HBXIP基因对乙肝病毒X蛋白诱导细胞凋亡的影响

探讨乙型肝炎病毒X蛋白结合蛋白(hepatitisBXinteractingprotein ,HBXIP)基因在乙型肝炎病毒X蛋白(HBX)诱导肝癌细胞凋亡时对细胞周期的影响.构建HBXIP基因真核表达载体pcDNA3 hbxip ,进行瞬时基因转染,将克隆有HBx基因的pCMV X (分别为1μg、2 μg和3μg)和pcDNA3 hbxip质粒分别和共转染至人H74 0 2肝癌细胞中(总体积分别为5 0 μl) .发现瞬时转染3μgpCMV X质粒后,肝癌细胞凋亡发生率为34 4 % ,肝癌细胞的细胞周期相关蛋白p2 7表达水平发生明显上调;与对照组相比,瞬时转染1μg、2 μg和3μg时,细胞周期蛋白D和细胞周期蛋白E的表达水平均发生明显上调,但随着HBX水平的增加细胞周期蛋白D和细胞周期蛋白E的表达水平发生明显下降;在稳定转染pCMV X质粒的H74 0 2 X肝癌细胞中无明显的细胞凋亡发生,研究发现p2 7的表达水平发生了明显下调,而细胞周期蛋白D和细胞周期蛋白E的表达水平发生了明显上调;当pcDNA3 hbxip质粒与pCMV X质粒进行共瞬时转染时,细胞凋亡发生率由pcDNA3质粒与pCMV X质粒共转染时的2 9 2 %下降为13 3% ,p2 7的表达水平发生了下调,但细胞周期蛋白D和细胞周期蛋白E的表达水平无明显变化.研究结果表明,瞬时转染一定剂量的x基因可导致肝癌细胞发生凋亡,细胞周期相关蛋白p2 7、细胞周期蛋白D和     

Ad-IL-24对SGC-7901胃癌细胞生长抑制的体外实验

旨在研究携带人IL-24基因的腺病毒表达载体(Ad-IL-24)对SGC-7901人胃癌细胞的生长抑制作用并分析其分子机制。以不同MOI(感染复数)的Ad空载体腺病毒感染SGC-7901人胃癌细胞,筛选出最佳感染剂量;Ad-IL-24以最佳感染剂量感染SGC-7901胃癌细胞,RT-PCR法和Western blotting法检测腺病毒介导的IL-24基因在SGC-7901胃癌细胞中的转录;MTT法检测Ad-IL-24对SGC-7901胃癌细胞的生长抑制作用,流式细胞仪检测其诱导SGC-7901人胃癌细胞凋亡和细胞周期改变的效应,Hoechst33258染色荧光显微镜检测其诱导胃癌细胞凋亡的核形态改变;RT-PCR半定量法进一步检测SGC-7901胃癌细胞中凋亡相关基因的转录。结果显示,100MOI为感染SGC-7901胃癌细胞的腺病毒最佳感染剂量;Ad-IL-24能成功介导IL-24基因在SGC-7901胃癌细胞中转录性表达;Ad-IL-24感染SGC-7901胃癌细胞后,能明显抑制胃癌细胞生长和诱导凋亡;Ad-IL-24能显著上调SGC-7901胃癌细胞中bax、caspase-3和p53的表达和下调bc...     

人重组Fas配体体外诱导细胞周期特异性凋亡模型的建立及其意义

以Molt-4、Jurkat细胞株和外周血淋巴细胞(peripheralbloodlymphocyte,PBL)为靶细胞,检测细胞膜上Fas的表达。人重组Fas配体(recombinanthumanFasligand,rhFasL)诱导细胞6～36h后用改良后的API等方法检测细胞凋亡及诱导凋亡过程中细胞周期蛋白的变化,探讨Fas介导的细胞凋亡与细胞周期的关系。结果显示:rhFasL诱导Molt-4、Jurkat细胞株和植物血凝素刺激进入细胞周期的PBL的凋亡具有细胞周期特异性并始动于G1期;而G0期PBL的细胞膜上虽然也有Fas的表达,但不能诱导细胞凋亡。研究还发现rhFasL诱导细胞凋亡时G1期的细胞周期蛋白D3明显升高,细胞周期蛋白E明显下降。以上结果表明rhFasL体外诱导的细胞凋亡发生在晚G1期,细胞凋亡的发生与细胞是否通过限制点进入细胞周期有关,细胞凋亡发生于晚G1期是G1期细胞周期蛋白E的下降和检测点的监督导致DNA受损的细胞不能通过G1/S交界的结果。     

