泛素特异性加工酶2的研究进展

泛素特异性加工酶2(ubiquitin-specific protease 2,USP2)是一种重要的去泛素化酶,通过特异性识别靶蛋白,使靶蛋白去泛素化并阻碍其降解,以调控细胞内靶蛋白数量和活性,从而参与细胞功能的调节.USP2-45及USP2-69是USP2基因选择性剪接而形成的两种不同亚型,在不同组织、细胞和不同发育阶段均有表达,且与细胞周期调控,骨骼肌纵向生长、肌细胞分化、离子通道、生精作用及生物钟调控等关系密切.本文结合当前研究进展,系统阐述USP2的两种亚型的结构、分布及功能,为进一步研究USP2与疾病的关系打下理论基础.     

放免法同时测定糜酶转基因小鼠心脏中血管紧张素转换酶及糜酶样活性

为研究糜酶的功能,本文利用蛋白酶抑制剂Lisinopril、Aprotinin及EDTA结合放射免疫分析的方法同时测定了糜酶转基因小鼠心脏组织中血管紧张素转换酶（angiotensinconvertingenzyme,ACE）及糜酶样活性。结果显示转基因小鼠心脏组织中康酶比活力（0．274±0．071U／mg蛋白）较对照（0.152±0.021U／mg蛋白）显著升高,而ACE比活力不变（分别为0.172±0．029U／mg蛋白和0．177±0．019U／mg蛋白）。同时测定出转基因小鼠心脏局部血管紧张素Ⅱ（angiotensinⅡ,AngⅡ）含量（1984±184pg／g心脏组织）为对照（568±88pg／g心脏组织）的3倍。表明糜酶转基因小鼠心脏中可表达糜酶且糜酶能催化AngⅡ的生成,而蛋白酶抑制剂结合放免分析是一种快速、灵敏的检测ACE及糜酶样活性的方法。     

托伐普坦对小鼠内髓集合管细胞系水通道蛋白及肾小管功能的影响

目的探讨托伐普坦对小鼠内髓集合管细胞系水通道蛋白2、细胞凋亡以及肾小管功能的影响。方法用不同浓度的托伐普坦刺激小鼠内髓集合管细胞后,Western blot检测水通道蛋白2的蛋白表达,caspase3活性检测试剂盒检测caspase3活性,MTS法及流式细胞计量术检测细胞凋亡;用不同浓度的托伐普坦治疗心肌梗死模型小鼠后,ELISA检测β_2-微球蛋白(β_2-MG)与N-乙酰-β-D-葡萄糖苷酶(NAG)含量。结果托伐普坦呈剂量依赖性,显著降低水通道蛋白2的蛋白表达(P0.05),但是不影响caspase 3的活性及细胞凋亡,托伐普坦不影响小鼠尿中β_2-MG与NAG的含量。结论托伐普坦显著降低内髓集合管细胞水通道蛋白2的蛋白表达,不影响其细胞凋亡及小鼠肾小管功能。     

铁调素对体外培养大鼠骨骼肌细胞铁代谢的影响

目的 探讨铁调素对骨骼肌铁代谢的调节机制.方法 培养大鼠骨骼肌L6细胞,随机分成4组,每组6个重复,每组各加入不同浓度的铁调素溶液(终浓度分别为0、0.05、0.1、0.15 mg/L)孵育12h,采用同位素示踪法,用55Fe测定细胞铁摄取及铁释放能力的变化;通过流式细胞仪检测细胞内铁池(LIP)铁含量;应用免疫印迹法检测膜铁转运蛋白1(FPN1)的变化.结果 与空白对照组比较,0.1mg/L 铁调素处理组肌细胞的铁释放能力明显下降(P<0.05),0.05及0.15mg/L铁调素处理组肌细胞的铁摄取和铁释放均无明显变化(P>0.05);0.1mg/L 铁调素处理组其细胞内LIP铁含量较对照组明显升高(P<0.05);0.1mg/L 铁调素处理组细胞膜上FPN1的表达较对照组明显降低(P<0.05).结论 铁调素可通过影响骨骼肌细胞FPN1的表达水平,降低骨骼肌细胞内铁的向外转运,增加细胞内铁池的铁含量,从而调节骨骼肌细胞的铁代谢.     

