大鼠肝细胞缺氧预处理后基因表达谱的改变

离体培养的肝细胞经缺氧预处理后能耐受长时间缺氧，表明缺血预处理保护肝脏的现象也存在于体外。缺氧预处理保护肝细胞是一个受体介导的信号传导过程，但其确切机制至今仍不明了。为此，我们利用基因芯片技术研究缺氧预处理后肝细胞基因表达谱的变化，探索缺氧预处理预防肝细胞缺氧再灌注损伤的可能作用机制。     

低氧预处理对大鼠BMSCs葡萄糖代谢的影响

目的观察低氧预处理对BMSCs葡萄糖代谢的影响,探讨其潜在机制,为干细胞疗法的优化提供理论依据。方法取1～3日龄SD大鼠骨髓,采用密度梯度离心法获得BMSCs,取第4代细胞用于实验。根据处理方法不同将细胞分为4组:A组采用常氧培养24 h;B组以1%低氧浓度培养24 h;C组于低氧预处理前用20μmol/L甲氧雌二醇处理24 h;D组于低氧预处理前用50μmol/L缺氧诱导因子1(hypoxia-inducible factor 1,HIF-1)特异的siRNA处理12 h。采用MTT法、生化分析仪及实时荧光定量PCR检测各组BMSCs活力、葡萄糖代谢以及HIF-1αmRNA及葡萄糖转运蛋白1(glucose transporter 1,Glut-1)mRNA的表达。结果 MTT检测示A、B、C、D组吸光度(A)值分别为387.67±58.92、322.50±50.60、297.00±53.00、286.00±41.00,各组间差异无统计学意义(P>0.05)。与A组相比,B组葡萄糖摄取量和乳酸生成量显著增加(P<0.05);C、D组略高于A组,但差异无统计学意义(P>0.05),但显著低于B组,差异有统计学意义(P<0.05);C、D组间比较差异无统计学意义(P>0.05)。与A组相比,B组HIF-1αmRNA和Glut-1 mRNA表达均显著上调(P<0.05);C、D组HIF-1αmRNA和Glut-1 mRNA表达较B组大幅度降低(P<0.05),但仍显著高于A组(P<0.05);C、D组间差异无统计学意义(P>0.05)。结论低氧预处理可上调大鼠BMSCs的葡萄糖摄取和代谢能力,其机制涉及HIF-1 mRNA及其下游基因Glut-1 mRNA的表达上调。     

低氧预处理对大鼠自体肝移植后胰腺的保护作用

目的探讨低氧预处理对大鼠自体肝移植后胰腺的保护作用及其机制。方法 90只大鼠随机分成假手术组(sham组,术后6h、24h、48h,每组10只)、自体肝移植组(术后6h、24h、48h,每组10只)和低氧预适应组(术前通过低氧装置给予低氧预适应90min,氧浓度为8%O2∶92%N2;12 h后行自体肝脏移植,6 h、24h、48 h,每组10只)。测定血淀粉酶、脂肪酶,了解胰腺外分泌功能;测定胰腺组织MDA的含量;光镜、电镜观察胰腺形态学改变。SP法检测HIF-1a在胰腺中的表达。结果低氧预适应组大鼠血淀粉酶、脂肪酶、胰腺组织MDA的含量与自体肝移植组比较显著降低(P0.05),光镜、电镜观察低氧预适应组大鼠组织学结构损害轻于自体肝移植组,相应时间点低氧预处理组HIF-1a表达增加(P0.05)。结论低氧预处理对大鼠自体肝移植后胰腺具有保护作用,其机制与胰腺组织HIF-1a表达增加有关。     

