靶向干扰睾丸支持细胞功能的重组避孕疫苗的构建与表达

目的 构建含小鼠卵泡刺激素(亚单位(FSH-β)与闭锁因子(Ocln )特定基因片段的重组质粒,以期获得靶向干扰睾丸支持细胞功能的重组避孕疫苗.方法 运用PCR 技术,分别扩增小鼠FSH-β亚单位特定基因序列和闭锁因子第二个胞外环序列片段;定向克隆到pET28b表达载体中;并运用免疫印迹法对重组蛋白的表达做初步鉴定.结果 通过双酶切分析、测序鉴定证实特定的FSH-β亚单位和Ocln基因片断被成功克隆到pET28b中,且诱导表达的重组蛋白能够被抗小鼠FSH-β及抗O cln抗体所识别.结论 重组分子具有两种靶分子的抗原性,为进一步研究其生物学活性及免疫避孕效应奠定基础.     

谷氨酰胺对大鼠肠缺血再灌注损伤后闭合蛋白的保护作用

目的:探讨谷氨酰胺(Gln)对大鼠肠缺血再灌注损伤后闭合蛋白(occludin)的保护作用及其可能机制。方法:成年雄性Wistar大鼠30只随机分为假手术组(sham组)、缺血再灌注损伤组(I/R组)和谷氨酰胺预处理组(Gln组)。Gln组给予谷氨酰胺1 g·kg-1·d-1连续灌胃7 d。Sham组和I/R组以同等剂量生理盐水灌胃7d。Sham组仅分离肠系膜上动脉而根部不夹闭。I/R组和Gln组均用无损伤血管夹夹闭SMA根部30 min后放松血管夹形成再灌注损伤模型。各组大鼠均于制模后24 h采集静脉血和回肠标本。酶联免疫吸附试验检测血清中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-10和IL-2水平。全自动生化仪检测超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽(GSH)和谷胱甘肽过氧化物酶(GSH-Px)水平。免疫组化及Western blotting法检测occludin蛋白的表达情况。结果:I/R组occludin蛋白表达明显低于正常对照组及谷氨酰胺处理组(P0.05);I/R组TNF-α和MDA水平均高于sham组和Gln组(P0.05)。I/R组血清的SOD活力、GSH、GSH-Px、IL-10和IL-2均低于sham组和Gln组(P0.05)。结论:谷氨酰胺对肠缺血再灌注损伤后的occludin蛋白具有保护作用,其机制可能与抑制炎症反应、抗氧化应激有关。     

c-Src激酶对脂多糖致炎过程中occludin蛋白表达的影响

目的 探讨在脂多糖(lipopolysaccharide,LPS)处理小鼠肺泡上皮细胞(mouse lung epithelial-12,MLE-12)过程中,c-Src激酶对occhdin蛋白表达的影响. 方法 将培养的MLE-12细胞按随机数字表法分3组(每组3孔细胞):对照组(C组)、LPS实验组(L组)、LPS+c-Src激酶抑制剂PP2组(L+P组).C组不做任何处理;L组用LPS(浓度为10 mg/L)处理,处理时间为1、3、6 h;L+P组用PP2(浓度为100 μmol/L)预处理60 min,然后LPS(浓度为10 mg/L)处理6h.Western blot法和免疫荧光染色法分别检测各组不同时间点occludin蛋白的表达. 结果 Western blot发现,与C组比较:L组中6h时点occludin蛋白表达下调明显、c-Src表达明显升高,差异均有统计学意义(P＜0.05);与L组6h比较,L+P组occludin蛋白表达上调(P＜0.05).免疫荧光染色显示,occludin蛋白主要表达于细胞膜上,LPS影响occludin蛋白在膜上分布,PP2可以减轻LPS的影响. 结论 LPS导致MLE-12细胞紧密连接蛋白occludin表达下调,c-Src激酶参与其调节.     

乳酸菌对感染肠上皮细胞通透性及紧密连接蛋白表达的影响

目的:研究乳酸菌对侵袭性大肠杆菌(EIEC) 感染肠上皮细胞(Caco-2)后,对其通透性和紧密连接(TJ)蛋白表达的影响. 方法:建立Caco-2 单层细胞感染模型.实验分为正常组、感染组、乳酸菌组和庆大霉素组.用电阻仪测定单层细胞的跨膜电阻值(TER),用高效液相法(HPLC)测定甘露醇透过率的变化,采用免疫组化法观察TJ相关蛋白,如Claudin-1蛋白,Occludin蛋白, JAM-1蛋白,ZO-1蛋白的分布和结构变化. 结果:EIEC感染Caco-2细胞后,单层细胞的TER值和甘露醇的透过率随时间延长而升高,而经乳酸菌处理后,TER值升高趋缓(P＜0.05),甘露醇的透过率降低(P＜0.05);EIEC感染后,相邻Caco-2细胞间TJ结构遭到破坏,TJ相关蛋白的表达亦减少,而乳酸菌处理后可使EIEC引起的TJ结构受损减轻、相关蛋白的表达增多. 结论:乳酸菌黏附于肠上皮细胞Caco-2后,可抑制EIEC破坏单层细胞的完整性,并改善TJ的结构变化和相关蛋白的表达分布.     