NAD(P)H:醌氧化还原酶1对多巴胺能细胞的保护作用

目的: 研究多巴胺(DA)对多巴胺能细胞的毒性作用、NAD(P)H:醌氧化还原酶1(NQO1)的保护作用及其机制。方法: 运用MTT法检测多巴胺对神经母细胞瘤细胞(SH-SY5Y)活性的影响;运用脂质体转染或Ⅱ相酶诱导剂预处理的方法对细胞进行预处理,并用免疫荧光方法检测转染效率;运用蛋白质印迹(Western blotting)方法检测细胞经过不同处理后胞内NQO1蛋白的表达情况;运用醌蛋白检测方法(硝基四氮唑蓝/甘氨酸法)检测细胞经不同处理后胞内醌化蛋白含量的变化。结果: 多巴胺对细胞具有剂量依赖性毒性,且与细胞内醌化蛋白的含量相关;脂质体转染和Ⅱ相酶诱导剂萝卜硫素(SF)均能使细胞内NQO1表达量增高;NQO1高表达能缓解多巴胺对细胞造成的毒性;细胞内NQO1表达量增高能减少多巴胺引起的细胞内醌化蛋白含量。结论: 细胞内NQO1的过表达可以减轻多巴胺对细胞产生的毒性作用。     

参麦对缺血再灌注性鼠肾小管细胞损伤的作用

目的 :参麦注射液对缺血再灌注小鼠肾小管细胞损伤作用的观察。方法 :采用离体培养的鼠肾小管细胞株 ,复制缺血再灌注损伤模型 ,用MTT法测定细胞增殖活性 ,酶组化法观察细胞膜和线粒体中ATP酶和LDH含量的变化 ,用生化法检测小管细胞内SOD活性和MDA含量 ,结果 :不同浓度的SMI对肾小管细胞有明显的促增殖作用 ,在SMI浓度为 5 0 μg·ml 1时 ,促增殖作用最强 ;SMI能显著提高IR后小管细胞的增殖活性和细胞存活率 ;缺血培养 4小时后 ,MDA含量明显增高 ,ATP酶、LDH含量明显降低 ,SMI降低IR培养的小管细胞MDA含量 ,增强SOD活性 ,ATP酶、LDH含量。结论 :SMI具有促进鼠肾小管细胞增殖和减轻肾小管细胞IR损伤的作用 ,其机制可能与增强SOD的活性、加速OFR的清除 ,增强细胞抗氧化的能力 ,减轻膜的脂质过氧化 ,抑制ATP酶含量的减少 ,进而维持生物膜的结构和功能的完整性有关。     

X线放射诱导大鼠心肌细胞系H9c2损伤中组蛋白乙酰化水平降低

目的研究辐射暴露对心肌细胞的影响及组蛋白乙酰化的修饰变化,分析其相关性并探讨其机制。方法将大鼠心肌细胞系H9c2分为对照组和放射组,放射组分别用X线给予2、4、6和8 Gy剂量照射。48 h后,通过比色法检测细胞培养上清乳酸脱氢酶(LDH)活性、细胞内丙二醛(MDA)含量及过氧化物歧化酶(SOD)活性;用annexin V-FITC/PI染色及流式细胞仪检测细胞凋亡率;Western blot检测凋亡相关蛋白Bcl-2、caspase3及组蛋白乙酰化相关蛋白H3K9ac、HDAC3表达。结果与对照组相比,不同剂量照射组细胞上清LDH相对活性增加(P0.05),细胞内MDA相对含量增加(P0.05),细胞内SOD相对活性降低(P0.05);此外放射组细胞凋亡率明显增高(P0.05),并且Bcl-2蛋白表达水平降低(P0.05),caspase-3蛋白表达水平增高(P0.05);同时与对照组相比,放射组组蛋白H3乙酰化水平降低(P0.05),HDAC3表达水平增高(P0.05)。结论辐射暴露导致H9c2细胞损伤并诱导其凋亡,此过程中细胞内组蛋白乙酰化水平降低,而HDAC3可能是调节组蛋白乙酰化水平的主要因子。     