异丙酚预处理对乳鼠肝细胞氧-糖缺失/恢复时凋亡的影响

目的探讨异丙酚预处理对乳鼠肝细胞氧-糖缺失/恢复时凋亡的影响及其机制。方法出生1～3 d健康SD乳鼠30只、体重7～11 g,雌雄不拘,提取乳鼠肝细胞,用10%胎牛血清的高糖DMEM培养基孵育6 d后,随机分为5组（n=6）,空白对照组（C组）;单纯氧-糖缺失/恢复组（O组）建立氧-糖缺失/恢复模型;不同浓度异丙酚预处理组（P25组、P50组、P100组）分别用含终末浓度为25、50、100μmol/L的异丙酚培养液预处理后建立氧-糖缺失/恢复模型。观察肝细胞凋亡情况,采用免疫组织化学方法测定肝细胞Bcl-2蛋白、p53蛋白的表达。结果与C组比较,O组、P25组、P50组、P100组的肝细胞凋亡率增加,Bcl-2蛋白表达下调,p53蛋白表达上调;与O组比较,P25组、P50组、P100组的肝细胞凋亡率降低,Bcl-2蛋白表达上调,p53蛋白表达下调（P〈0.01）,且呈浓度依赖性。结论异丙酚25、50、100μmol/L预处理对氧-糖缺失/恢复的乳鼠肝细胞具有保护作用,且呈浓度依赖性,其机制可能与下调p53蛋白的表达和上调Bcl-2蛋白的表达有关。     

Delta阿片受体参与低氧预处理减轻心肺复苏脑损伤的研究

目的研究delta阿片受体(DOR)是否参与了低氧预处理(HPC)对心肺复苏大鼠脑损伤的保护作用。方法 90只大鼠接受为期7d的HPC后,建立大鼠窒息性心肺复苏脑损伤模型,随机均分为五组:心跳停止(CA)组(A组)、HPC+CA组(B组)、Naltrindole(NTI,DOR特异性拮抗药)+CA组(C组)、HPC+人工脑脊液(ACSF)+CA组(D组)及HPC+NTI+CA组(E组)。A组仅建立心肺复苏模型;C组大鼠在窒息前30min经侧脑室注入含50nmolNTI的ACSF10μl;B、D、E组均接受连续7d的HPC,HPC结束后24h,B组大鼠接受气管插管、8min窒息以及心肺复苏;而D组和E组大鼠则在窒息前30min分别经侧脑室注入含0或50nmolNTI的ACSF10μl。观察复苏成功率并对存活大鼠的神经功能缺损进行评分,观察复苏后海马CA1区神经元损伤及凋亡情况。结果 HPC改善心肺复苏脑损伤后的神经功能缺损,抑制早期海马神经元的凋亡,增加海马CA1区存活神经元的数量,而DOR拮抗药Naltrindole明显消除了HPC的神经保护作用。结论在HPC减轻心肺复苏大鼠脑损伤的过程中,DOR发挥着重要作用。     

特发性高草酸尿症大鼠空肠蛋白质组二维电泳可行性研究

目的：在正式进行双向凝胶电泳实验前需要进行二维电泳的可行性评价,为双向凝胶电泳实验的顺利进行奠定实验基础。方法：取1只特发性高草酸尿大鼠（IH）和正常对照大鼠的空肠组织300mg,匀浆提取组织总蛋白,以双向电泳技术分离蛋白质,银染后扫描获得图谱,并以ImageMaster^TM 2D Platinum software（Version5．0）软件进行分析。结果：获得了清晰、稳定的凝胶蛋白图谱,正常大鼠凝胶图谱可检测到2541个蛋白点,IH大鼠凝胶图谱可检测到2654个蛋白点。结论：从获得的二维电泳图谱来看,2个蛋白质样品的蛋白质点的迁移基本稳定,从而保证后续正式的双向凝胶电泳实验有较好的重复稳定性和可行性。     

