成年大鼠脊髓厚片自发荧光特异性消除新方法

目的改良成年大鼠脊髓厚片自发荧光的特异性消除方法。方法利用染色前0.01 mol/L柠檬酸钠缓冲液(pH=6.0)80℃水浴热抗原修复45 min和染色后醋酸铵缓冲的0.01 mol/L硫酸铜溶液(p H=5.0)浸润的独立或者联合处理,特异性消除成年大鼠脊髓厚片漂片法免疫荧光染色中的自发荧光。结果染色前柠檬酸钠酸性缓冲液水浴热抗原修复能够显著消除成年大鼠脊髓厚片自发荧光。在此基础上,染色后短时间醋酸铵缓冲的酸性硫酸铜溶液浸润,即可完全消除这些自发荧光,并不减弱漂片法免疫荧光染色的目标荧光信号。结论柠檬酸钠酸性缓冲液水浴热抗原修复-醋酸铵缓冲的酸性硫酸铜溶液浸润的联合处理比较理想地特异性消除成年大鼠脊髓厚片漂片法免疫荧光染色中的自发荧光。     

PPARγ配体罗格列酮预防MNNG诱导的大鼠胃癌发生的实验研究

目的： 探讨PPARγ配体罗格列酮（RSG）对化学致癌剂N-甲基-N’-硝基-亚硝基胍(MNNG)诱导的大鼠胃癌发生的影响,并探讨罗格列酮防治胃癌的可能机制。方法: 二级Wistar大鼠90只,随机分为5组：A组(对照组),B组（MNNG诱癌组）,C－E组（RSG处理组,分别灌喂不同浓度罗格列酮）,以上处理连续10个月,比较各组动物胃癌发生率的差异。同时采用微阵列技术对罗格列酮体内干预的大鼠腺胃癌进行基因表达谱的分析研究,筛选PPARγ配体抗肿瘤新的靶基因并予验证。结果: A、B、C、D、E组动物胃癌的发生率分别为0%（0/10）、70%（14/20）、15%（3/20）、30%（6/20）和30%（6/20）, 病理组织学证实为腺癌。与B组相比,C、D、E组动物腺胃癌的发生率均明显低于B组动物（P<0.01）。采用微阵列技术在大鼠腺胃癌组织中筛选出79个上调的差异表达基因,其中RSG处理组大鼠胃癌组织中HCaRG基因表达显著高于MNNG诱癌组,同时应用荧光定量PCR证实在RSG处理组其它大鼠胃癌组织中HCaRG表达明显高于MNNG诱癌组（P<0.05）,而在人胃癌组织中HCaRG表达明显低于正常胃黏膜组织（P<0.05）。 结论: PPARγ配体罗格列酮能显著降低MNNG诱导的大鼠腺胃癌发生率,诱导HCaRG表达可能是罗格列酮预防胃癌发生机制之一。     

PTEN在肝纤维化大鼠肝组织中的动态表达

目的： 探讨大鼠肝纤维化过程中肝组织PTEN的动态表达。方法: 采用胆总管结扎法建立大鼠肝纤维化模型,HE及Masson三色染色检测肝脏组织学变化,免疫组织化学染色、Western blotting及实时荧光定量PCR技术检测大鼠肝组织的PTEN蛋白及mRNA表达。结果: 大鼠肝纤维化模型成功建立,随着造模时间延长,肝纤维化程度逐渐加重,造模不同时间均可见不同程度的肝细胞变性坏死而导致正常肝细胞逐渐减少;免疫组织化学染色显示正常大鼠肝组织中PTEN广泛表达,主要表达于细胞浆,随着肝纤维化的进展,PTEN阳性表达细胞逐渐减少（P<0.01）;Western blotting及实时荧光定量PCR显示造模1周、2周、3周及4周不同时间大鼠纤维化肝组织中PTEN蛋白及mRNA表达均显著低于假手术组（P<0.01）,并随着肝纤维化的进展逐渐降低（P<0.01）。结论: 大鼠纤维化肝组织中PTEN蛋白及mRNA表达均随着肝纤维化的进展逐渐降低,其下降程度与肝纤维化程度一致。     

