乙型肝炎病毒对载脂蛋白B表达的影响及其机制探讨

目的 探讨乙型肝炎病毒(HBV)对载脂蛋白B(ApoB)表达的影响,并探讨其调节机制.方法 采用RT-PCR和Western blot法检测HepG2和HepG2.2.15中ApoB mRNA和蛋白的表达,全自动生化分析仪Olympus 5400检测HBV患者和健康对照者ApoB血清学水平,分析健康对照者、慢性乙型肝炎、肝纤维化和肝癌患者中ApoB表达水平的差异,将HBV感染性克隆pHBV1.3转染HepG2细胞,RT-PCR和Western blot法检测ApoB和微粒体甘油三酯转移蛋白(MTP)表达水平的变化.结果 HepG2.2.15细胞中ApoB mRNA和蛋白的表达水平较HepG2低;ApoB在慢性乙型肝炎患者和肝纤维化患者的血清学水平明显低于健康对照者(P＜0.05);HBV能够在mRNA和蛋白水平抑制ApoB和MTP的表达.结论 HBV可能通过抑制MTP的表达抑制ApoB的合成和分泌.

Abstract：

Objective To explore the effect of hepatitis B virus(HBV) on the expression of apolipoprotein B(ApoB) and its regulatory mechanism. Methods mRNA and protein expression of ApoB in HepG2 and HepG2.2.15 cells was measured by RT-PCR and Western blot, serum ApoB levels in patients with HBV infection and in healthy individuals were measured by biochemical analyzer Olympus 5400, the expression of ApoB difference among healthy individuals, patients with chronic hepatitis B, liver cirrhosis, and hepatocellular carcinoma were analyzed, HBV infectious clone pHBV1.3 was tranfected into HepG2 cells,and expression of ApoB and microsomal triglyceride transfer protein(MTP) was measured by RT-PCR and Western blot. Results Expression of ApoB mRNA and protein was lower in HepG2.2.15 cells than in HepG2 cells, serum apoB levels was much lower in patients with chronic hepatitis B and liver cirrhosis as compared to healthy individuals( P ＜0.05 ), HBV could inhibit the expression of ApoB and MTP at mRNA and protein levels. Conclusion HBV may downregulate the synthesis and secretion of ApoB via inhibits the expression of MTP.     

乙型肝炎病毒在体内外抑制补体C3和C4表达的研究

目的 探讨乙型肝炎病毒(HBV)对补体C3和C4表达的影响,并探讨其调节机制.方法 采用基因芯片筛选HepG2和HepG2.2.15细胞的差异表达基因,免疫比浊法检测HBV患者和健康对照者补体C3和C4血清学水平,将HBV感染性克隆pHBV1.3转染HepG2细胞,RT-PCR和Western blot法检测C3和C4表达水平的变化.结果 补体C3和C4 mRNA在HepG2.2.15细胞中水平降低;C3和C4在慢性乙型肝炎患者和肝癌患者的血清学水平明显低于健康对照者(P＜0.05);HBV能够在mRNA和蛋白水平下调C3和C4的表达.结论 HBV能够在体内外抑制补体C3和C4的表达.     

HBV通过增强DNAJB4启动子活性调节其在HepG2.2.15细胞中的表达

目的 初步探讨HBV与DNAJB4基因的转录调节作用机制.方法 用RT-PCR及Real-time PCR检测HepG2.2.15细胞和HepG2细胞中DNAJB4在mRNA水平上的表达差异.构建DNAJB4启动子虫荧光素酶报告质粒,分别与HBV、HBs、HBp、HBc、HBx表达质粒共转染HepG2细胞,比较虫荧光素酶活性.结果 在mRNA水平上,DNAJB4在HepG2.2.15细胞中的表达量是其在HepG2细胞中表达量的2.59倍,且二者有显著性差异(P＜0.01).DNAJB4启动子虫荧光素酶报告质粒与HBV表达质粒共转染组的相对荧光素酶活性是其对照组的2.28倍.转染HBs和HBc表达质粒组的相对荧光素酶活性分别是其对照组的2.11倍和1.77倍,而HBx、HBp表达质粒对荧光素酶活性没有影响.结论 在HepG2.2.15细胞中,HBV能通过增强DNAJB4启动子活性增加DNAJB4转录水平的表达,其中HBs和HBc蛋白起主要作用.     