选择性iNOS抑制剂氨基胍减缓小鼠BCG免疫性肝损伤造成的P450降低

探讨免疫性肝损伤对肝脏细胞色素P4 5 0药物代谢酶系 (CYP4 5 0 )的影响及免疫损伤条件下NO在CYP 4 5 0药物代谢酶系活性调节中可能的作用机制。采用尾静脉注射BCG诱发小鼠产生免疫性肝损伤 ,用紫外分光光度法测定肝组织匀浆中CYP4 5 0总含量 ,HE染色法观察肝脏病理组织学变化 ,免疫组化法检测肝组织iNOS蛋白表达。免疫刺激条件下小鼠肝组织内可见大量炎性细胞浸润所致肉芽肿及淤血 ,iNOS呈团块状棕色强阳性表达。肝脏中CYP4 5 0总含量亦降低至对照组 [(1 93± 0 2 6 ) μmol g]的 5 6 9% [(1 10± 0 4 5 ) μmol g](P <0 0 5 )。iNOS选择性抑制剂氨基胍不仅可使BCG所致肝脏肉芽肿形成及iNOS表达减少 ,亦可逆转BCG所致小鼠肝脏CYP4 5 0酶总含量降低 (P <0 0 5 )。以上结果提示iNOS表达参与了BCG免疫损伤条件下CYP4 5 0药物代谢酶总含量下调机制。     

高通量筛选肿瘤转移相关基因的研究

目的利用高通量双萤光素酶报告基因系统筛选与肿瘤转移相关的人类功能基因。方法利用与肿瘤转移相关的重要因子——血管内皮生长因子（VEGF）,将含有VEGF反应序列的萤光素酶报告基因（VEGF—LUC）质粒与409个待筛人类功能基因真核表达质粒共转染Hela细胞以筛选影响VEGF．LUC表达的人类功能基因,进一步通过Westernblot在蛋白质水平验证初筛阳性的基因对细胞内VEGF蛋白表达量的影响。结果染色质重塑蛋白4A（CHMP4A）明显增强VEGF．LUC相对萤光素酶值（P〈0．05）,Westernblot结果表明CHMP4A明显增强Hela细胞内VEGF的表达。结论初步筛选到一个可能通过影响VEGF活性增强肿瘤侵袭性的基因CHMP4A。     

Amiloride通过抑制缺氧诱导的NHE1表达而延缓calpain介导的ABCA1降解

目的:研究缺氧对钠氢交换体1(NHE1)表达、细胞内钙离子浓度([Ca2+]i)和钙蛋白酶(calpain)活性的影响,探讨NHE1抑制剂阿米洛利(amiloride)对ABCA1降解的影响以及与calpain相关的机制。方法:RAW264.7细胞缺氧0、12、24和48 h。MTT法检测细胞活力,real-time PCR及Western blot检测NHE1的表达。流式细胞术检测[Ca2+]i,荧光素法检测细胞内calpain活性。进而,经缺氧24 h处理的细胞,cycloheximide干预条件下,NHE1抑制剂amiloride处理6 h及12 h,检测ABCA1蛋白含量。最后,给予calpain抑制剂ALLN及细胞内钙螯合剂BAPTA共孵育12 h,检测ABCA1含量及calpain活性。结果:缺氧呈时间依赖方式抑制细胞增殖。缺氧促进NHE1表达上调,增加[Ca2+]i及calpain活性。缺氧加速ABCA1蛋白降解,而amiloride减缓ABCA1蛋白降解。ALLN及BAPTA升高ABCA1蛋白含量,降低calpain活性。结论:Amiloride延缓calpain介导的ABCA1降解,提示缺氧诱导NHE1表达可能至少部分参与ABCA1蛋白降解。     

Novel role of protein disulfide isomerase in the regulation of NADPH oxidase activity: pathophysiological implications in vascular diseases