常温下缺血预处理对肝细胞凋亡调节基因表达影响的实验研究

目的 探讨常温下大鼠肝脏缺血预处理对细胞凋亡调节基因C-jun和Bcl-X_L的表达及其对肝细胞的保护作用。方法 将30只Wistar大鼠随机分成假手术组(S组,10只)、缺血再灌注组(IR组,10只)、缺血预处理组(IP组,10只)。S组在游离肝蒂后、IR组和IP组在再灌30 min后0、1、3、6、20 h点采集肝组织标本切片行肝细胞凋亡,C-jun和BcI-X_L基因表达及形态学改变等检测。各组均在3、6、20 h点采集血标本检测肝损害标记酶(ALT、AST、LDH)。结果 ALT、AST及LDH值各时间点IR组和IP组均明显高于S组(P<0.01),IP组明显低于IR组(P<0.01)。IR组和IP组与S组比较,细胞凋亡指数(AI)有显著性增加(P<0.01),IP组与IR组比较,AI明显减少(P<0.01)。C-jun基因在S组和IP组各时间点表达不明显;在IR组表达明显,3h点达高峰,并持续至6h点。Bcl-X_L基因在S组、IR组各时间点表达不明显,在IP组3、6、20h点表达明显(P<0.05或P<0.01)。形态学研究显示,IR组有肝组织大片坏死及不可逆超微结构损害;IP组未见明显肝细胞坏死,且超微结构损害为可逆性。结论 ①缺血预处理通过调节肝细胞凋亡调节基因C-jun和Bcl-X_L的表达,发挥其对大鼠常温下肝脏缺血再灌注损伤的保护作用;②IR损伤可能通过激活凋亡诱导基因C-jun表达而促发大鼠肝细胞的过度凋亡;③IP可能通     

缺血预处理对大鼠冷保存供肝细胞HMG-CoA还原酶活力的影响

目的 观察缺血预处理对大鼠冷保存供肝细胞HMG-CoA还原酶(HMGCR)活力的影响,探讨缺血预处理减轻大鼠供肝冷保存损伤的机制.方法 将25只大鼠随机分为对照组(C组)、冷保存组(Ⅰ组)、缺血预处理组(IP组)、阿托伐他汀30 μmol/L和100μmol/L处理组(A30组和A100组),每组5只.C组为正常大鼠肝脏,4℃UW液灌注;Ⅰ组为灌注后冷保存8 h;IP组先给予缺血预处理,灌注后冷保存8 h;A30组和A100组先给予缺血预处理,用终质量浓度为30μmol/L和100μmol/L的阿托伐他汀UW液灌注后再置入含30μmol/L和100μmol/L的阿托伐他汀UW液中4℃冷保存8 h.分别测定5组大鼠肝脏的HMGCR活力.结果 供肝经过8 h冷保存后(Ⅰ)HMGCR活性为(1872±157)nmol/(min·mg),与对照组(C)(3298±224)nmol/(min·mg)比较明显下降(P＜0.05),给予缺血预处理后再给予冷保存(IP),则HMGCR活性为(3746±231)nmol/(min·mg),明显升高(P＜0.05);给予缺血预处理同时给予HMGCR抑制剂阿托伐他汀则HMGCR活性则受到明显抑制,并随着阿托伐他汀浓度的增加而抑制效应更为明显,A30组、A100组HMGCR酶活力分别为(2010±193)nmol/(min·mg)、(1469±132)nmol/(min·mg),同IP组比较明显下降(P＜0.05).结论 缺血预处理可以明显提高HMGCR活力,而阿托伐他汀则可消除缺血预处理升高HMGCR活性的作用;缺血预处理升高HMGCR活力的机制是增加HMGCR蛋白的表达.

Abstract：

Objective To investigate the effects of ischemic preconditioning on the activity of 3-hydroxy-3-methylglutary coenzyme A reductase (HMGCR) of hepatocytes following cryopreservation in rats. Methods Twenty-five rats were randomly divided into five groups: control group ( C), cryopreservation group (Ⅰ), ischemic preconditioning group (IP), atorvastatin (30 γμmol/L) treatment group (A30) and atorvastatin (100 μmol/L) treatment group (A100). In control group, normal donor livers were flushed with 4 ℃ UW solution. In group I, donor livers were cryopreserved for 8 h after UWs flushing. In group IP, donor livers were subjected to ischemic preconditioning, and then flushed and cryopreserved in UWs. In group A30, donor livers were treated as in group I except that 30 μmol/L atorvastatin was added to the UWs. In group A100, donor lovers were treated as in group I with the exception that 100 μmol/L atorvastatin was added to the UWs. The activity of HMGCR was assessed. Results HMGCR activity in the donor cantly increased after ischemic preconditioning[(3746±231 ) nmol/(min-mg)]. The enhanced HMGCR activity induced by ischemic preconditioning was inhibited by atorvastatin in a concentration-dependent mantivity by up-regulating its protein expression and this effect can be abrogated by atorvastatin.     