地塞米松对大鼠内毒素急性肺损伤肺泡灌洗液中白细胞介素-1β和肿瘤坏死因子-α含量的影响

目的 探讨地塞米松对大鼠内毒素（LPS）急性肺损伤（ALI）肺泡灌洗液中白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）含量的影响.方法 将48只雄性SD大鼠随机分为正常对照组、急性肺损伤组（内毒素ALI模型组）、地塞米松干预组,每组16只.每组大鼠依据不同的观察时间点（以大鼠气管内滴注LPS的时刻为起始时刻后的1、2、4、8 h 4个时间点）分为4个亚组（n=4）.给予正常对照组气管内滴注0.9%氯化钠溶液 0.3 mL,10 min后股静脉注射0.9%氯化钠溶液 1 mL;急性肺损伤组气管内滴注LPS 0.2 mg/kg（溶于0.3 mL 0.9%氯化钠溶液）,10 min后股静脉注射0.9%氯化钠溶液 1 mL;地塞米松干预组气管内滴注LPS 0.2 mg/kg,10 min后股静脉注射地塞米松注射液3 mg/kg（溶于1 mL 0.9%氯化钠溶液）.各组于1、2、 4、8 h 4个时间点于心脏抽血检测动脉血氧分压（PaO2）;取肺组织进行肺湿/干质量比值（W/D）测定,并采用苏木精-伊红（HE）染色观察肺组织形态学变化;用酶联免疫吸附测定（ELISA）法检测肺泡灌洗液（BALF）中IL-1β和TNF-α的含量.结果 给予气管内滴注LPS后于1、2、4、8 h观察大鼠动脉血PaO2,急性肺损伤组、地塞米松干预组较正常对照组显著降低,两组PaO2均在4 h时达最低点,各时间点动脉血PaO2对比,地塞米松干预组均显著高于急性肺损伤组,差异均有统计学意义（P〈0.05）.在LPS致炎后1、2、4、8 h 4个时间点急性肺损伤组、地塞米松干预组各时间点W/D比值均较正常对照组显著增加,两组间比较地塞米松干预组较急性肺损伤组降低,差异具有统计学意义（P〈0.05）.病理形态学观察可见急性肺损伤组与地塞米松干预组均出现肺水肿、出血、炎性细胞浸润,而地塞米松干预组肺损伤程度较急性肺损伤组减轻.ELISA试验结果显示急性肺损伤组在气管内滴入LPS 1 h后BALF中IL-1β、TNF-α含量迅速升高,4 h时达峰值,地塞米松干预后IL-1β、TNF-α表达在相同时间点均较模型组显著降低,差异具有统计学意义（P〈0.05）;而正常对照组BALF中IL-1β、TNF-α在不同时间点无明显变化.结论 地塞米松可通过抑制内毒素性大鼠ALI肺组织中IL-1β、TNF-α的表达,改善呼吸氧合功能,减轻肺损伤.     

饮食钾缓解盐诱导的冠状动脉损伤*

 目的：探讨饮食钾对盐诱导的冠状动脉损伤的保护机制。方法: 将4周龄SD大鼠随机分为3组,每组10只,分别是对照组（NS,蒸馏水）、高盐组（HS,含1.5% NaCl蒸馏水）和高盐补钾组（HS+HP,含1.5% NaCl和0.5% KCl蒸馏水）干预16周。每2周监测各组大鼠尾动脉血压。干预16周后,硝酸还原酶法检测大鼠血清中NO的含量;硫代巴比妥酸法检测各组大鼠血清中丙二醛（MDA）的含量; HE染色观察各组大鼠左冠状动脉的大体形态;免疫荧光染色观察各组大鼠冠状动脉内皮型一氧化氮合酶（eNOS）的表达;二氢乙啶荧光探针染色/Western blotting法观察各组大鼠冠状动脉氧化应激的程度。结果：高盐干预16周后,高盐组大鼠根据尾动脉血压的变化分为盐敏感性大鼠和盐抵抗性大鼠。本实验只研究HS组中盐敏感性大鼠。在HS组中,大鼠血压较NS组显著升高,给予补钾后可以缓解血压的升高。HS组较NS组大鼠血清中NO降低,MDA升高,冠状动脉eNOS表达降低,还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶的gp91亚基表达升高,冠状动脉管壁厚度显著增加,且 DHE荧光探针染色发现其超氧阴离子增加。高盐摄入的同时给予补钾可以缓解高盐摄入引起的有害效应。结论：高盐摄入通过氧化应激引起冠状动脉结构和功能的改变,补钾可以缓解这一效应的发生。     

