大鼠额叶损伤后诱导型一氧化氮合酶的表达变化

为了探讨大鼠额叶损伤后不同时间诱导型一氧化氮合酶(iNOS)的表达变化及意义,本研究采用大鼠额叶锐器损伤模型,经Nissl和H.E.染色观察伤后的病理变化过程,RT-PCR及免疫组织化学染色方法检测伤后iNOSmRNA和iNOS阳性细胞的表达变化。结果显示:伤后12、24h创伤区炎症细胞大量浸润;创伤区及周边iNOSmRNA的表达3h开始上升,24h达到高峰;伤后iNOS阳性细胞数量也增多,伤后3、6h主要由神经细胞表达iNOS,在12、24h主要由巨噬细胞表达iNOS,而72、120h则主要由胶质细胞表达iNOS。上述结果说明大鼠额叶锐器伤后iNOS的表达增加,iNOS阳性细胞的种类和数量变化与伤后时程有关。

泮托拉唑对应激损伤大鼠胃黏膜修复作用的研究

目的:观察急性损伤的胃黏膜雌激素诱导基因相关肽(TFF1)表达及应用泮托拉唑后的变化,探讨TFF1在应激胃黏膜损伤中的作用机制。方法:56只大鼠随机分为正常组、单纯造模组及造模干预组,各造模组制作大鼠水浸-束缚应激(WRS)模型,其中单纯造模组分为单纯造模1组(造模后即刻)、单纯造模2组(造模后4h)、单纯造模3组(造模后8h),造模干预组分为造模干预1组(造模后即刻)、造模干预2组(造模后4h)、造模干预3组(造模后8h),观察胃黏膜损伤指数(UI)及组织学变化,采用免疫组化法检测TFF1蛋白的表达。结果:水浸束缚应激后胃黏膜广泛损伤,单纯造模组UI升高,TFF1表达降低。予泮托拉唑干预后,与相应单纯造模组相比UI降低(单纯造模1组与造模干预1组69.13±1.97vs23.38±1.30,单纯造模2组与造模干预2组57.50±8.81vs10.38±3.02,单纯造模3组与造模干预3组43.50±6.76vs5.88±1.25,均P<0.01)。TFF1染色计分增加(单纯造模1组与造模干预1组0.55±0.11vs0.92±0.15,单纯造模2组与造模干预2组0.76±0.24vs1.36±0.21,单纯造模3组与造模干预3组1.12±0.16vs1.65±0.11,均P<0.01)。结论:TFF1可能参与胃黏膜保护,促进溃疡修复;泮托拉唑还可能通过上调雌激素诱导基因参与胃黏膜的防御屏障。

VGluT1样阳性终末在大鼠三叉神经本体觉中枢通路的第三级核团-“带状区”内的分布及来源

本文综合运用免疫组织化学、逆行束路追踪和免疫荧光组织化学双标技术,对Ⅰ型囊泡膜谷氨酸转运体(VGluT1)样阳性终末在大鼠三叉神经本体觉中枢通路的第三级核团-“带状区”内的分布和来源、以及与向丘脑腹后内侧核(VPM)投射的神经元之间的联系进行了研究。结果显示:(1)在组成“带状区”的四个核团,即三叉神经感觉主核背内侧部(Vpdm)、三叉上核尾外侧部(Vsup-CL)、三叉神经运动核腹侧区(AVM)和上橄榄核背侧区(ADO)内,均可观察到大量的VGluT1样阳性终末呈密集分布;(2)将逆行追踪剂四甲基罗达明(TMR-DA)注入丘脑VPM后,在上述核团内均可观察到大量的TMR逆标神经元的胞体和突起;(3)在激光共聚焦显微镜下可观察到部分VGluT1样阳性终末包绕在TMR逆标神经元的胞体或树突周围,并与之形成密切接触;(4)当切断三叉神经感觉根7d后,光学显微镜下可观察到手术侧Vpdm内的VGluT1样阳性产物明显下降,其他三个核团内无明显变化,但当切断三叉神经运动根8周后,则在手术侧Vsup内观察到VGluT1样阳性产物几乎完全消失,而Vpdm、AVM和ADO内并无变化。以上结果提示:(1)大鼠三叉神经本体觉中枢通路的第三级核团-“带状区”的四个核团内均有大量的VGluT1样阳性终末分布,但它们的来源有所不同,其中Vpdm内的VGluT1样阳性终末来自于外周的三叉神经节细胞,Vsup内的VGluT1样阳性终末来自于三叉神经中脑核神经元,而AVM和ADO内的VGluT1样阳性终末可能来自于中枢其他核团;(2)口面部本体感觉信息从“带状区”向丘脑VPM传递的过程中,谷氨酸发挥着重要作用。