DNA甲基化在中枢神经系统发育中的研究进展

DNA甲基化是最早被发现的表观遗传修饰之一。近年来,大量的研究显示DNA甲基化在中枢神经系统(CNS)发育中发挥了重要作用。不同种类的DNA甲基转移酶(Dnmt)和DNA甲基结合蛋白(MBD)在CNS发育的不同阶段发挥不同的作用。DNA甲基化促进神经干细胞向神经元方向分化,抑制其向胶质细胞分化。Dnmt和MBD主要在神经元中表达,而在胶质细胞不表达或表达较少。DNA甲基化调节神经发生和突触的形成,参与学习记忆。星型胶质细胞的标志物GFAP去甲基化促进早期神经上皮分化为星型胶质细胞。少突胶质细胞相关基因MAG和Sox10等也受甲基化的调节。本文主要从以上方面综述了DNA甲基化在中枢神经系统发育中的作用。     

HCMV感染对胶质瘤细胞U87自噬的影响

研究人巨细胞病毒(HCMV)感染对神经胶质瘤U87细胞自噬的影响。通过观察微管相关蛋白1轻链3(LC3)、自噬相关基因Beclin1及其蛋白表达的变化,从而探讨HCMV与神经胶质瘤发生、发展的关系及意义。用HCMV AD169(MOI=5)感染神经胶质瘤U87细胞,同时将未感染HCMV的U87细胞作为对照组。分别在6、12、24、48 h用RT-PCR检测Beclin1的表达,Western-blot和免疫荧光检测Beclin1和LC3编码蛋白的表达,最后用CCK-8检测细胞的增殖活性。结果显示,HCMV感染的U87细胞LC3-II蛋白表达水平逐渐下降(P<0.05);同时,HCMV感染的U87细胞Beclin1基因及蛋白的表达水平也逐渐下降(P<0.01),且HCMV感染U87细胞增殖显著(P<0.01)。以上结果表明,HCMV感染抑制胶质瘤U87细胞自噬,并会引起Beclin1表达水平下调,进而导致胶质瘤细胞增殖。     

VHL综合征遗传学研究

VHL综合征(von Hippel-Lindau syndrome,VHL;MIM 193300)是一种常染色体显性遗传的多系统肿瘤综合征,最常见临床表现是视网膜或中枢神经系统(central nervous system,CNS)血管母细胞瘤.CNS血管母细胞瘤和肾细胞癌(renal cell carcinoma,RCC)的并发症是VHL患者最主要的死因.VHL综合征主要因VHL基因(the vonHipple-Lindau gene,VHL)突变所致,细胞周期素D1基因(the cyclin D1 gene,CCND1)突变和蛋白异常也可能参与其发生.目前已建立了多个VHL基因缺陷动物模型.在此就VHL综合征的遗传学研究进展作一概述.     

巨细胞病毒对人胚成纤维细胞骨架的影响及其凋亡诱导作用

人胚成纤维细胞是人巨细胞病毒(human cytomegalovirus,CMV)的容许细胞,CMV可在该细胞内活化复制[1].CMV感染后对细胞骨架及细胞凋亡有何影响尚不明确.国外有学者在透射电镜下观察感染CMV的人胚成纤维细胞,发现其微丝解聚,细胞骨架破坏[2],但结果尚不完善.我们的研究分别从细胞形态学、核酸水平、细胞超微水平等方面观察了感染CMV后的人胚成纤维细胞形态、肌动蛋白基因(β-actin)mRNA以及微丝的变化,并进一步观察了其对细胞凋亡的影响.     

选择性影响抗原呈递细胞CD86表达的鼠巨细胞病毒基因的鉴定[英]／Loewendorf A…／／J Virol．

巨细胞病毒(CMV)感染抗原呈递细胞(APC)可导致其功能损害,如表面协同刺激分子表达水平的降低,迄今其机制尚不清楚。本文中,作者分析并鉴定了鼠CMV(MCMV)基因组中影响APC表面CD86分子表达的1个基因。     