乙型肝炎病毒对超敏C反应蛋白表达的影响及其临床意义

目的 探讨乙型肝炎病毒( HBV)对超敏C反应蛋白(hs-CRP)表达的影响及其临床意义.方法 采用RT-PCR检测人肝癌细胞系HepG2和HepG2.2.15中hs-CRP mRNA的表达,通过Olympus5400全自动生化分析仪检测HBV患者和健康对照者hs-CRP血清学水平,分析hs-CRP在慢性乙型肝炎、肝硬化和肝癌患者中hs-CRP表达水平的差异.结果 HepG2.2.15细胞中hs-CRPmRNA的表达水平较HepG2高;hs-CRP在乙肝感染者血清学水平显著升高(P＜0.05);hs-CRP在肝硬化患者和肝癌患者明显高于慢性肝炎患者.结论 HBV能够上调hs-CRP的表达,其血清学水平与疾病进程相关.     

shRNA沉默HBx下调HepG2.2.15细胞基质金属蛋白酶2的表达

目的探讨HBx小发夹RNA(shRNA)对HepG2.2.15细胞中基质金属蛋白酶2(MMP-2)表达的影响。方法采用psiHBV/X质粒转染HepG2.2.15细胞,反转录PCR评估沉默效率;MTT法检测转染后HepG2.2.15细胞增殖情况;实时定量PCR(qRT-PCR)检测MMP-2 mRNA表达;Western blot法检测MMP-2蛋白表达的变化。结果反转录PCR检测HBx基因的沉默效率为53.6%;MTT检测结果显示转染24、48、72 h后HepG2.2.15细胞的增殖受抑制,与对照组比较,差异有统计学意义(P0.05);psiHBV/X质粒转染HepG2.2.15细胞后,qRT-PCR和Western blot检测结果显示,HepG2.2.15细胞中MMP-2基因的mRNA水平和蛋白水平表达分别有不同程度的下调,与对照组比较差异有统计学意义(P0.05)。结论 RNA干扰技术抑制HepG2.2.15细胞中HBx基因的表达,可抑制细胞增殖,下调HepG2.2.15细胞中MMP-2的表达。     

APOBEC3F的克隆、真核表达及其抗HBV效应的初步研究

目的克隆、体外真核表达载脂蛋白B mRNA编辑酶催化多肽样3F(APOBEC3F)基因并研究其抗病毒效应。方法应用RT-PCR的方法从人PBMC细胞中扩增APOBEC3F基因,构建HA-APOBEC3F融合蛋白真核表达载体pXFA3F,pXFA3F和具有复制能力的1.3倍HBV载体pHBV1.3共转染HepG2细胞,Western印迹检测APOBEC3F融合蛋白在HepG2细胞中的表达,ELISA方法检测细胞培养上清液中的HBsAg和HBeAg水平,实时定量PCR检测HBV相关mRNA水平变化。结果克隆了APOBEC3F基因,其经测序与GeneBank公布序列一致;构建的APOBEC3F真核表达载体pXFA3F经转染可在HepG2细胞中瞬时表达;APOBEC3F可抑制乙型肝炎表面抗原和e抗原的分泌,可使转染细胞内HBV相关mRNA水平下降。结论成功克隆并真核表达了APOBEC3F基因,其在体外具有抗HBV生物学效应。     

设计特异性识别HBV核心启动子的锌指蛋白以抑制HepG2.2.15细胞中HBV的转录

目的:设计特异性结合乙肝病毒核心启动子(HBVCp)的锌指蛋白(ZFP),观察ZFP在体外对HBV转录的抑制情况。方法:利用Zinc finger tools设计特异性结合HBV Cp的ZFP。将ZFP核苷酸序列人工合成后克隆至pEGFP-N1和pcDNA3.1(+),构建真核表达质粒pEGFP-N1/ZFP-Flag和pcDNA3.1(+)/ZFP。通过倒置荧光显微镜、RT-PCR和Western blot鉴定pEGFP-N1/ZFP-Flag在COS-7中的表达情况;将pcDNA3.1(+)/ZFP转入HepG2.2.15细胞后24 h,采用ELISA和FQ-PCR检测上清液中HBeAg和HBV DNA含量,RT-PCR检测细胞内HBV mRNA水平。结果:ZFP在COS-7细胞中能正常表达。与空质粒组相比,ZFP组细胞培养上清液中HBV DNA拷贝量和HBeAg含量明显降低(P<0.05),细胞内HBV mRNA也显著减少。结论:人工设计的ZFP可以在真核细胞内正常表达,并能有效抑制HBV的转录。     