内毒素诱导大鼠肝细胞白蛋白表达下降的分子机制

目的　探讨内毒素诱导肝细胞白蛋白表达下降的分子机制。方法　 1μg/ml内毒素刺激肝细胞后 ,分别在 0 ,2 ,8,12和 2 4h时留取肝细胞及上清检测白蛋白mRNA及其蛋白水平的变化。细胞内信号蛋白p38激酶和ERK激酶的特异性阻断剂SB2 0 35 80和PD980 5 9预处理肝细胞后检测上清中白蛋白的浓度。结果　内毒素刺激后 2 4h白蛋白mRNA下降约为 30 % ,与此同时白蛋白浓度下降约 5 0 %。SB2 0 35 80和PD980 5 9可以在体外抑制内毒素诱导肝细胞白蛋白表达的下降。结论　内毒素在转录水平抑制肝细胞白蛋白mRNA的表达来抑制白蛋白的合成。这一过程与细胞内信号传导通路p38和ERK激酶密切相关 ,进一步阐明了感染时低白蛋白血症的分子机制。     

肝细胞缺氧预处理细胞保护作用中热休克蛋白70表达的调控机制

目的　研究热休克蛋白 70 (HSP70 )表达与肝细胞缺氧预处理的关系及其调控机制。方法　建立肝细胞缺氧预处理模型 ,应用蛋白激酶C(PKC)抑制剂、激动剂和丝裂原激活的蛋白激酶 (MEK)的抑制剂 ,通过检测PKC磷酸化水平、p44 /4 2丝裂原激活蛋白激酶 (p44 /4 2MAPKs)、HSP70表达量、细胞存活率 ,同时在透射电镜下观察肝细胞超微结构改变 ,研究缺氧预处理与HSP70表达的关系及HSP70表达的影响因素。对相关数据进行统计学处理。结果　和缺氧复氧组 (HR)比较 ,预处理组 (HP)和PKC激动剂组的HSP70和 p44 /4 2MAPKs的表达量显著增加 ,同时PKC磷酸化水平显著增高 ,三者分别为每分钟 (4 2 .63± 4.73 )、(10 9.42± 16.0 9)、(15 2 .47± 19.5 9)pmol/g(P均 <0 .0 1) ,肝细胞结构改变较小 ;和缺氧预处理组相比 ,PKC抑制剂组相应的观察指标呈相反的变化 ,PKC磷酸化活性显著降低 ,为每分钟 (65 .2 8± 5 .3 6) pmol/g(P <0 .0 1) ;MEK抑制剂组HSP70表达量和磷酸化激活的p44 /4 2MAPKs表达量降低 ,肝组织细胞结构出现较明显的改变。结论　效应保护蛋白HSP70在缺氧预处理中参与对肝细胞的保护 ,HSP70受到PKC介导p44 /4 2MAPKs信号通路的调控。PKC处在p44 /4 2MAPKs上游 ,PKC对 p44 /4 2MAPKs起正性调控作用 ;p44 /4 2MAP     

人睾丸组织蛋白质组双向凝胶电泳技术的建立及其优化

目的 建立并优化人睾丸组织蛋白质组分析所需的双向凝胶电泳技术,提高其分辨率及重复性.方法 利用固相pH梯度双向凝胶电泳技术进行分离,凝胶经银染显色后,用Melanie 4软件分析2-DE图谱.对样品处理、上样量,胶条的选择等步骤进行了一系列的优化.结果 优化后成功地获得了人睾丸组织分辨率高、重复性好的双向电泳图谱.结论 应用双向凝胶电泳技术建立了人睾丸组织蛋白质组图谱,为其及相关疾病的蛋白质组学的进一步研究奠定了基础.     

