可显著抑制LLC细胞的生长和增值,促进细胞凋亡,这与其大量分泌NO有关。该磁性纳米质粒复合物可诱导TAM发挥抗肿瘤作用,为研究开发有效的抗肺癌基因治疗措施奠定基础。     

靶向干扰Chkl增强阿霉素抗人乳腺癌MCF-7/adr细胞的作用机制研究

Chkl的高表达可能是肿瘤对化疗药物的敏感性降低的重要因素之一,本研究的目的是观察siRNA干扰Chk1对人乳腺癌耐药细胞株MCF-7/adr(耐阿霉素)生长及细胞周期的影响,探讨Chk1在乳腺癌细胞耐药中的作用机制。采用RNAi技术抑制MCF-7/adr细胞中Chk1的表达。Westernblot检测转染前后细胞内Chk1蛋白表达情况,经阿霉素作用后,流式细胞术(FCM)检测其细胞周期分布及细胞凋亡率,MTT法检测细胞增殖。Western blot结果显示,Chk1 siRNA转染24h后,MCF-7/adr细胞中Chk1蛋白表达下降了67%,明显低于对照组和空载体转染组(P<0.05)。FCM法检测结果显示,同时,抑制Chk1的表达可解除阿霉素引起的G_2/M期阻滞;使阿霉素诱导的细胞凋亡率由转染前的(5.54±0.15)%上升到(22.24±0.13)%(P<0.05);在阿霉素浓度为0.4mg/L、4mg/L时,细胞的增殖活性分别下降13%、34%。提示siRNA干扰Chk1能够通过调控MCF-7/adr细胞周期及增殖从而增强乳腺癌细胞对阿霉素的敏感性,为临床上克服乳腺癌化疗耐药提供了新的作用靶点。     

阳离子脂质体Lipofectamine2000对PC12细胞转染效率的研究

通过改变转染试剂及DNA用量和转染时间等影响转染效率的重要因素来实现阳离子脂质体lipofectamineTM2000(lipo2000)对PC12细胞的高效转染.结果表明,lipo2000转染PC12细胞的最佳转染条件:lipo2000用量为5μL,DNA 2μg,转染时间为6 h,这种条件下的PC12细胞转染率高达40%,且未影响细胞的正常分泌,这为应用PC12细胞进行神经生物学研究提供了基础资料.     

半边旗二萜类成分PAG通过ROS诱导肝癌细胞凋亡的作用研究

探讨半边旗二萜类成分Pteisolic acid G(PAG)对人肝癌细胞HepG2增殖和凋亡的影响及作用机制。用不同浓度的PAG处理HepG2细胞后,采用MTT法检测细胞存活率;采用PI单染法检测细胞周期分布;采用Annexin V-FITC/PI双染法检测细胞凋亡率;采用RT-PCR和Western Blotting检测细胞内mRNA和蛋白表达情况;采用DCFH-DA法检测细胞内ROS水平,采用ROS抑制剂乙酰半胱氨酸(NAC)评价PAG细胞增殖抑制作用对ROS的依赖性。结果表明,在24 h、48 h和72 h时,PAG可剂量依赖性地抑制HepG2细胞的增殖(p0.05),IC_(50)分别为64.8μmol/L,38.5μmol/L和24.8μmol/L;用药24 h时PAG可剂量依赖性地使HepG2细胞阻滞在G_2/M期,同时增加HepG2细胞凋亡率(p0.05);PAG可剂量依赖性地降低HepG2细胞内Bcl-2 mRNA和caspase 3、PARP、Bcl-2蛋白的表达(p0.05),增加Bax mRNA和actived-caspase 3、cleaved-PARP、Bax蛋白的表达(p0.05)。当使用1 mmol/L的ROS抑制剂NAC预处理HepG2细胞时,PAG对HepG2细胞增殖抑制作用被显著阻断。上述结果表明,半边旗二萜类成分PAG可提高Bax/Bcl-2的基因和蛋白表达比值,从而诱导肝癌细胞HepG2凋亡,该作用可能是通过升高细胞内ROS水平来实现的。     

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.     

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.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.