神经激肽A在大鼠食管发育中的表达变化

目的 探讨神经激肽 A(NKA)在大鼠食管发育中的表达及变化。方法 应用免疫组织化学 PAP法和图象分析术系统研究大鼠胚胎 13d至成年食管中 NKA的表达情况。结果 胚胎 2 1d食管粘膜肌层和环肌层内可观察到 NKA免疫反应 (NKA- IR)阳性膨体纤维 ,出生后 ,随幼鼠的生长发育 ,相继在上皮 ,粘膜下层 ,纵肌层、肌间丛和粘膜下丛出现 NKA- IR表达 ,30 d时已和成年鼠相似 ;定量分析的结果与 NKA- IR在食管各层的变化一致。结论 NKA在食管中的发生发育主要在临出生前至生后 4周内 ;

卡托普利对肾性高血压大鼠左室肥厚及心脏核因子-κ B表达的影响

研究表明，促炎性细胞因子与高血压的发生、发展存存密切的联系，而促炎性细胞因子不仅可引发高血压而且其水平升高与高血压的并发症如动脉硬化、心室肥厚、等的发生、发展密切相关。因此，降低岛血压病人的炎性细胞因子水下可能会减绥甚至防止高血压并发症的发生。

卡托普利对肾性高血压大鼠左室肥厚及心脏核因子-kB表达的影响

研究表明．促炎性细胞因子与高血压的发生、发展存在密切的联系，而促炎性细胞因子不仅可引发高血压而且其水平升高与高血压的并发症如动脉硬化、心室肥厚、等的发生、发展密切相关。因此．降低高血压病人的炎性细胞因子水平可能会减缓甚至防止高血压并发症的发生。核因子－kappaB可高效诱导多种细胞因子，黏附分子，趋化因子的编码基因的表达，故对其有效的控制是降低炎性细胞因子不利影响的主要步骤之一。本研究采用肾性高血压大鼠模型，观察了卡托普利对炎性细胞因子合成的关键转录因子一核因子－kabppaB（NP－kB）在心肌组织内表达的影响，探讨了AcEI在高血压中的抗炎作用机制，为更好地防治高血压提供理论依据，现报告如下。

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.

Increased expression of chondroitin sulphate proteoglycans in rat hepatocellular carcinoma tissues

AIM:To investigate the expression of chondroitin sulphate proteoglycans(CSPGs)in rat liver tissues of hepatocellular carcinoma(HCC).METHODS:Thirty male Sprague Dawley rats were randomly divided into two groups:control group(n=10) and HCC model group(n=20).Rats in the HCC model groups were intragastrically administrated with 0.2%(w/v)N-diethylnitrosamine(DEN)every 5 d for 16 wk,whereas 0.9%(w/v)normal saline was administered to rats in the control group.After 16 wk from the initiation of experiment,all rats were killed and livers were collected and fixed in 4%(w/v)paraformaldehyde.All tissues were embedded in paraffin and sectioned.Histological staining(hematoxylin and eosin and Toluidine blue)was performed to demonstrate the onset of HCC and the content of sulphated glycosaminoglycan(sGAG).Immunohistochemical staining was performed to investigate the expression of chondroitin sulphate(CS)/dermatan sulphate(DS)-GAG,heparan sulphate(HS)-GAG,keratan sulphate(KS)-GAG in liver tissues.Furthermore,expression and distribution of CSPG family members,including aggrecan,versican,biglycan and decorin in liver tissues,were also immunohistochemically determined.RESULTS:After 16 wk administration of DEN,malignant nodules were observed on the surface of livers from the HCC model group,and their hepatic lobule structures appeared largely disrupted under microscope.Toluidine blue staining demonstrated that there was an significant increase in sGAG content in HCC tissues when compared with that in the normal liver tissues from the control group[0.37±0.05 integrated optical density per stained area(IOD/area)and 0.21± 0.01 IOD/area,P<0.05].Immunohistochemical studies demonstrated that this increased sGAG in HCC tissues was induced by an elevated expression of CS/DS(0.28±0.02 IOD/area and 0.18±0.02 IOD/area,P< 0.05)and HS(0.30±0.03 IOD/area and 0.17±0.02 IOD/area,P<0.01)but not KS GAGs in HCC tissues.Further studies thereby were performed to investigate the expression and distribution of several CSPG components in HCC tissues,including aggrecan,versican,biglycan and decorin.Interestingly,there was a distinct distribution pattern for these CSPG components between HCC tissues and the normal tissues.Positive staining of aggrecan,biglycan and decorin was localized in hepatic membrane and/or pericellular matrix in normal liver tissues;however,their expression was mainly observed in the cytoplasm,cell membranes in hepatoma cells and/or pericellular matrix within HCC tissues.Semi-quantitative analysis indicated that there was a higher level of expression of aggrecan(0.43± 0.01 and 0.35±0.03,P<0.05),biglycan(0.32±0.01 and 0.25±0.01,P<0.001)and decorin(0.29±0.01 and 0.26±0.01,P<0.05)in HCC tissues compared with that in the normal liver tissues.Very weak versican positive staining was observed in hepatocytes near central vein in normal liver tissues;however there was an intensive versican distribution in fibrosis septa between the hepatoma nodules.Semi-quantitative analysis indicated that the positive rate of versican in hepatoma tissues from the HCC model group was much higher than that in the control group(33.61%and 21.28%,P <0.05).There was no positive staining in lumican and keratocan,two major KSPGs,in either normal or HCC liver tissues.CONCLUSION:CSPGs play important roles in the onset and progression of HCC,and may provide potential therapeutic targets and clinical biomarkers for this prevalent tumor in humans.

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.

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.