Vascular cell NADPH oxidase complexes are key sources of signaling reactive oxygen species (ROS) and contribute to disease pathophysiology. However, mechanisms that fine-tune oxidase-mediated ROS generation are incompletely understood. Besides known regulatory subunits, upstream mediators and scaffold platforms reportedly control and localize ROS generation. Some evidence suggest that thiol redox processes may coordinate oxidase regulation. We hypothesized that thiol oxidoreductases are involved in this process. We focused on protein disulfide isomerase (PDI), a ubiquitous dithiol disulfide oxidoreductase chaperone from the endoplasmic reticulum, given PDI's unique versatile role as oxidase/isomerase. PDI is also involved in protein traffic and can translocate to the cell surface, where it participates in cell adhesion and nitric oxide internalization. We recently provided evidence that PDI exerts functionally relevant regulation of NADPH oxidase activity in vascular smooth muscle and endothelial cells, in a thiol redox-dependent manner. Loss-of-function experiments indicate that PDI supports angiotensin II-mediated ROS generation and Akt phosphorylation. In addition, PDI displays confocal co-localization and co-immunoprecipitates with oxidase subunits, indicating close association. The mechanisms of such interaction are yet obscure, but may involve subunit assembling stabilization, assistance with traffic, and subunit disposal. These data may clarify an integrative view of oxidase activation in disease conditions, including stress responses.     

ERp29, an unusual redox-inactive member of the thioredoxin family

Oxidative folding in the endoplasmic reticulum is accomplished by a group of oxidoreductases where the protein disulfide isomerase (PDI) plays a key role. Structurally, redox-active PDI domains, like many other enzymes utilizing cysteine chemistry, adopt characteristic thioredoxin folds. However, this structural unit is not necessarily associated with the redox function and the current review focuses on the interesting example of a loss-of-function PDI-like protein from the endoplasmic reticulum, ERp29. ERp29 shares a common predecessor with PDI; however in the course of divergent evolution it has lost a hallmark active site motif of redox enzymes but retained the characteristic structural fold in one of its domains. Although the functional characterization of ERp29 is far from completion, all available data point to its important role in the early secretory pathway and allow tentative categorization as a secretion factor/escort protein of a broad profile.     

Fine structural localization of glucose-6-phosphatase activity in the pancreatic islets of two amphibian species (Salamandra salamandra L. and Rana esculenta L.).

The ultrastructural distribution of glucose-6-phosphatase activity has been investigated in the salamander and frog pancreas. In the pancreas of salamander the enzyme was located in the A-cells, while in frog it occurred in all main types of islet cells B-, A-, and D-cells). As a rule, the reaction intensity was higher in the frog islet cells. No reaction was recorded in the exocrine pancreatic tissue of both species. The glucose-6-phosphatase activity was constantly detected in the lumen of rough endoplasmic reticulum and between the nuclear envelopes. Other enzyme localizations, observed especially in the A-cells of the salamander pancreas, were considered possible diffusion artifacts ro remnants of other phosphatase activity. The enzyme distribution in different types of islet cells, as well as its functional significance are discussed in relation to the findings of other authors.     

DNA, RNA and total protein content of leg and breast muscles of White Rock chickens selected for 56-day body weight

Pectoralis and gastrocnemius muscles from White Rock chickens divergently selected for 29 generations for high or low 56-day body weight were analyzed for DNA, RNA and protein (mg/g) content at 12 ages between hatch and 273 days of age. Dimorphism between lines was maximum at day 18 for both muscle types and then declined with age. High-line chickens generally deposited relatively more muscle tissue than those from the low line. Although nonadditive genetic variation was evident for absolute muscle weights, it was more frequent for muscle weight relative to body weight. For both muscle types, DNA unit number, as measured by DNA content (mg/g), was larger for the high line than the low immediately after hatch and smaller in the high than in the low line from day 10 to 56 after which lines were similar. RNA and protein/g muscle were similar for both lines at most ages. Between days 4 and 56, a period of rapid muscle growth, DNA unit size (protein/DNA) of both muscle types was larger in the high than in the low line. Heterosis was positive for protein, DNA unit number and size, and negative for RNA content. While weight of pectoralis muscle was similar to that of one gastrocnemius muscle on day 1, by day 273 its weight was over 3-fold greater. DNA unit number was higher in pectoralis than gastrocnemius muscle from hatch to day 4, similar on days 7 and 10 and lower for pectoralis muscle beyond day 10. RNA content was similar at all ages except 4, 7 and 10 days. DNA unit size followed the same pattern as DNA unit number; however, greater nuclei number at hatch for the high line corresponded with low DNA unit size. This pattern suggests a higher rate of cellular filling for pectoralis than gastrocnemius muscle.     