TGF-β1、CTGF基因的过表达与早期糖尿病肾病关系的研究

目的:探讨转化生长因子(TGF)-β1、结缔组织生长因子(CTGF)在早期糖尿病肾病(DN)中的作用。方法:建立链脲佐菌素(STZ)诱导的糖尿病大鼠模型,随机分为正常对照组(NC)和糖尿病组(DM)。每周测量体质量,每2周测定血糖,于实验第8周末处死大鼠后测定相关的生化指标及肾功能指标,同时留取肾皮质液氮冻存,应用实时定量PCR方法检测肾皮质TGF-β1、CTGF的mRNA水平,用免疫组化法检测肾小球中TGF-β1和CTGF的蛋白表达情况,并进行半定量分析。结果:与NC组相比,DM组血尿素氮、24h尿量及尿微量蛋白、肾脏肥大指数明显升高(P<0.01),但血肌酐明显下降(P<0.01)。实时定量PCR及免疫组化结果显示,DM组较NC组TGF-β1、CTGFmRNA及蛋白表达量均明显增高。结论:TGF-β1与CTGF在肾病早期表达明显升高,对于评价DN进程具有重要意义。

丹参素冰片酯干预TLR4信号通路抗动脉粥样硬化的机制研究

目的:探讨丹参素冰片酯干预TLR4信号通路抗动脉粥样硬化的作用及其机制。方法:选取SD大鼠60只,随机分为空白组(10只)和实验组(50只),实验组建造AS模型,模型成功后随机将实验组分为模型组,辛伐他汀组,丹参素冰片酯高、中、低剂量组。药物干预4周,采用自动生化分析仪测定血脂,通过免疫组化技术检测大鼠腹主动脉中TLR4、MyD88及NF-κB的表达。结果:丹参素冰片酯可显著降低高脂血症大鼠血清TC、TG、LDL-C水平(P<0.05或P<0.01);与模型组相比,辛伐他汀组及各丹参素冰片酯组均能减少TLR4、MyD88、NF-κB的表达(P<0.05或P<0.01),以丹参素冰片酯高剂量组与辛伐他汀组最为明显(P<0.01)。结论:丹参素冰片酯干预能降低血脂、抑制TLR4信号通路中TLR4、MyD88和NF-κB的表达,其抗AS的作用机制与阻断TLR4/MyD88/NF-κB信号传导通路中相关信号分子的过度激活,抑制炎症细胞在动脉管壁集聚有关。

Inhibition of high-mobility group box 1 expression by siRNA in rat hepatic stellate cells

AIM:To explore the role of high-mobility group box 1 (HMGB1) protein during liver fibrogenesis and investigate the functional effects of HMGB1 gene silencing in hepatic stellate cells (HSCs) using siRNA.METHODS:Hepatic fibrosis in rats was induced through serial subcutaneous injections of dimethylnitrosamine,and expression of HMGB1 was detected by immunohistochemistry.HMGB1 siRNAs were developed and transiently transfected into HSC-T6 cells using Lipofectamine 2000.HMGB1 expression was evaluated by real-time polymerase chain reaction (PCR) and Western blotting analysis.Expression of α-smooth muscle actin (α-SMA) and collagen typesⅠand Ⅲ was evaluated by real-time PCR.Cell proliferation and the cell cycle were determined using the methyl thiazolyl tetrazolium method.Finally,collagen content in HSC supernatant was evaluated by an enzyme-linked immunosorbent assay.RESULTS:The results showed that HMGB1 was upregulated during liver fibrosis and that its expression was closely correlated with the deposition of collagen.siRNA molecules were successfully transfected into HSCs and induced inhibition of HMGB1 expression in a time-dependent manner.Moreover,HMGB1 siRNA treatment inhibited synthesis of α-SMA and collagen types Ⅰ and Ⅲ in transfected HSCs.CONCLUSION:This study suggests a significant functional role for HMGB1 in the development of liver fibrosis.It also demonstrates that downregulation of HMGB1 expression might be a potential strategy to treat liver fibrosis.