乙型肝炎病毒影响肝细胞对双链DNA诱导产生的Ⅰ型干扰素应答

目的:了解乙型肝炎病毒(HBV)是否影响肝细胞对双链DNA(dsDNA)分子诱导产生的Ⅰ型干扰素应答。方法:以肝癌细胞HepG2以及整合有HBV基因的HepG2.2.15细胞为模型,转染dsDNA分子poly(dA-dT),实时定量RT-PCR检测IFN-β、干扰素应答基因IFIT1与炎症因子TNF-α的表达,Western blot检测NF-κB、IRF3的核转位以及pTBK1的表达。HBV复制型质粒HBV1.3转染HepG2、HBV siRNA转染HepG2.2.15后,实时定量RT-PCR检测细胞中IFIT1的表达。结果:poly(dA-dT)能够诱导HepG2细胞产生Ⅰ型干扰素,在转染后6小时,IFN-β与IFIT1表达达高峰,而后下降,TNF-α的表达与对照组相比没有明显变化。Poly(dA-dT)转染后6小时能检测到TBK1的磷酸化与IRF3的核转位,但NF-κB的核转位不明显。HepG2.2.15细胞在poly(dA-dT)转染后12小时能检测到TNF-α的一过性表达,而IFN-β与IFIT1的表达在72小时才达到高峰,与之对应的:在转染后6小时,没有检测到TBK1的磷酸化与IRF3的核转位。poly(dA-dT)与HBV1.3共转HepG2后,HBV的蛋白表达受到抑制,而将HBV siRNA转染HepG2.2.15后,细胞对poly(dA-dT)的应答明显增强。结论:HepG2与HepG2.2.15能够应对ds-DNA的刺激,通过NF-κB与IRF3信号通路产生Ⅰ型干扰素或炎症因子,但HBV的存在影响了肝细胞对dsDNA产生的固有免疫应答。     

SAMHD1 对HBV DNA 形成的抑制作用及其机制研究

目的:研究不同肝细胞SAMHD1 表达水平及其对HBV DNA 形成的抑制作用和相关机制。方法:通过实时荧光定量PCR 和Western blot 法检测Jurkat、THP-1、HepG2、HepG2.2.15 和Huh7 细胞等细胞SAMHD1 mRNA 和蛋白表达水平;通过对HepG2.2.15 细胞转染过表达质粒和shRNA,升高或者降低SAMHD1 的表达水平,观察不同SAMHD1 表达水平对HBV DNA 水平的影响;补充不同浓度的dNTP,比较不同dNTP 浓度对HBV DNA 水平的影响。结果:肝癌细胞系有较高的SAMHD1表达水平;降低SAMHD1 可以使HBV DNA 水平升高2.3 ~ 2.8 倍,升高SAMHD1 可以使HBV DNA 降低至33% 左右(P<0.01);升高dNTP 浓度可以促进HBV DNA 的合成水平,抵消SAMHD1 的抑制作用。结论:SAMHD1 可以抑制肝癌细胞内HBV DNA 的生成,可能是通过降低细胞内dNTP 浓度实现的。     

巯基化Ras相关蛋白7a抑制人肝癌细胞系HepG2.2.15中HBV复制

目的探究人肝癌细胞HepG2.2.15中Ras相关蛋白7a (Rab7a)的巯基化机制及其对乙型肝炎病毒(HBV)复制的影响。方法通过生物素转换实验检测H2S对HepG2.2.15细胞中Rab7a巯基化水平的影响并验证Rab7a的巯基化位点;通过酶联免疫法、RT-qPCR和Western blot检测巯基化的Rab7a对HBV复制标志物水平的影响。结果在HepG2.2.15细胞中H2S可增强内源性Rab7a的巯基化水平(P<0.01);NaHS(H2S速释供体)可增强外源性Rab7a的巯基化表达,但其巯基化位点突变后则不受NaHS影响(P<0.01);Rab7a巯基化后,可抑制HepG2.2.15细胞上清中HBsAg、HBeAg和HBV-DNA及细胞内HBV-cccDNA、3.5kb RNA、total RNA和HBcAg蛋白的表达(P<0.05)。结论 Rab7a的巯基化抑制HBV阳性细胞系HepG2.2.15细胞中HBV的复制水平。     