海水浸泡对火器伤兔肢体骨骼肌组织脂质过氧化反应影响的研究

目的:观察海水浸泡对火器伤伤口组织脂质过氧化反应的影响。方法:以滑膛枪发射质量250mg钢珠,致伤兔后肢,伤后将致伤兔随机分为两组:一组为海水浸泡组(SIG),将致伤兔浸泡于粗制海盐配制的人造海水中30min;另一组为单纯致伤组(SWG),伤后不浸泡海水。伤后3、6、12、24h手术取距伤道边缘0.5cm(A区)、1.5cm(B区)和2.5cm(C区)处肌组织,测定ATP、丙二醛(MDA)含量和超氧化物歧化酶(SOD)活力。取伤前骨骼肌组织作为对照。结果:伤后SIG兔伤肢骨骼肌MDA含量明显高于对照,伤后3、6h升高,12h略降,24h有再次升高的趋势。SWG的MDA含量变化与SIG相似,但升幅低于SIG;2组SOD活力和ATP含量的改变与MDA含量变化相似。结论:海水浸泡的火器伤伤口脂质过氧化反应增强,从而加重了肢体火器伤的过氧化反应。     

Curcumin inhibits the proliferation of human hepatocellular carcinoma J5 cells by inducing endoplasmic reticulum stress and mitochondrial dysfunction

Curcumin (diferuloylmethane), which is obtained from turmeric, the rhizome of Curcuma longa (L.), inhibits many human cancer cells. However, the molecular mechanisms responsible for curcumin-induced endoplasmic reticulum stress in human hepatic cellular carcinoma J5 cells, are not yet clearly understood. J5 cells were treated with various concentrations of curcumin for different durations. The cell viability was detected by MTT assay. The protein expressions of caspase-12, ATF6, GADD153, Calnexin, Calreticulin, PDI and Ero1-Lα, which are associated with endoplasmic reticulum stress and the unfolding protein response pathway, were examined by Western blot analysis. The cell cycle was analyzed by flow cytometry. The protein expressions of TCTP, Mcl-1, Bcl-2 and Bax, which are related to mitochondrial dysfunction, were detected by Western blot analysis. We also detected the ATF6 protein location by immunocytochemistry. The results showed that curcumin inhibits the proliferation of J5 cells in a time- and dose-dependent manner. Curcumin induced the unfolding protein response by down-regulating the protein expressions of Calnexin, PDI and Ero1-Lα and up-regulating the Calreticulin expression. Curcumin induces the GADD153 expression by cleaving caspase-12 and ATF6, and then by translocating ATF6 to the nucleus. Curcumin also down-regulates the protein expressions of TCTP, Mcl-1 and Bcl-2, in order to induce mitochondrial dysfunction. Curcumin induced cell cycle arrest at the G2/M phase by decreasing the Cdc2 expression. In conclusion, the present study showed that curcumin inhibits the proliferation of J5 cells by inducing endoplasmic reticulum stress and mitochondrial dysfunction.     

IL-8在兔动脉粥样硬化模型中斑块处的蛋白和基因表达

白细胞介素- 8(Interleukin- 8,IL- 8)是趋化因子CXC亚家族的一员,对中性粒细胞有趋化作用,可以诱导平滑肌细胞的增殖和迁移,是一个促血管生成因子,在动脉粥样硬化慢性炎症过程中起着重要作用。本文通过建立兔高脂血症动脉粥样硬化斑块模型,动态监测了在动脉粥样硬化发展过程中IL- 8的蛋白和基因表达,以了解IL- 8在动脉粥样硬化中的作用。采用免疫组化的方法定位IL- 8在高脂血症兔动脉粥样硬化斑块模型主动脉升弓部血管壁的蛋白表达,8、12周模型组内膜显著增厚并有明显阳性染色,半定量分析AS模型组阳性染色的面积分别是对照组的4 .4 8、8.76倍,平均积分光密度(IOD)值分别是对照组的4 .16、4 .36倍。采用双抗夹心EL ISA法测定兔血管组织匀浆液中的IL- 8蛋白表达量,8、12周AS模型组IL- 8蛋白含量与总蛋白比值和对照组比较均有显著性差异,分别是对照组的1.84、2 .0 6倍。用原位杂交的方法定位IL- 8m RNA的表达,8周模型组血管内膜有强的阳性染色,IL- 8m RNA的表达增加。本研究通过建立动脉粥样硬化模型,动态监测了整个动脉粥样硬化过程中IL-8的蛋白和基因表达。IL- 8的蛋白和基因上调主要是在高脂血症兔动脉粥样硬化模型的脂纹期     