参麦注射液对糖尿病心肌病大鼠心肌纤维化的干预作用及相关机制探讨

 目的：探讨参麦注射液对糖尿病心肌病心肌纤维化的干预作用并初步探讨其机制。方法：将30只Wistar大鼠分为对照组、糖尿病组以及治疗组,每组各10只。采用链脲佐菌素单次腹腔注射的方法制作大鼠糖尿病模型,以枸橼酸缓冲液腹腔注射为对照,治疗组则采用腹腔注射的方式给予参麦注射液干预治疗。采用心室插管对大鼠心功能进行评估;Masson染色观察大鼠心脏组织中胶原水平;二氢乙啶染色观察大鼠心脏组织中活性氧（ROS）水平;Western blotting观察大鼠心肌组织中I 型胶原、基质金属蛋白酶2（MMP-2）以及金属蛋白酶组织抑制剂2（TIMP-2）的表达水平。结果：与对照组相比,糖尿病组心功能显著下降（P＜0.05）;而治疗组心功能则显著恢复（P＜0.05）。与对照组相比,糖尿病组心肌组织ROS及胶原生成水平显著升高（P＜0.05）,但经参麦注射液治疗后均显著降低（P＜0.05）。与对照组相比,糖尿病组I 型胶原及TIMP-2表达水平显著升高（P＜0.05）,而MMP-2水平显著降低（P＜0.05）;与糖尿病组相比,治疗组I 型胶原及TIMP-2水平显著降低（P＜0.05）,而MMP-2水平则显著升高（P＜0.05）。结论:参麦注射液可能通过抑制氧化应激损伤而减轻糖尿病心肌病心肌纤维化程度。     

真武汤对阿霉素所致大鼠肾损伤的治疗作用

 目的：研究真武汤对阿霉素肾病模型（adriamycin nephropathy,AN）大鼠足细胞裂孔隔膜蛋白分子（podocin和nephrin）表达的影响,探讨其防治阿霉素肾病大鼠蛋白尿的机制。方法：采用常规生化、病理方法（包括HE染色、Masson染色及电镜）观察真武汤对AN模型所致纤维化大鼠肾功能、肾组织形态学变化及羟脯氨酸（Hyp）含量的改善作用;采用蛋白免疫印迹等方法探索真武汤对足细胞标志蛋白podocin和nephrin信号分子表达的影响。结果：模型组大鼠尿蛋白（TP）、血尿素氮(BUN)和血清肌酐（SCr）显著增加,肌酐清除率（CCr）显著下降（P<0.05）;Hyp显著增加（P<0.05）;肾组织podocin和nephrin蛋白表达水平显著降低（P<0.05）;肾小管萎缩,基底膜增厚;足突扁平、融合、消失。肾小球集中现象明显;部分肾小管扩张,肾小管上皮细胞变性,蛋白管型明显;肾间质纤维组织增生和较多炎症细胞浸润。与模型组相比,各治疗组TP、BUN和SCr均有一定程度的下降,CCr显著提高;Hyp明显下降（P<0.05）;真武汤组肾组织podocin和nephrin蛋白表达水平显著提高（P<0.05）;肾组织病变程度轻于模型组。结论：真武汤能减少阿霉素肾病大鼠肾组织羟脯氨酸含量,改善肾功能及减轻病理损伤。 真武汤降低模型大鼠蛋白尿的作用可能与其维持足细胞podocin和nephrin的表达有关。     