间断低氧预适应对大鼠肝切除术后残余肝脏再生的影响

目的 研究间断低氧预适应对大鼠肝切除术后残余肝脏再生的影响。方法 54只SD大鼠用SPSS软件随机分为3组：假手术组（SO组）、肝部分切除组（PH组）和间断低氧预适应组（IHP组）。PH组切除大鼠的左叶和中叶肝脏,约占70％。IHP组大鼠每日在低氧环境下暴露1h,连续进行1周后行肝切除术。于术后第1、3、5天每组随机选取6只SD大鼠处死进行检测。称取肝脏重量,计算肝脏再生度和再生指数。取下腔静脉血用全自动生化分析仪检测血清谷丙转氨酶（ALT）和谷草转氨酶（AST）水平。采用免疫组化方法检测残余肝组织增生细胞核抗原（PCNA）阳性率。结果 IHP组术后第1天和第3天残余肝脏的再生度和再生指数明显高于PH组（P＜0.05）,而术后第5天两组差异无统计学意义。虽然IHP组和PH组大鼠术后血清ALT和AST水平开始下降,但二者均明显高于SO组,且术后第1天IHP组明显低于PH组（P＜0.05）。术后各个时间点IHP组残余肝脏的PCNA阳性细胞比例明显高于SO组和PH组（P＜0.05）。结论 间断低氧预适应能够在一定程度上防止肝切除术后残余肝组织中肝细胞破坏,并且能促进肝脏的早期再生。但其机制需进一步研究。     

人胰液蛋白质组的双向电泳方法初步研究

目的 在已有实验基础上进一步建立优化的用于人胰液差异蛋白质组研究的双向凝胶电泳(two-dimensional electrophoresis,2-DE)方法;探讨利用混合胰液进行胰腺疾病的胰液差异蛋白质组学的可行性.方法 通过ERCP术中放置鼻胰管引流收集5例胰腺癌(其中2例手术病理分别为胰腺中分化导管腺癌及导管内乳头状瘤癌变,另外3例经影像学检查结合临床诊断)和6例慢性胰腺炎胰液标本,取其中一例慢性胰腺炎胰液标本,通过改进胰液标本的处理方法、泡胀液的组成和一向等电聚焦条件后进行双向凝胶电泳,并和先前实验条件下的胰液2-DE图谱进行比较分析;同时利用优化后的双向凝胶电泳方法比较单份胰液和同病种混合胰液标本的2-DE图谱.结果 应用优化的2-DE方法,在胰液蛋白质加样量为200 μg时,可见凝胶上约有280个蛋白质点,有较好分辨率,较原条件下电泳图谱有较大改进.单份胰液和同病种混合胰液标本的2-DE图谱有较高程度的相似性(>75%).结论 优化后的胰液2-DE方法切实可行,通过各不同胰腺疾病混合胰液的2-DE图谱差异比较可为胰腺疾病的胰液差异蛋白质组学研究建立良好的基础.     

应用双向电泳分析精子冻融前后蛋白改变的初步研究

目的：精子蛋白双向电泳的建立，并利用其初步研究冻融前后精子蛋白的改变，为进一步筛选差异表达的蛋白奠定基础。方法：来源于同一个人的冻融前后精子样品经溶解液溶解，并以等电聚焦（IEF）作为第一向电泳，以变性的聚丙烯酰胺凝胶电泳（SDS-PAGE）作为第二向，对上述样品进行分离，凝胶银染后进行比较。结果：建立了精子蛋白的双向电泳，并用其分析了冻融前后精子蛋白的改变。结论：冻融前后精子蛋白发生不止一种蛋白的改变，本研究的实验方法是一种研究精子冻融蛋白改变的有效手段，但仍有待进一步优化。     

Protein changes in human sperm demonstrated by two-dimensional electrophoresis before and after cryopreservation

Protein changes in human sperm demonstrated by two-dimensional electrophoresis before and after cryopreservation     

Identification of the noncollagenous proteins of bovine bone by two-dimensional gel electrophoresis