氯丙烯中毒微循环及超微结构观察

氯丙烯中毒可引起周围微循环障碍,微血管出现痉挛→痉挛或扩张→扩张。微动脉及微静脉自律运动频率及幅度减弱,血流速度减缓,组织单位面积灌流量减少;微血管胆碱酯酶量减少。脊髓及脊神经根神经纤维微丝、微管及线粒体变性;心肌、肝细胞、肾近曲小管上皮细胞线粒体嵴减少,内质网空泡样变,部分心肌细胞断裂,肌原纤维之间间隙增大。本文讨论了氯丙烯中毒的毒性机理,认为微循环障碍是重要因素之一。     

In vivo reduction-oxidation state of protein disulfide isomerase: the two active sites independently occur in the reduced and oxidized forms

Thiol-disulfide oxidoreductases of the human protein disulfide isomerase (PDI) family promote protein folding in the endoplasmic reticulum (ER), while also assisting the retrotranslocation of toxins and misfolded ER proteins to the cytosol. The redox activity of PDI-like proteins is determined by the redox state of active-site cysteines found in a Cys-Xaa-Xaa-Cys motif. Progress in understanding redox regulation of the mammalian enzymes is currently hampered by the lack of reliable methods to determine quantitatively their redox state in living cells. We developed such a method based on the alkylation of cysteines by methoxy polyethylene glycol 5000 maleimide. With this method, we showed for the first time that in vivo PDI is present in two semi-oxidized forms in which either the first active site (in the a domain) or the second active site (in the a' domain) is oxidized. We report a steady-state redox distribution of endogenous PDI in HEK-293 cells of 50 +/- 5% fully reduced, 18 +/- 2% a-oxidized/a' -reduced, 15 +/- 2% a-reduced/a' -oxidized, and 16 +/- 4% fully oxidized. These results suggest that neither of the two domains in human PDI exclusively catalyzes substrate oxidation or reduction in vivo.     

1.8%AVM乳油对小鼠肝微粒体CYP450酶系与GST影响的初探

目的观察1.8%阿维菌素（AVM）乳油对小鼠肝微粒体细胞色素P450（CYP450）酶系与谷胱甘肽S-转移酶（GST）的影响,初步探讨1.8%AVM在肝脏的可能的代谢过程和毒性机理。方法 40只清洁级昆明种小鼠（雌雄各半）灌胃给予1.8%AVM乳油（70、35、17.5mg/kg）,连续7d,以0.9%氯化钠溶液作对照。末次给药后处死小鼠,采用差速离心法制备大鼠肝微粒体,Brandford法测定微粒体蛋白浓度,CO还原差示光谱法检测肝微粒体CYP450含量,差示光谱法则定肝微粒体b5（Cyt-b5）含量,紫外分光光度法则定肝微粒红霉素N-脱甲基酶活性（ERD）和氨基比林-N-脱甲基酶（ADM）和GST的活性。结果各1.8%AVM乳油染毒剂量组ERD、ADM活性均低于对照组,差异有统计学意义（P〈0.05）;各剂量组小鼠肝脏指数、CYP450与b5的含量及GST的活性与对照组比较差异均无统计学意义（P〉0.05）,且不同剂量组间差异亦无统计学意义（P〉0.05）。结论 1.8%AVM乳油可抑制小鼠肝微粒体ERD（主要反映CYP3A活性）和ADM（主要反映CYP2E1）的活力,而对CYP450和b5含量及GST活性影响小,未观察到1.8%AVM乳油对小鼠肝微粒体重要的Ⅰ相酶CYP450和Ⅱ相酶GST的诱导或抑制作用。