Occludin在非小细胞肺癌组织中的表达及其临床意义

目的:检测非小细胞肺癌（NSCLC）组织中闭合蛋白（Occludin）的表达水平及其临床意义。方法:应用免疫组织化学方法检测73例NSCLC组织及其配对正常肺组织中Occludin蛋白表达水平,分析Occludin蛋白的表达差异及其与临床病理参数的相关性。结果:Occludin蛋白在肿瘤组织中的阳性表达率显著低于配对正常肺组织（P＜0.05）。Occludin蛋白的表达水平与患者性别、肿瘤大小、淋巴结转移、组织学类型、Ki-67表达水平显著相关。结论:Occludin蛋白表达下调与NSCLC细胞的增殖、转移能力相关,在肿瘤进展中发挥重要作用。     

黄体酮对脑缺血再灌注大鼠紧密连接蛋白ZO-1和occludin表达的影响

目的探讨黄体酮对大鼠局灶性脑缺血再灌注后血脑屏障紧密连接蛋白ZO-1、occludin表达及血脑屏障通透性的影响。 方法将42只健康雄性SD大鼠按随机数字表法分为假手术组（6只）和缺血再灌注组,后者再按再灌注时间分为缺血2h再灌注3h、6h、12h、24 h、48 h及72h组（各6只）。缺血再灌注组用线栓法制备成大鼠大脑中动脉缺血再灌注模型。采用荧光分光光度法测定缺血侧脑组织中伊文氏蓝(EB)含量来评价血脑屏障的通透性,Western blotting法检测脑组织ZO-1和occludin的表达。取EB漏出最多组的时间点,增设黄体酮干预组和溶剂对照组（各6只）,与相同时间点的缺血再灌注组比较,观察黄体酮对ZO-1、occludin表达及血脑屏障通透性的影响。 结果 缺血2h再灌注3h时脑组织EB含量开始增加,再灌注24 h时达高峰;ZO-1、occludin的表达在缺血2h再灌注3h时开始下降,再灌注24 h时达最低。黄体酮干预组EB含量明显低于缺血2h再灌注24 h组,差异有统计学意义(P＜0.05)。黄体酮干预组ZO-1和occludin的表达水平均明显高于缺血2h再灌注24 h组,差异有统计学意义(P＜0.05)。 结论 黄体酮町抑制缺血再灌注大鼠紧密连接蛋白ZO-1和occludin表达的降低,从而起到保护血脑屏障的作用。     

清胰颗粒对重症急性胰腺炎大鼠肠黏膜上皮紧密连接的保护作用

目的：探讨清胰颗粒对重症急性胰腺炎（SAP）大鼠肠黏膜上皮紧密连接的保护作用。方法：24只健康雄性Wistar大鼠随机分为假手术组、SAP组和清胰颗粒组。采用3.5%牛磺胆酸钠胆胰管逆行注射制备SAP大鼠模型,清胰颗粒组于造模前2 h、造模后6 h和18 h分别给予清胰颗粒灌胃,SAP组以同样的方法给予安慰剂。各组分别于造模后24 h取末端回肠,HE染色,光镜下观察病理组织学改变,采用Chiu’小肠组织损伤评价标准对肠黏膜损伤进行评价,应用Western blot和实时荧光定量PCR法检测回肠黏膜上皮紧密连接蛋白（Occludin）表达。结果：与假手术组相比,SAP组大鼠Chiu’小肠组织损伤评分（4.6±0.6） vs （0.0±0.0）明显升高,与SAP组相比,清胰颗粒组Chiu’小肠组织损伤评分（2.0±0.4）vs（4.6±0.6）明显降低;Western blot和实时荧光定量PCR结果显示,与假手术组相比SAP组大鼠Occludin蛋白表达（1.2±0.4）vs（3.2±0.4）和mRNA表达（1.3±0.3）vs（2.9±0.5）明显降低（P＜0.05）;与SAP组相比,清胰颗粒组 Occludin蛋白表达（4.1±0.4）vs（1.2±0.4）和mRNA表达（4.2±0.3）vs（1.3±0.3）明显升高（P＜0.05）。结论：清胰颗粒能减轻SAP大鼠肠黏膜损伤程度,这一作用可能与增强肠黏膜紧密连接蛋白Occludin表达有关。     