Asymmetry of the internal connections of the striate cortex of the cat in the projection zone of the center of the field of vision

Studies were carried out on the organization of the internal connections of the striate cortex in cats in the projection zone of the center (0–5°) of the field of vision by microintophoretic application of horseradish peroxidase to electrophysiologically identified orientational columns. The area containing neurons showing retrograde labeling in most cases extended in the mediolateral direction. Labeled cells were located in the upper (II, III) and lower (V, VI) layers of the cortex, and the shapes and orientations of the areas containing labeled neurons in these layers coincided. Spatial asymmetry was detected in the distribution of labeled neurons relative to the orientational column studied. Labeled cells were located predominantly medial to the columns, regardless of the distance from the projection of the area centralis. Considering the visuotopical map of field 17, the asymmetry detected here provides evidence that neurons in orientational columns have more extensive connections with neurons of the peripheral part of the cortex. An asymmetrical distribution of “silent” zones around the receptive fields of neurons in orientational columns is suggested, and that these appear to receive influences from the periphery of the visual field. Laboratory of Visual Physiology and Laboratory of Central Nervous System Morphology, I. P. Pavlov Institute of Physiology, Russian Academy of Sciences, 6 Makarov Bank, 199034 St. Petersburg, Russia. Translated from Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 82, No. 12, pp. 23–29, December, 1996.     

Effects of the lesion of the postcommissural part of the septum on the behavior of the rat.

The effects of the lesion of the postcommissural part of the septum on behavior of the rat has been studied. Results may be summarized as follows. An increase in the exploratory behavior in the open field which decreases rapidly; a decrease in the number of defecations in this test and a decrease in time leaving a dark environment for exploration. In the shuttle box test, no facilitation of the acquisition, but a permanent and quite significant increase in the intertrial activity has been found. We conclude that the lesions tend to decrease the emotivity of the subjects. An interpretation on the basis of the species -- specific defensive reactions explains the transitory and permanent effects of the lesions on the spontaneous activity.     

Counterpointing the functional role of the forebrain and of the brainstem in the control of the sleep-waking system