Localization of vesicular glutamate transporters in the peripheral vesti-bular system of rat

Objective To examine the vesicular glutamate transporters （VGluTs： VGluT 1-VGluT3） in the peripheral vestibular system. Methods The vestibular structures, including Scarpa＇s ganglion （vestibular ganglion, VG）, maculae of utricle and saccule, and ampullary cristae, from normal Sprague-Dawley rats were processed immunohistochemically for VGluTs, by avidin-biotinylated peroxidase complex method, with 3-3＇-diaminobenzidine （DAB） as chromogen. Results （1） VGluT 1 was localized to partial neurons of VG and to the putative primary afferent fibers innervating vestibular end-organs. （2） Intense VGluT3 immunoreactivity was detected in large number of sensory epithelia cells, and weak labeling of VGluT3- positive afferent fibers was in the maculae and ampullary cristae. （3） No or very weak VGluT2 immunoreactivity was observed in the VG and acoustic maculae. Conclusion These results provide the morphological support that glutamate exists in the peripheral vestibular system, and it may play an important role in the centripetal vestibular transmission.

Protective effect of nitric oxide on hepatopulmonary syndrome from ischemia-reperfusion injury

AIM： To evaluate immunological protection of nitric ox- ide （NO） in hepatopulmonary syndrome and probable mechanisms of ischemia-reperfusion （IR） injury in rat liver transplantation, METHODS： Sixty-six healthy male Wistar rats were randomly divided into three groups （11 donor/recipi-ent pairs）. In group 11, organ preservation solution was lactated Ringer＇s solution with heparin 10 000/μL at 4℃. In groups I and 111, the pLeservation solution added, respectively, L-arginine or Ng-L-arginine methyl ester （L-NAME） （1 mmol/L） based on group 11, and recipients were injected with L-arginine or L-NAME （50 mg/kg） in the anhepatic phase. Grafted livers in each group were stored for 6 h and implanted into recipi- ents. Five rats were used for observation of postopera- tive survival in each group. The other six rats in each group were used to obtain tissue samples, and execut- ed at 3 h and 24 h after transplantation. The levels of alanine aminotransferase （ALT）, tumor necrosis factor （TNF）-a and NO metabolites （NOx） were detected, and expression of NO synthase, TNF-α and intercellular adhesion molecule 1 （ICAM-1） was examined by tri- phosphopyridine nucleotide diaphorase histochemical and immunohistochemical staining. RESULTS： By supplementing L-arginine to strengthen the NO pathway, a high survival rate was achieved and hepatic function was improved. One-week sur- viral rate of grafted liver recipients in group I was significantly increased （28.8 4±36.6 d ys 4 4±1.7 d, P 〈 0.01） as compared with groups 11 and Ill. Serum levels of ALT in group ] were 2-7 times less than those in groups 11 and 11I （P 〈 0.01）. The cyclic guanosine monophosphate （cGMP） levels in liver tissue and NOx in group I were 3-4 times higher than those of group 11 after 3 h and 24 h reperfusion, while in group ]]], they were significantly reduced as compared with those in group ]1 （P 〈 0.01）. The levels of TNF-（z in group I were significantly lower than in group Ⅱ after 3 h and 24 h reperfusion （P 〈 0.01）, while being sig- nificantly higher in group Ⅲ than group Ⅱ （P 〈 0.01）. Histopathology revealed more severe tissue damage in graft liver and lung tissues, and a more severe in- flammatory response of the recipient after using NO synthase inhibitor, while the pathological damage to grafted liver and the recipient＇s lung tissues was signifi-cantly reduced in group I after 3 h and 24 h reperfu- sion. A small amount of constitutive NO synthase （cNOS） was expressed in liver endothelial cells after 6 h cold storage, but there was no expression of inducible NO synthase （iNOS）. Expression of cNOS was particularly significant in vascular endothelial cells and liver cells at 3 h and 24 h after reperfusion in group Ⅱ but expres- sion of iNOS and ICAM-1 was low in group I. There was diffuse strong expression of ICAM-1 and TNF-α in group Ⅱ at 3 h after reperfusion. CONCLUSION： The NO/cGMP pathway may be critical in successful organ transplantation, especially in treat- ing hepatopulmonary syndrome during cold IR injury in rat orthotopic liver transplantation.