细胞自噬基因Atg6在涡虫中枢神经系统再生中的功能研究

细胞自噬基因Atg6在细胞自噬过程中发挥重要作用,其功能缺陷影响神经发生。涡虫是研究中枢神经系统(central nervous system, CNS)再生的良好模型,其头部切除后1周就能再生出一个新的头部。因此,研究Atg6基因在涡虫CNS再生中的作用对探究自噬调控神经发生具有重要意义。本研究首次报道了日本三角涡虫(Dugesia japonica) Atg6基因(DjAtg6)的分子特征,并利用RNAi技术研究了其在涡虫CNS再生中作用。结果显示:DjAtg6 cDNA全长1366 bp,编码423个氨基酸。DjATG6含有ATG6/Beclin 1蛋白家族的Coil-Coil结构域和β折叠α螺旋自噬功能结构域。涡虫沿咽前咽后切割后,DjAtg6表达量显著增加,其转录本主要在新再生的脑神经节表达。RNAi-DjAtg6引起涡虫头部再生迟缓、脑神经结构偏小,并下调神经相关基因的表达。此外,本研究还发现,RNAi-DjAtg6不影响涡虫干细胞的增殖,但下调细胞迁移相关基因mmp1和mmp2的表达,且干扰mmp1和mmp2的表达影响涡虫头再生。因此,本研究结果表明,DjAtg6在涡虫CNS再生的组织重构中发挥重要作用,干扰DjAtg6影响涡虫CNS再生可能与细胞迁移有关,其详细的分子机制尚需进行深入研究。     

Chk2与细胞周期调控

细胞周期检测点激酶2(Chk2)是近来新发现的一个细胞周期调控蛋白。在发生DNA损伤或复制阻滞后,细胞通过不同途径激活Chk2,进而作用于下游不同的靶蛋白,最终激活G1、S和(或)G2／M期检测点机制,使细胞周期进程发生阻滞,同时激活修复相关基因的转录,促进细胞对损伤进行修复。Chk2基因突变在肿瘤发病中具有一定意义,但其发生率较低。肿瘤细胞可通过激活Chk2来加强损伤修复,导致耐药表型产生。     

Systemic immune deficiency necessary for cytomegalovirus invasion of the mature brain

Cytomegalovirus (CMV) is a significant opportunistic pathogen associated with AIDS and immunosuppressive therapy. Infection of the mature central nervous system (CNS) can cause significant pathology with associated neurological deficits, mental disorders, and cognitive impairment and may have potentially fatal consequences. Using genetically immunocompromised mice, we studied mechanisms of CMV invasion into, and behavior within, the CNS. Adult immunodeficient (nude and SCID) and control mice were peripherally infected with recombinant mouse CMV expressing a green fluorescent protein reporter gene. Control mice actively eliminated acute peripheral infection and were resistant to invasion of CMV into the brain. In contrast, virus infected brains of immunodeficient mice but only after a minimum of 21 days postinoculation. After inoculation, CMV was found in circulating leukocytes (MAC-3/CD45⁺) and in leukocytes within the brain, suggesting these cells as a possible source of CMV entry into the CNS. CNS infection was observed in many different cell types, including neurons, glial cells, meninges, ependymal cells, and cells of cerebral vessels. Infection foci progressively expanded locally to adjacent cells, resulting in meningitis, choroiditis, encephalitis, vasculitis, and necrosis; clear indication of axonal transport of CMV was not found. Regional distribution of CMV was unique in each brain, consisting of randomly distributed, unilateral foci. Testing whether CMV gained access to brain through nonspecific vascular disruption, vascular injections of a tracer molecule revealed no obvious disruption of the blood brain barrier in mice with CMV in the brain. Results indicate the importance of host adaptive immunity (particularly T cells) in controlling entry and dissemination of CMV into the brain and are consistent with the view that virus may be carried into the brain by circulating mononuclear cells that traffic through the blood brain barrier.     

CD8+ T lymphocytes control murine cytomegalovirus replication in the central nervous system of newborn animals

Human CMV infection of the neonatal CNS results in long-term neurologic sequelae. To define the pathogenesis of fetal human CMV CNS infections, we investigated mechanisms of virus clearance from the CNS of neonatal BALB/c mice infected with murine CMV (MCMV). Virus titers peaked in the CNS between postnatal days 10-14 and infectious virus was undetectable by postnatal day 21. Congruent with virus clearance was the recruitment of CD8(+) T cells into the CNS. Depletion of CD8(+) T cells resulted in death by postnatal day 15 in MCMV-infected animals and increased viral loads in the liver, spleen, and the CNS, suggesting an important role for these cells in the control of MCMV replication in the newborn brain. Examination of brain mononuclear cells revealed that CD8(+) T cell infiltrates expressed high levels of CD69, CD44, and CD49d. IE1(168)-specific CD8(+) T cells accumulated in the CNS and produced IFN-gamma and TNF-alpha but not IL-2 following peptide stimulation. Moreover, adoptive transfer of brain mononuclear cells resulted in decreased virus burden in immunodepleted MCMV-infected syngeneic mice. Depletion of the CD8(+) cell population following transfer eliminated control of virus replication. In summary, these results show that functionally mature virus-specific CD8(+) T cells are recruited to the CNS in mice infected with MCMV as neonates.     