This paper reviews the lifetime contributions of the author to the field of sleep-wakefulness (S-W), reinterprets results of the early studies, and suggests new conclusions and perspectives. Long-term cats with mesencephalic transection show behavioral/polygraphic rapid eye movement sleep (REMS), including the typical oculo-pupillary behavior, even when the section is performed in kittens prior to S-W maturation. REMS can be induced as a reflex. Typical non-rapid eye movement S (NREMS) is absent and full W/arousal is present only after a precollicular section. The isolated forebrain (IF) rostral to the transection exhibits all features of W/arousal and NREMS [with electroencephalographic (EEG) spindles and delta waves], arousal to olfactory stimuli, and including the appropriate oculo-pupillary behaviors. These features also mature normally after neonatal transection. REMS is absent from the IF. After deprivation there is NREMS pressure and rebound in the IF, but the decerebrate cat only shows pressure for REMS. Most IF reactions to pharmacologic agents are within expectations, except for the tolerance/withdrawal effects of long-term morphine use which are absent. In contrast, these effects are supported by the brainstem (i.e. seen in the decerebrate cat). In cats with ablation of the telencephalon, or diencephalic cats, delta waves are absent in the thalamus. EEG thalamic spindle waves are seen triggering S for only 4-5 days after ablation. Therefore, true NREMS is absent in chronic diencephalic cats although pre- and postsomniac behaviors persist. These animals are hyperactive and show a pronounced, permanent insomnia; however, a low dose of barbiturate triggers a dramatic REMS/atypical NREMS rebound. Cats without the thalamus (athalamic cats), initially show a dissociation between behavioral hyperactivity/insomnia and the neocortical EEG, which for 15-20 days exhibits only delta and slower oscillations. Fast, low-voltage W rhythms appear later on, first during REMS, but spindle waves and S postures are absent from the start, such that these cats also display only atypical NREMS. Athalamic cats also show barbiturate-sensitive insomnia. Cats with ablation of the frontal cortices or the caudate nuclei remain permanently hyperactive. They also show a mild, but significant hyposomnia, which is permanent in afrontal cats, but lasts for about a month in acaudates. The polygraphic/behavioral features of their S-W states remain normal. We conclude and propose that: (a) the control of the S-W system is highly complex and distributed, but is organized hierarchically in a well-defined rostro-caudal manner; the rostral-most or highest level (telencephalon), is the most functionally complex/adaptative and regulates the lower levels; the diencephalic/basal forebrain, or middle level, has a pivotal role in inducing switching between S and W and in coordinating the lowest (brainstem) and highest levels; (b) W can occur independently in both the forebrain and brainstem, but true NREMS- and REMS-generating mechanisms exist exclusively in the forebrain and brainstem, respectively; (c) forebrain and brainstem S-W processes can operate independently from each other and are preprogrammed at birth; this helps understanding normal and abnormal polygraphic/behavioral dissociations in humans and normal dissociations/splitting in aquatic mammals; (d) NREMS homeostasis is present in the IF, but only REMS pressure after deprivation persists in the decerebrate cat; (e) the thalamus engages in both NREMS and W; (f) insomnia in diencephalic cats is the result of an imbalance between antagonistic W- and S-promoting cellular groups in the ventral brain (normally modulated by the telencephalon); (g) the EEG waves, which are signature for each S-W state, appear to truly drive the concomitant behaviors, e.g. a hypothetical human IF could alternate between behavioral NREMS and W/arousal/awareness; (h) a role for REMS is to keep the individual sleeping at the end of the self-limiting NREMS periods. The need for accelerating research on telencephaling NREMS periods. The need for accelerating research on telencephalic S-W processes and downstream control of the lower S-W system levels is emphasized.     

The development of the ligament of the head of the femur

The hip joints of 30 human male and female fetuses and stillborns between 20 mm and 350 mm crown-rump length were studied by light microscopy. The ligament of the head of the femur developed in situ as a condensation of mesenchyme at the end of the second month of intra-uterine life (IUL), and was vascularized by branches of acetabular vessels early in the fourth month. In the majority of fetuses older than 5.5 months IUL, vessels in the ligament passed a short way into the femoral head within cartilage canals, to supply a small region around the fovea capitis. The remainder of the head was supplied by vessels in canals from around the upper part of the neck. The ligament changed from predominantly cellular to fibrous during the last 4 months of IUL. This increase in strength suggested significant mechanical functions in utero: limitation of adduction-flexion and opposition to postero-superior dislocation were the most likely.     

Topical organization of the projections of neurons of the substantia nigra and the ventral field of the tegmentum of the midbrain to the head of the caudate nucleus of the cat

Neuroscience and Behavioral Physiology -     

Involvement of the striatum in the central regulation of the hormonal functions of the gonads

Studies reported here show that intrastriatal administration of corticoliberin to rats decreases the blood testosterone level. However, in conditions of chemical deficiency of dopaminergic transmission in the dorsal striatum induced by injection of 6-hydroxydopamine, the effect of this neurohormone did not appear. It is concluded that extrahypothalamic corticoliberin is involved in regulating the hormonal reproductive system acting via dopaminergic mechanisms. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 85, No. 4, pp. 594–597, April, 1999.     

Ultrastructure of the endothelium of the drainage system of the eye

The endothelium of the ocular drainage system (Schlemm’s canal, collector tubules, and aqueous veins) in primary juvenile glaucoma undergoes degenerative dystrophic changes with compensatory hypertrophy and proliferation at the initial stages of the glaucomatous process and atrophy and desquamation at advanced and terminal stages. Progressive decrease in the pinocytous function of endotheliocytes, reduction of the protein-synthesizing and mitochondrial compartments of the cytoplasm, and formation of autophagosomes reflect the process of endotheliocyte degeneration in general. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 5, pp. 574–577, May, 2008