PTEN在肝纤维化大鼠肝组织中的动态表达

目的： 探讨大鼠肝纤维化过程中肝组织PTEN的动态表达。方法: 采用胆总管结扎法建立大鼠肝纤维化模型,HE及Masson三色染色检测肝脏组织学变化,免疫组织化学染色、Western blotting及实时荧光定量PCR技术检测大鼠肝组织的PTEN蛋白及mRNA表达。结果: 大鼠肝纤维化模型成功建立,随着造模时间延长,肝纤维化程度逐渐加重,造模不同时间均可见不同程度的肝细胞变性坏死而导致正常肝细胞逐渐减少;免疫组织化学染色显示正常大鼠肝组织中PTEN广泛表达,主要表达于细胞浆,随着肝纤维化的进展,PTEN阳性表达细胞逐渐减少（P<0.01）;Western blotting及实时荧光定量PCR显示造模1周、2周、3周及4周不同时间大鼠纤维化肝组织中PTEN蛋白及mRNA表达均显著低于假手术组（P<0.01）,并随着肝纤维化的进展逐渐降低（P<0.01）。结论: 大鼠纤维化肝组织中PTEN蛋白及mRNA表达均随着肝纤维化的进展逐渐降低,其下降程度与肝纤维化程度一致。     

肝纤维化大鼠肝脏中内质网应激相关分子CHOP和TRB3的表达变化

 目的： 观察内质网应激相关分子CCAAT/增强子结合蛋白同源蛋白(CHOP）和Tribbles同源蛋白3（TRB3）在四氯化碳（CCl4）致大鼠肝纤维化过程中的表达变化,探讨其在肝纤维化过程中的可能作用。方法： 体重180～200　g的雄性Wistar大鼠随机分为正常4周组、正常8周组、肝纤维化4周组和肝纤维化8周组,肝纤维化组大鼠皮下注射40%CCl4制备肝纤维化模型,分别在4周和8周处死大鼠,观察肝组织病理改变,Western blotting检测肝脏活化转录因子6（ATF6）蛋白,免疫组化、Western blotting和real-time PCR分别检测肝脏CHOP和TRB3蛋白和mRNA表达变化,TUNEL法检测肝脏细胞凋亡。结果： 肝纤维化组大鼠肝脏可见假小叶形成,p90ATF6蛋白表达量较正常组明显减少（P<0.01）,p50ATF6蛋白表达量较正常组明显增加（P<0.01）,肝细胞胞浆CHOP和TRB3蛋白及mRNA表达量较正常组显著增加（P<0.01）,细胞凋亡率较正常组显著增加（P<0.01）。结论： 内质网应激相关分子CHOP和TRB3在CCl4致大鼠肝纤维化过程中蛋白及mRNA表达水平明显增加,其变化趋势与大鼠肝细胞凋亡率一致,提示内质网应激可能通过CHOP和TRB3促进肝细胞凋亡,参与肝纤维化发生发展。     

钙中性蛋白酶2及Bax在肝纤维化大鼠肝组织中的表达变化及意义

 目的：探讨钙中性蛋白酶2（calpain 2）及Bcl-2相关X蛋白(Bax)在肝纤维化过程中的表达变化及其在肝纤维化过程中的可能作用。方法:  雄性Wistar大鼠40只,随机分为正常对照4周、8周组和肝纤维化模型4周、8周组,每组各10只。肝纤维化组按3 mL/kg体重的剂量皮下注射40% CCl4植物油溶液,每隔3 d注射1次,造模时间分别为4周和8周;对照组大鼠皮下注射等量植物油溶液。采用TUNEL法检测肝组织中肝细胞凋亡情况;real-time PCR检测肝组织中calpain 2及bax mRNA的表达变化。免疫组织化学法及Western blotting检测肝组织中calpain 2及Bax蛋白的表达情况。 结果:  Real-time PCR检测发现肝纤维化4周和8周组大鼠肝组织中calpain 2及bax mRNA表达较相应的正常对照组显著增加。免疫组化及Western blotting检测显示肝纤维化4周组大鼠肝组织中calpain 2蛋白的表达与正常4周组比较无显著差异;随着肝纤维化程度的加重,肝纤维化8周时大鼠肝组织中calpain 2的表达显著增加;而肝组织中Bax的表达从肝纤维化4周时就显著增加,肝纤维化8周时达到高峰。此外,通过TUNEL法检测发现肝纤维化4周和8周组大鼠肝组织中肝细胞凋亡的数目较正常组大鼠显著增加。结论:  Calpain 2与Bax可能参与了肝纤维化的发生发展过程。     