Ghrelin对糖尿病大鼠下丘脑弓状核胃牵张敏感神经元放电活动及胃运动的调控

 目的：观察链脲佐菌素(STZ)所致糖尿病（DM）大鼠下丘脑弓状核（Arc）胃牵张（GD）敏感神经元放电活动及胃运动改变,探讨ghrelin对DM大鼠下丘脑Arc GD敏感神经元放电活动和胃运动的影响。方法：采用STZ腹腔注射诱导DM大鼠模型;通过细胞外记录神经元单位放电和在体胃运动方法,观察ghrelin及其受体阻断剂［D-Lys3］-GHRP-6对DM大鼠下丘脑Arc GD敏感神经元自发放电活动和胃运动的影响;应用real-time PCR和荧光免疫组化方法,探讨DM大鼠Arc内ghrelin受体（GHS-R1a）mRNA及其免疫阳性物的表达。结果：在正常大鼠Arc记录到的98个GD敏感神经元中,64.3%为GD兴奋性（GD-E）神经元,35.7%为GD抑制性（GD-I）神经元。在63个GD-E神经元中,Arc微量注射ghrelin可使其中73.0%神经元兴奋,其放电频率与生理盐水组比较显著增加（P<0.05）;而在35个GD-I神经元中,Arc微量注射ghrelin可抑制其中60.0%神经元,放电频率显著降低（P<0.01）;ghrelin改变GD神经元放电效应可被ghrelin 受体阻断剂［D-Lys3］-GHRP-6阻断（P<0.05）;在DM大鼠,Arc记录到的66个GD敏感神经元中有56.1%为GD-E神经元,43.9%为GD-I神经元。Arc注射ghrelin可兴奋其中35.1%GD-E神经元,放电频率与生理盐水比较显著增加（P<0.05）;而在29个GD-I神经元中,ghrelin可抑制其中21个神经元（72.4%）,放电频率显著降低（P<0.01）。与正常大鼠比较,DM大鼠Arc GD敏感神经元中的GD-E和GD-I比例无显著改变（P>0.05）,但ghrelin使GD-E神经元兴奋的比率明显降低（P<0.05）,放电频率平均增加率也显著下降（P<0.05）;但ghrelin使GD-I 神经元抑制比率和放电频率平均减少率均无显著改变（P>0.05）。在体胃运动研究结果显示,Arc微量注射ghrelin,可显著促进正常和DM大鼠胃运动,且呈显著量效关系（P<0.05,P<0.01）,但ghrelin对正常大鼠的促胃运动作用显著强于其对DM大鼠的作用（P<0.05）,［D-Lys3］-GHRP-6可完全阻断ghrelin该作用。Real-time PCR研究结果显示,DM大鼠下丘脑Arc GHS-R1a mRNA表达较正常大鼠明显减少（P<0.05）;免疫荧光研究进一步证实DM大鼠下丘脑Arc GHS-R1a 免疫阳性物表达较正常大鼠明显减少（P<0.05）。结论：下丘脑Arc ghrelin参与DM大鼠GD敏感神经元自发放电活动,并参与胃运动的调控,该效应可能是通过作用于ghrelin受体而实现的。     

缺氧诱导因子-1α基因在严重烫伤延迟复苏大鼠肺组织中的表达

目的观察大鼠严重烫伤延迟复苏后肺组织缺氧诱导因子-1α（HIF-1α）的表达变化,探讨其与肺组织损伤的相关性。方法雄性SD大鼠120只,随机分为延迟复苏组（DF,n=60）、即时复苏组（IF,n=50）和正常对照组（NC,n=10）,建立Ⅲ度烫伤面积30％TBSA的模型,分别于伤后1、6、12、24、48和72h取材,采用组织病理学、组织芯片技术、免疫组织化学染色与图象分析技术,观察肺组织病理改变与HIF-1α的表达。结果DF组伤后6—24h肺组织病理损伤严重,IF组相对较轻。HIF-1α的阳性表达位于肺泡上皮细胞胞核,DF组表达明显强于IF组,而NC组表达阴性。HIF-1α表达的灰度值定量分析结果显示：DF组与IF组在伤后72h各时相点表达均升高,与NC组比较差异有统计学意义（P〈0．05）;DF组在各时相点HIF-1α表达均高于相对应的IF组各时相点,差异有统计学意义（P〈0．05）。结论严重烫伤延迟复苏后大鼠肺组织HIF-1α的高表达与肺组织的损伤的程度是一致的,推测其参与了严重烫伤延迟复苏后大鼠肺组织的损伤。     