Enhanced cytomegalovirus infection of developing brain independent of the adaptive immune system

Cytomegalovirus (CMV) has been suggested as the most prevalent infectious agent causing neurological dysfunction in the developing brain; in contrast, CMV infections are rare in the adult brain. One explanation generally given for the developmental susceptibility to the virus is that the developing immune system is too immature to protect the central nervous system from viral infection, but as the immune system develops it can protect the brain. We suggest an alternate view: that developing brain cells are inherently more susceptible to CMV infection, independent of the immune system. We used a recombinant mouse CMV that leads to green fluorescent protein expression in infected cells. Control experiments demonstrated a high correlation between the number of cells detected with the viral GFP reporter gene and with immunocytochemical detection of the virus. After intracerebral inoculation, the number of CMV-infected cells in neonatal brains was many times greater than in mature control or mature immunodepressed SCID mice, and the mortality rate of neonates was substantially greater than SCID or control adults. Parallel experiments with live brain slices inoculated in vitro, done in the absence of the systemic immune system, generated similar data, with immature hippocampus, hypothalamus, cortex, striatum, and cerebellum showing substantially greater numbers of infected cells (100-fold) than found in adult slices in these same regions. Interestingly, in the cerebellar cortex, CMV-infected cells were more prevalent in the postmitotic Purkinje cell layer than in the mitotic granule cell layer, suggesting a selective infection of some cell types not dependent on cell division. Together, these data support the view that CMV has an intrinsic preference for infection of developing brain cells, independent, but not mutually exclusive, of the developmental status of the systemic immune system in controlling CMV infection.     

先天性巨细胞病毒感染的研究进展

巨细胞病毒(cytomegalovirus, CMV)在世界范围内均有较高的感染率,是最常见的先天性感染。先天性CMV感染可继发于母体原发性感染或非原发性感染。高达40%～50%的感染新生儿是在妊娠早期原发感染, 之后出现长期后遗症,主要包括先天性CMV感染相关听力损伤和神经后遗症。血清学检查对于确定原发性CMV感染至关重要。产前超声检查发现胎儿异常要警惕先天性CMV感染的可能性,磁共振成像有助于发现CMV相关脑异常。羊膜穿刺术是诊断胎儿CMV感染的金标准。加强育龄妇女及孕妇的卫生健康知识教育、减少CMV感染及抗病毒治疗是目前预防先天性CMV感染的主要措施。更昔洛韦及缬更昔洛韦是目前治疗CMV感染最有效的药物。高免疫球蛋白及CMV疫苗预防先天性 CMV 感染的作用尚无明确结论。     

Glucocortiocoid Treatment of MCMV Infected Newborn Mice Attenuates CNS Inflammation and Limits Deficits in Cerebellar Development

Infection of the developing fetus with human cytomegalovirus (HCMV) is a major cause of central nervous system disease in infants and children; however, mechanism(s) of disease associated with this intrauterine infection remain poorly understood. Utilizing a mouse model of HCMV infection of the developing CNS, we have shown that peripheral inoculation of newborn mice with murine CMV (MCMV) results in CNS infection and developmental abnormalities that recapitulate key features of the human infection. In this model, animals exhibit decreased granule neuron precursor cell (GNPC) proliferation and altered morphogenesis of the cerebellar cortex. Deficits in cerebellar cortical development are symmetric and global even though infection of the CNS results in a non-necrotizing encephalitis characterized by widely scattered foci of virus-infected cells with mononuclear cell infiltrates. These findings suggested that inflammation induced by MCMV infection could underlie deficits in CNS development. We investigated the contribution of host inflammatory responses to abnormal cerebellar development by modulating inflammatory responses in infected mice with glucocorticoids. Treatment of infected animals with glucocorticoids decreased activation of CNS mononuclear cells and expression of inflammatory cytokines (TNF-α, IFN-β and IFNγ) in the CNS while minimally impacting CNS virus replication. Glucocorticoid treatment also limited morphogenic abnormalities and normalized the expression of developmentally regulated genes within the cerebellum. Importantly, GNPC proliferation deficits were normalized in MCMV infected mice following glucocorticoid treatment. Our findings argue that host inflammatory responses to MCMV infection contribute to deficits in CNS development in MCMV infected mice and suggest that similar mechanisms of disease could be responsible for the abnormal CNS development in human infants infected in-utero with HCMV.     