胆汁外引流对梗阻性黄疸大鼠肠黏膜紧密连接影响的实验研究

目的探讨胆汁在保护小肠黏膜屏障中的作用及其机理。方法 50只Wistar大鼠随机分为对照组(n=10)、梗阻性黄疸(阻黄)组(n=20)及外引流组(n=20)。造模10 d后测定血浆内毒素水平;取末端回肠黏膜组织,光镜下观察黏膜形态改变并测量相关指标,采用免疫组化及Western blot法检测紧密连接相关蛋白(闭锁小带蛋白-1及闭锁蛋白)的表达。结果阻黄组小肠黏膜萎缩明显,其肠绒毛高度、黏膜厚度和隐窝深度与对照组相比分别下降27.8%、21.7%和25.4%(P=0.001、0.001、0.040);外引流组各指标较对照组无明显变化(P=0.050、0.070、0.080),而明显优于阻黄组(均P=0.001)。阻黄组的血浆内毒素水平高达(1.49±0.27)EU/ml,明显高于对照组的(0.27±0.09)EU/ml(P=0.001);外引流组为(0.91±0.25)EU/ml,高于对照组而低于阻黄组(P=0.001)。免疫组化结果显示,闭锁小带蛋白-1的强阳性表达从对照组的7/10例降至阻黄组的6/20例(P=0.040),闭锁蛋白则从8/10例降至7/20例(P=0.020);而外引流组分别为8/20例(P=0.100、0.210)和9/20例(P=0.060、0.200),与前2组相比差异均没有统计学意义。Western blot分析显示,阻黄组闭锁小带蛋白-1和闭锁蛋白的表达水平较之对照组均明显下降(P=0.001、0.010);外引流组高于阻黄组(P=0.005、0.014),而闭锁小带蛋白-1的表达水平低于对照组(P=0.001),闭锁蛋白的表达与对照组比较差异无统计学意义(P=0.062)。结论肠道内胆汁缺乏会破坏肠紧密连接相关蛋白的成分,损伤肠黏膜屏障;胆道梗阻时由于存在其他因素,肠黏膜屏障损伤更严重。胆汁在保护肠黏膜屏障中具有重要作用。     

Tyrosine phosphatase inhibition induces loss of blood-brain barrier integrity by matrix metalloproteinase-dependent and -independent pathways

Tight junctions between endothelial cells of brain capillaries form the structural basis of the blood-brain barrier (BBB), which controls the exchange of molecules between blood and CNS. Regulation of cellular barrier permeability is a vital and complex process involving intracellular signalling and rearrangement of tight junction proteins. We have analysed the impact of tyrosine phosphatase inhibition on tight junction proteins and endothelial barrier integrity in a primary cell culture model based on porcine brain capillary endothelial cells (PBCEC) that closely mimics the BBB in vitro. The tyrosine phosphatase inhibitor phenylarsine oxide (PAO) induced increased matrix metalloproteinase (MMP) activity, which was paralleled by severe disruption of cell-cell contacts and proteolysis of the tight junction protein occludin. ZO-1 and claudin-5 were not affected. Under these conditions, the transendothelial electrical resistance (TEER) was markedly reduced. PAO-induced occludin proteolysis could be prevented by different MMP inhibitors. Pervanadate (PV) reduced the TEER similar to PAO, but did not increase MMP activity. Cell-cell contacts of PV-treated cells appeared unaffected, and occludin proteolysis did not occur. Our results suggest that tyrosine phosphatase inhibition can influence barrier properties independent of, but also correlated to MMPs. Evidence is given for a role of MMPs in endothelial tight junction regulation at the BBB in particular and probably at tight junctions (TJs) in general.     

Effects of cardiopulmonary bypass on tight junction protein expressions in intestinal mucosa of rats

AIM： To investigate the tight junction protein expressions of intestinal mucosa in an experimental model of cardiopulmonary bypass （CPB） in rats. METHODS： Thirty anesthetized rats were randomly divided into two groups： Group S （n = 10） served as sham operation and group C （n = 20） served as CPB which underwent CPB for 1 h. Expression of occludin and zonula occludens-1 （ZO-1） were determined by Western blotting and immunotochemistry, respectively. Plasma levels of diamine oxidase （DAO） and d-lactate were determined using an enzymatic spectrophotometry. RESULTS： Immunohistochemical localization of occludin and ZO-1 showed disruption of the tight junctions in enterocytes lining villi at the end of CPB and 2 h after CPB. The intensities of the occludin and ZO-i at the end of CPB were lower than those of control group （76.4% ± 22.5% vs 96.5% ± 28.5% and 62.4% ± 10.1% vs 85.5% ±25.6%, P 〈 0.05） and were further lower at 2 h after CPB （50.5% ± 10.5% and 45.3% ± 9.5%, P 〈 0.05）. Plasma d-lactate and DAO levels increased significantly （8.688 ± 0.704 vs 5.745 ± 0.364 and 0.898 ± 0.062 vs 0.562 ± 0.035, P 〈 0.05） at the end of CPB compared with control group and were significantly higher at 2 h after CPB than those at the end of CPB （9.377 ± 0.769 and 1.038 ± 0.252, P 〈 0.05）. There were significant negative correlations between occludin or ZO-1 expression and DAO （r^2 = 0.5629,r^2 = 0.5424, P 〈 0.05） or d-lactate levels （r^2 = 0.6512,r^2 = 0.7073, P 〈 0.05） both at the end of CPB and 2 h after CPB. CONCLUSION： CPB markedly down-regulates the expression of occludin and ZO-1 proteins in intestinal mucosa of rats. The close correlation between expression of tight junctions （TJs） and plasma levels of DAO or d-lactate supports the hypothesis that intestinal permeability increases during and after CPB because of decreases in the expressions of TJs.