Embryonic hepatocyte transplantation for hepatic cirrhosis:Efficacy and mechanism of action

AIM： To investigate the efficacy and mechanism of action of allogeneic embryonic hepatocyte transplantation for the treatment of hepatic cirrhosis. METHODS： Rat embryonic hepatocytes were characterized by examining cell markers. Wistar rats with CCl4-induced cirrhosis were randomly divided into two groups： a model group receiving continuous CCl4, and a cell transplantation group receiving continuous CCh and transplanted with embryonic fluorescent-labeled hepatocytes. In addition, a normal control group was composed of healthy rats. All rats were sacrificed after 2 wk following the initiation of the cell transplant. Ul- trasound, pathological analyses and serum biochemical tests were used to evaluate the efficacy of embryonic hepatocyte transplantation. To analyze the recovery status of cirrhotic hepatocytes and the signaling pathways influenced by embryonic hepatocyte transplantation, real-time polymerase chain reaction was performed to examine the mRNA expression of stellate activation-associated protein （STAP）, c-myb, ~ smooth muscle actin （^-SMA） and endothelin-1 （ET-1）. West- ern blotting and immunohistochemistry were employed to detect ^-SMA and ET-1 protein expression in hepatic tissues. RESULTS： Gross morphological, ultrasound and his- topathological examinations, serum biochemical tests and radioimmunoassays demonstrated that hepatic cir- rhosis was successfully established in the Wistar rats. Stem cell factor receptor （c-kit）, hepatocyte growth factor receptor （c-Met）, Nestin, ~ fetal protein, albu- min and cytokeratin19 markers were observed in the rat embryonic hepatocytes. Following embryonic hepa- tocyte transplantation, there was a significant reversal in the gross appearance, ultrasound findings, histo- pathological properties, and serum biochemical param- eters of the rat liver. In addition, after the activation of hepatic stellate cells and STAP signaling, ^-SMA, c-myb and ET-1 mRNA levels became significantly lower than in the untreated cirrhotic group （P 〈 0.05）. These levels, however, were not statistically different from those of the normal healthy group. Immunohisto- chemical staining and Western blot analyses revealed that ^-SMA and ET-1 protein expression levels in the transplantation group were significantly lower than in the untreated cirrhotic group, but being not statistically different from the normal group. CONCLUSION： Transplantation of embryonic hepatocytes in rats has therapeutic effects on cirrhosis. The described treatment may significantly reduce the expression of STAP and ET-1.

Different morphologic features of rat cochlea progenitor spheres and their implications

Objective: To detect the different morphologic features, developmental regulation, potential of proliferation and differentiation of neonatal rat cochlea progenitor spheres. Methods: We isolated the cochlea sensory epithelium cells from neonatal rats and cultured them in nonadherent conditions to acquire different morphologic spheres. Then we observed the diameter and compositional change of cell colonies in distinct sphere types on day 3, 6, 9 and 12, and summarized the regularity of development and their conversion. We also detected the proliferative activity of distinct spheres by immunohistochemical staining of Abcg2, Nestin and BrdU. After induced spontaneous differentiation, the spheres were detected in the changes of the marker of hair cell, MyosinVIIA; by immunocytochemical staining, we revealed the potential of how different spheres were converted into hair cell-like cells. Results: The acquired three types of suspended spheres are solid, transitional, and hollow. There's morphologic significance among them and they can covert into the other type of spheres among them. The ability of self-renewing and proliferation in distinct spheres vary and all of them have the potential of spontaneously differentiation into hair cell-like cells. Conclusion: All the type of spheres not only has the potential of proliferation and differentiation, but also hasthe potential of spontaneous differentiation into hair cell-like cells. Distinct types of cell spheres neither originate from different progenitor cell subcolonies nor are different stages of the same cell spheres. Solid spheres are most practically useful.

Effects of Graded Hypothermia on Hypoxic-ischemic Brain Damage in the Neonatal Rat

Objective To investigate the effect of graded hypothermia on neuropathologic alterations of neonatal rat brain after exposed to hypoxic-ischemic insult at 37℃, 33℃, 31℃, and 28℃, respectively, and to observe the effect of hypothermia on 72-kDa heat shock protein （HSP72） expression after hypoxic-ischemic insult. Methods Seven days old Wistar rats were subjected to unilateral common carotid artery ligation followed by exposure to hypoxia in 8% oxygen for 2 hours at 37℃, 33℃, 31℃, and 28℃, respectively. The brain temperature was monitored indirectly by inserting a mini-thermocouple probe into the temporal muscle during hypoxia. After hypoxia-ischemia their mortality was assessed. Neuronal damage was assessed with HE staining 72 hours after hypoxia. HSP72 expression at 0.5, 24, and 72 hours of recovery was immunohistochemically assessed using a monoclonal antibody to HSP72. Results Hypoxia-ischemia caused 10.5% （2 / 19） of mortality in rat of 37℃ group, but no death oc- curred in 33℃, 31℃ or 28℃ groups. HE staining showed neuropathologic damage was extensive in rats exposed to hypoxia-ischemia at 37℃ （more than 80.0%）. The incidence of severe brain damage was significantly decreased in 33℃ （53.3%） and 31℃ groups （44,4%）, and no histologic injury was seen in the 28℃ group of rats. Expression of HSP72 was manifest and persistent in the rat brain of 37℃ group, but minimum in the rat brain of 28℃ group. Conclusion Mild and moderate hypothermia might prevent cerebral visible neuropathologic damage associated with hypoxic-ischemic injury by decreasing stress response.