Murine cytomegalovirus infection of neural stem cells alters neurogenesis in the developing brain

Background

Congenital cytomegalovirus (CMV) brain infection causes serious neuro-developmental sequelae including: mental retardation, cerebral palsy, and sensorineural hearing loss. But, the mechanisms of injury and pathogenesis to the fetal brain are not completely understood. The present study addresses potential pathogenic mechanisms by which this virus injures the CNS using a neonatal mouse model that mirrors congenital brain infection. This investigation focused on, analysis of cell types infected with mouse cytomegalovirus (MCMV) and the pattern of injury to the developing brain.

Methodology/Principal Findings

We used our MCMV infection model and a multi-color flow cytometry approach to quantify the effect of viral infection on the developing brain, identifying specific target cells and the consequent effect on neurogenesis. In this study, we show that neural stem cells (NSCs) and neuronal precursor cells are the principal target cells for MCMV in the developing brain. In addition, viral infection was demonstrated to cause a loss of NSCs expressing CD133 and nestin. We also showed that infection of neonates leads to subsequent abnormal brain development as indicated by loss of CD24(hi) cells that incorporated BrdU. This neonatal brain infection was also associated with altered expression of Oct4, a multipotency marker; as well as down regulation of the neurotrophins BDNF and NT3, which are essential to regulate the birth and differentiation of neurons during normal brain development. Finally, we report decreased expression of doublecortin, a marker to identify young neurons, following viral brain infection.

Conclusions

MCMV brain infection of newborn mice causes significant loss of NSCs, decreased proliferation of neuronal precursor cells, and marked loss of young neurons.     

Growth retardation and microcephaly induced in mice by placental infection with murine cytomegalovirus

BACKGROUND: The placenta is regarded as a site of congenital cytomegalovirus (CMV) infection. The placental infection of fetuses with murine CMV (MCMV) was investigated in a mouse model. METHODS: The placentas and fetuses were examined using the polymerase chain reaction (PCR) and Southern blotting for viral DNA and immunostaining for viral antigen. Since the transplacental infection rarely occurs, the placentas were directly injected with MCMV at day 12.5 of gestation; the embryos were then allowed to develop until day 18.5 of gestation. RESULTS: Formation of infected foci at day 18. 5 of gestation was found in more than 60% of the injected placentas. Infection of about 50% of the embryos occurred from the infected placentas. The frequency of infection in the brain was 27%, which was the same as that in the liver and higher than that in the lungs. In the brains, infected cells were often observed in the ventricular zone of the cerebrum and sometimes in the cortical plate and the hippocampus. Developmental retardation with microcephaly was observed in about 25% of offspring exposed to infection in utero. CONCLUSIONS: These results suggest that formation of infected foci in the placenta is important for embryonic congenital infection, and that the cerebral ventricular zone is one of the most susceptible sites for CMV infection in the embryonic stage.     

A recombinant rhesus cytomegalovirus expressing enhanced green fluorescent protein retains the wild-type phenotype and pathogenicity in fetal macaques

To facilitate identification of rhesus cytomegalovirus (RhCMV)-infected cells, a recombinant virus expressing enhanced green fluorescent protein (EGFP), designated RhCMV-EGFP, was constructed. An expression cassette for EGFP under the control of the simian virus 40 (SV40) early promoter was inserted into the intergenic region between unique short 1 (US1) and US2 of the RhCMV genome by homologous recombination. RhCMV-EGFP exhibited comparable growth kinetics to that of wild-type virus in rhesus fibroblast cultures and retained its pathogenicity in monkey fetuses. Typical neurologic syndromes caused by CMV infection were observed in all fetuses experimentally inoculated with RhCMV-EGFP, as evidenced by sonographic and gross examinations. Systemic RhCMV infections were established in all fetuses, as viral antigen was detected in multiple organs and virus was isolated from fetal blood samples. The engineered viral genome was stable following rapid serial passages in vitro and multiple rounds of replication in vivo. Infected cells could be readily distinguished by green fluorescence both in tissue cultures and in the fetuses. In addition, EGFP expression was detected in various cell types that were permissive to RhCMV infection, consistent with a broad tissue tropism of the SV40 promoter. These results demonstrate that RhCMV can be successfully engineered without loss of wild-type replication and pathogenic potential. Further, the spectrum of cortical anomalies and the distribution of infected cells in the brain tissues indicated that RhCMV may have preferentially targeted immature neuronal cells. The pattern of RhCMV infection in the central nervous system may offer an explanation for the severe developmental outcomes associated with congenital human CMV infection early in gestation.