Summary We have characterized the noncollagenous proteins of bovine bone using two high-resolution gel electrophoretic techniques. Proteins were extracted from bone tissue by extended dialysis against 0.5 M EDTA. In some cases, a preextraction was done in guanidine HCl. Bovine plasma was also examined to identify the proteins in bone that might also be present in blood. We have scored 160 major, reproducible spots on our standard preparation bone map. These comprise about 40 individual protein groups. There are many more minor spots present which puts the total number present over 200. Of these groups, 15 are not present on plasma maps. Bone proteins identified in this way include actin, bone Gla-protein, and osteonectin. The remainder are unknown. Bone Gla-protein is present in bovine bone in four isoelectric forms, pI=3.95−4.50. Electroblotting analysis of EDTA and guanidine HCl extracted material failed to reveal any higher molecular weight immunologically reactive species. The plasma proteins found in bone include, but are not limited to albumin, apo A-I lipoprotein, IgG, IgM, transferrin, α-2-HS-glycoprotein, and hemoglobin. Extraction with guanidine HCl plus EDTA significantly enriches the yield for the nonplasma proteins but does not appear to extract any additional bone proteins.     

Proteins of the rat prostate: I. Preliminary characterization by two-dimensional electrophoresis

The rat prostate consists of three distinct lobes: the ventral, lateral, and dorsal. Proteins in the three lobes of the prostate were studied with the ISO-DALT system for high-resolution two-dimensional electrophoresis. Proteins were detected with ammoniacal silver stain. Comparison of patterns from the three lobes of the prostate of control, noncastrated rats revealed that while there was a remarkable overall similarity, six groups of proteins showed lobe-specific differences. When prostatic regression was induced by castration, androgen-dependent proteins showed a decrease in staining intensity. A group of proteins, with pI 5.0-6.0 and MW 65,000-70,000, was consistently observed only during the active phase of prostatic regression (days 3-7 postcastration). Their presence during this specific interval may play a role in tissue involution.     

In-vitro influence of germ cells on Sertoli cel l-secreted proteins: a two-dimensional gel electrophoresis analysis

Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) was used to analyse [³⁵S]-methionine-labelled proteins secreted in vitro by Sertoli cells when cultured in the presence or absence of enriched preparations of pachytene spermatocytes (SPC), early spermatids (SPT) or residual bodies/cytoplasts from elongated spermatids (RB/CES). The presence of germ cells modified the pattern of Sertoli cell secreted proteins in co-culture. Out of 21 Sertoli cell secreted polypeptide families visualized by 2D PAGE, one (referred to as number 12) was stimulated, whereas the secretion of polypeptides 1 and 3 was inhibited by all of the germ cell populations tested. Early spermatids and RB/CES both enhanced the secretion of protein number 10 and inhibited the production of protein 11. The RB/CES fraction specifically inhibited secretion of polypeptide 13. Of particular note was the finding that co-culture with early spermatids or RB/CES induced the secretion of a novel polypeptide, termed GIP (germ cell-induced protein), with an apparent molecular weight of 72 kDa and an isoelectric point of 5.9. Under the present experimental conditions, media conditioned by the different germ cell fractions inhibited the secretion of polypeptide 2 but enhanced the secretion of polypeptides 10 and 18; of note also was the finding that media conditioned by early spermatids or RB/CES induced the appearance of GIP. This study confirms and extends the concept that germ cells influence Sertoli cell function and that the effects observed differ according to the stage of development of the germ cells. However, the sensitivity of the 2D gel electrophoresis technique, and to some extent its reproducibility, limit its use for studying the paracrine control of Sertoli cells in culture.     

Identification of prostate and other cancers by two-dimensional protein electrophoresis

Some cancers are so undifferentiated as to preclude their identification, and this hinders selection of an appropriate therapy. The systematic analysis and cataloguing of electrophoresed proteins from identified cancers and their comparison with the protein patterns from histologically indeterminate cancers should provide a general means of identifying the latter.     

Patterns of human epididymal fluid proteins on polyacrylamide gel electrophoresis

Proteins were analysed by sodium dodecylsulphate polyacrylamide gel electrophoresis in fluid taken from six epididymal regions in 10 men undergoing microsurgery to bypass epididymal obstructions resulting from various disorders. Some major proteins common to most samples were identified with apparent molecular weights of 95, 67, 56, and 44 kilodaltons. A degree of regional specificity in the synthesis and secretion of epididymal proteins was indicated. There appeared to be no correlation between protein pattern or the abundance of individual proteins and the cause of obstruction, although methodological constraints may have partially obscured any such relationship.