大鼠海马星形胶质细胞对突触可塑性的影响

目的研究星形胶质细胞在神经系统发育成熟过程中对突触可塑性的调控规律。方法取健康初生、幼年和成年大鼠各10只,每只取脑切片,用免疫组织化学方法观察其海马CA1区的S100、胶质纤维酸性蛋白(GFAP)和P38免疫反应产物强度;HE染色法显示神经元胞体。结果初生大鼠海马CA1区中神经元大量存在,但S100、GFAP和P38表达均少,幼鼠的表达增加,但仍显著少于成鼠的表达(P<0.01)。结论大鼠海马CA1区星形胶质细胞增殖与突触出现时间、突触数目增加及功能成熟有关。

Effect of Wuziyanzong pill on levels of sex hormones, and expressions of nuclear- associated antigen Ki-67 and androgen receptor in testes of young rats

OBJECTIVE: To evaluate the effects of Wuziyanzong pill on the levels of serum follicle stimulating hormone(FSH), luteinizing hormone(LH), testosterone(T) and the expressions of nuclear-associated antigen Ki67(Ki67), androgen receptor(AR) in testes of young rats.METHODS: Sixteen 20-day-old Wistar male rats were randomly divided into control and treatment group(n = 8). Rats in treatment group were administered Wuziyanzong pill by gavage; rats in control group administered the same volume of saline. After 10 days of treatment, the rats were killed, and then serum and testes were taken. The levels of FSH, LH and T were measured by radioimmunoassay(RIA). The histology of seminiferous tubule was observed by hematoxylin-eosin(HE) staining. The expression of Ki67 was detected by immunohistochemical assay(IHC). The m RNA level of Ki67, ARand CK-18 was detected with quantitative real-time polymerase chain reaction(Q-RT-PCR), the protein level of AR and CK-18 were tested by Western blot.RESULTS: Compared with control group, T level in treatment group increased significantly(P < 0.05).HE staining showed that both leydig cells and germ cells increased in treatment group. Expressions of Ki67 and AR became higher after treatment. There were no changes in CK-18 expression.CONCLUSION: Wuziyanzong pill can up-regulate AR level to promote germ cell proliferation and differentiation in young male rats.

Functional recovery of sciatic nerve through inside-out vein graft in rats

Objective： Present study aimed at further comprehensive functional, histomorphometrical and immunohistochemical assessment of peripheral nerve regeneration using rat sciatic nerve transection model.Methods： The 10-mm rat sciatic nerve gap was created in rats. In control group nerve stumps were sutured to adjacent muscle and in treatment group the gap was bridged using an inside-out vein graft. In sham-operated group the nerve was manipulated and left intact. All animals underwent walking track analysis test 4, 8, and 12 weeks after surgery.Subsequently, muscle mass measurement was performed to assess reenervation, histological examination to observe the sciatic nerve regeneration morphologically and immunohistochemistry to detect Schwann cells using anti S-100. Results were analyzed using a factorial ANOVA with two between-subjects factors. Bonferroni test for pairwise comparisons was used to examine the effect of treatments.Results： Functional analysis ofmyelinated nerve fibers showed that nerve function improved significantly in the time course in treatment group. However, quantitative morphometrical analysis of myelinated nerve fibers showed that there was no significant difference between 8 and 12 weeks in treatment group. Muscle weight ratio was bigger and weight loss of the gastrocnemius muscle was ameliorated by inside-out vein grafting. The position of positive immunohistochemical reactions further implied that regenerated axons and Schwann cell-like cells existed after vein grafting was performed, and was accompanied by the process of myelination and structural recovery of regenerated nerves.Conclusion： Functional analysis of peripheral nerve repair is far more reliable than quantitative morphometrical analysis