PI3K/Akt信号通路及内质网应激在肝纤维化大鼠肝细胞凋亡中的作用

目的观察磷脂酰肌醇-3激酶/蛋白激酶(PI3K/Akt)信号通路及葡萄糖调节蛋白78(GRP78)、生长停滞及DNA损伤基因(CHOP/GADD153)在四氯化碳(CCl4)诱导的肝纤维化中的表达并探讨其可能的作用。方法将30只SD大鼠随机分为正常组、肝纤维化模型(皮下注射40%CCl4橄榄油溶液)4及8周组。HE染色法观察肝组织病理形态学;用real-time PCR技术检测肝脏内GRP78及CHOP mRNA的表达;用Western blot检测肝脏内PI3K/Akt信号通路中Akt1、磷酸化Akt1及内质网应激相关蛋白GRP78及CHOP的表达;用原位末端转移酶标记(TUNEL)检测细胞凋亡。结果与正常组大鼠比较,肝纤维化模型4及8周组大鼠肝脏内GRP78及CHOP mRNA和蛋白表达均明显升高(P0.05),而肝脏内Akt1和磷酸化Akt1蛋白的表达则较正常大鼠显著降低(P0.05);与正常组大鼠比较,肝纤维化模型4及8周组大鼠肝细胞凋亡显著升高(P0.05)。结论 PI3K/Akt信号通路及内质网应激可能在肝纤维化大鼠肝细胞凋亡中发挥了重要作用。     

大鼠肝纤维化组织内ROCK-I、磷酸化MBS Thr-697和α-SMA表达增强

 目的 探讨ROCK-I、磷酸化MBS Thr-697、α-SMA蛋白及其mRNA在实验性大鼠肝纤维化形成过程中的动态表达及肌动蛋白细胞骨架的变化。方法 结扎胆总管制备肝纤维化模型,用免疫组织化学、Western blot与RT-PCR检测ROCK-I、α-SMA蛋白及其mRNA的表达,用特异性荧光染色检测肌动蛋白细胞骨架。结果 随着肝纤维化的发展,大鼠肝脏中ROCK-I及α-SMA阳性细胞明显增多;造模1~4周肝组织ROCK-I、α-SMA的阳性面积及ROCK-I、磷酸化MBS Thr-697、α-SMA蛋白表达显著性高于正常组（P<0.05）;ROCK-I基因表达与α-SMA表达呈正相关(r=0.718,P<0.05);随着造模时间延长,肝组织肌动蛋白荧光信号增强。结论 ROCK-I在肝纤维化发生过程中有表达上调和功能活化,可能通过诱导肌成纤维细胞表型形成而参与肝纤维化的发生。     

姜黄素和γ-干扰素对肝纤维化大鼠肝组织病理变化及TIMP-1表达的作用

目的:观察肝纤维化大鼠肝组织病理学变化及金属蛋白酶组织抑制因子-1(TIMP-1)的表达水平,探讨姜黄素和γ-干扰素及两者联合治疗抗肝纤维化的作用。方法:将40只雄性SD大鼠随机分为正常对照组、模型组和治疗组,治疗组中又分为姜黄素治疗、γ-干扰素治疗及联合治疗3个亚组,采用CCl4腹腔注射制作大鼠肝纤维化模型。各治疗组采取相应药物和剂量平行治疗4周,第10周末将各组大鼠处死,取肝组织切片HE染色观察组织病理学变化(包括肝纤维化程度);用半定量RT-PCR方法检测各组肝组织TIMP-1mRNA相对表达量。结果:肝组织病理学观察显示,模型组大鼠肝索排列紊乱,肝细胞变性坏死,汇管区和小叶内出现局限性纤维化,部分区域出现纤维纵隔,甚至假小叶形成,纤维化分级为Ⅳ～Ⅴ级(Ⅴ级为主),TIMP-1mRNA相对表达量较正常对照组明显升高(P<0.01);各治疗组肝组织病理变化明显改善,肝细胞变性坏死减少,肝纤维化程度明显减轻(以Ⅲ级为主),TIMP-1mRNA表达量明显低于模型组(P<0.01)。结论:姜黄素和γ-干扰素及两者联合治疗均能减轻肝纤维化大鼠组织病理学变化并下调肝组织TIMP-1mRNA表达,改善和逆转肝纤维化进程。     

DcR3 对肝纤维化动物模型的影响

目的:研究DcR3 基因对肝纤维化大鼠的预防性治疗的作用。方法:SPF 级健康雄性Wistar 大鼠30 只,体重范围180 ~220 g,随机分为3 组,每组10 只,分别为正常对照组、DcR3 基因预防性治疗组(1% DMN+DcR3 组)、造模组(1%DMN 组)。采用1%DMN 诱导大鼠肝纤维化模型,腹腔注射DcR3 质粒进行预防性干预。分别采用HE 染色和Masson 染色观察肝组织病理情况;qRT-PCR 和Western blot 法检测DcR3、Fas、FasL、-SMA 和TGF-1 的mRNA 和蛋白表达水平。结果:与模型组相比,DcR3 预防治疗组大鼠肝组织炎性细胞浸润及胶原类物质沉积有所改善;DcR3 基因可显著降低肝纤维化大鼠Fas、FasL、 SMA 和TGF-1 mRNA 和蛋白表达水平,DcR3 基因预防性治疗组与对照组和造模组有显著性差异(P<0.05);同时DcR3 基因预防性治疗组DcR3 mRNA 和蛋白表达水平显著升高(P<0.05)。结论:DcR3 基因可有效防治大鼠肝纤维化,其作用机制可能是DcR3 通过降低肝脏炎症反应,减少胶原类物质沉积,抑制 SMA 和TGF-1 表达,从而抑制HSC 活化;下调Fas和FasL 表达,抑制Fas/ FasL 途径诱导的肝细胞凋亡。     

肝纤维化大鼠肝细胞内质网形态及GRP78表达的研究

目的: 观察四氯化碳(CCl₄)致大鼠肝纤维化过程中内质网形态及内质网应激标志性蛋白——葡萄糖调节蛋白78(GRP78)的表达变化, 探讨内质网应激在肝纤维化发病机制中可能的作用。方法: 雄性Wistar大鼠皮下注射CCl₄制备肝纤维化模型,分别在4周及8周处死大鼠测定肝脏指数、血清丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)活性, 观察肝组织病理改变和肝细胞内质网形态, 免疫组化和real-time PCR分别检测肝组织GRP78蛋白及mRNA表达变化。结果: 肝纤维化组大鼠肝脏指数、血清ALT和AST活性显著高于正常对照组(P<0.01),肝纤维化明显, 电镜下见肝细胞内质网扩张,数量明显减少; 肝细胞胞浆中GRP78蛋白表达量及mRNA表达量较正常组显著增加(P<0.01)。结论: 在CCl₄诱导的肝纤维化发生过程中肝细胞内质网形态有明显损伤性变化, 内质网应激蛋白GRP78蛋白及基因表达水平明显增加, 提示内质网应激参与肝纤维化发生发展。     

四氯化碳诱导性肝硬化大鼠肝组织葡萄糖调节蛋白78表达的研究

目的研究四氯化碳(CCl4)诱导性肝硬化大鼠肝组织内葡萄糖调节蛋白78(GRP78)表达的变化。方法将健康雄性Wistar大鼠随机分为对照组(n=5)和肝硬化组(n=5),对照组皮下注射植物油0.12ml/100g体重,肝硬化组以0.3ml/100g体重皮下注射60%CCl4植物油。在实验第21周的第1d杀死鼠,右心室取血检测肝功能指标。鼠肝中叶取材,石蜡切片行苏木精-伊红(HE)染色和天狼猩红组织化学染色,光镜观察形态学改变,行GRP78免疫组织化学显色,无菌下取肝组织行Western blotting及RT-PCR检测肝组织GRP78的表达。结果肝硬化组的血清ALT含量(278.2±88.42)明显高于对照组(154.8±9.94,t=3.10,P<0.05);ALB含量(1.68±0.62)明显低于对照组(3.02±1.96,t=2.62,P<0.05)。对照组肝小叶结构和肝细胞状态正常,仅在中央静脉及汇管区可见少量胶原纤维。20周肝硬化组的肝细胞脂肪变性明显,胶原纤维大量增生,假小叶形成。GRP78免疫组织化学呈色显示,肝硬化组肝组织中阳性细胞数明显多于对照组,棕黄色着色定位于细胞质中。Western blotting和RT-PCR显示肝硬化组肝组织GRP78的表达明显增强。结论在CCl4诱导性肝硬化的肝组织中,GRP78表达明显增强,这可能与肝细胞的内质网应激有关。     

蓝莓对大鼠肝纤维化模型中转录因子KLF11表达的影响

目的:观察蓝莓对肝纤维化模型中抗纤维化转录因子Krüppel样因子11(KLF11)的影响,探讨其可能的机制。方法:40只健康同周龄SD大鼠被随机分为正常对照组、蓝莓对照组、模型组和蓝莓预防组,每组10只,使用四氯化碳制作肝纤维化模型。测定各组大鼠血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、Ⅳ型胶原、Ⅲ型前胶原(PCⅢ)、透明质酸(HA)和层粘连蛋白水平;Masson和HE染色对肝组织进行病理学检查;Western blot和免疫组织化学染色检测肝组织中KLF11的蛋白表达,Western blot检测肝组织中α-平滑肌肌动蛋白(α-SMA)的蛋白表达;real-time PCR检测肝组织中KLF11和Ⅰ型胶原(ColⅠ)的mRNA表达情况。结果:与正常对照组比较,模型组KLF11蛋白表达显著降低(P<0.01),而α-SMA蛋白表达显著升高(P<0.01);与模型组比较,蓝莓预防组KLF11的mRNA表达显著升高(P<0.01)而ColⅠ的mRNA表达显著降低(P<0.01);与模型组比较,其它各组大鼠血清ALT、AST、HA和PCⅢ水平均显著降低(P<0.01),蓝莓预防组大鼠肝纤维化分级显著降低(P<0.01)。结论:蓝莓对四氯化碳所致大鼠肝纤维化有一定的预防作用,其机制可能与其影响KLF11表达有关。     

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.     

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.     

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.     

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