大鼠肝脏星形细胞的分离培养及鉴定

目的　建立大鼠肝星形细胞 (hepaticstellatecells,HSC)的体外分离及培养方法 ,为进行肝纤维化的实验研究提供技术支持。方法　选用SD大鼠经消化酶灌注肝脏后 ,用调整密度后的淋巴细胞分离液分离HSC。通过观察其自发荧光及显著其特征性结构的脂肪染色、细胞免疫化学染色等方法鉴定。结果　分离得到HSC ,细胞得率为 1～ 1 5× 10 7/只 ,存活率为98%。结论　成功分离及培养HSC ,且方法经济、简便、稳定     

A procedure for the simultaneous demonstration of neurosecretory and mucosubstances in tissue sections.

Many well known histochemical techniques have been employed to study the neurosecretory products in invertebrates and vertebrates. Among these aldehyde-fuchsin (AF) has been one of the two specific stains to demonstrate the presence of neurosecretory cells. Various counterstains have been employed with AF. However, AF is also useful in demonstrating the presence of mucosubstances or elastin. A method is described here for a simultaneous demonstration of neurosecretory substances (NSS) and mucosubstances (MS) by mercurochrome counterstaining. For this method sections of material fixed in methanol-formaldehyde-acetic acid (MFA) or alcoholic Bouin's or Susa were oxidized in 0.3% permanganate with 0.3 ml concentrated H2SO4 for 1 to 2 minutes, decolorized in 1% oxalic acid, stained in 0.5% aqueous basic fuchsin containing 1 ml concentrated HCl and 1 ml paraldehyde for 10 min and counterstained with 0.5% aqueous mercurochrome. After mercurochrome counterstaining the characteristic AF staining (purple colour) of NSS is dramatically swamped (becoming brick red) but the purple developed by MS remains unaltered in vertebrate and invertebrate material. Eosin or other counterstains do not behave in this way.     

Threshold of detection of Cryptosporidium oocysts in human stool specimens: evidence for low sensitivity of current diagnostic methods.

To determine the minimum number of Cryptosporidium oocysts that can be detected in stool specimens by diagnostic procedures, stool samples seeded with known numbers of Cryptosporidium parvum oocysts were processed by the modified Formalin-ethyl acetate (FEA) stool concentration method. FEA concentrates were subsequently examined by both the modified cold Kinyoun acid-fast (AF) staining and fluorescein-tagged monoclonal antibody (immunofluorescence [IF]) techniques. Oocysts were more easily detected in watery diarrheal stool specimens than they were in formed stool specimens. For watery stool specimens, a 100% detection rate was accomplished at a concentration of 10,000 oocysts per g of stool by both the AF staining and IF techniques. In formed stool specimens, 100% of specimens seeded with 50,000 oocysts per gram of stool were detected by the IF technique, whereas 500,000 oocysts per g of stool were needed for a 100% detection rate by AF staining. Counting of all oocysts on IF slides indicated a mean oocyst loss ranging from 51.2 to 99.6%, depending on the stool consistency as determined by the FEA concentration procedure. Our findings suggest that the most commonly used coprodiagnostic techniques may fail to detect cryptosporidiosis in many immunocompromised and immunocompetent individuals.     

The detection of respiratory syncytial virus in nasopharyngeal aspirates: assessment, formulation, and evaluation of monoclonal antibodies as a diagnostic reagent

Comparisons were made between standard methods of cell culture, indirect immunofluorescence (IF) using hyperimmune respiratory syncytial virus (RSV) antiserum, and indirect IF using mouse monoclonal antibodies directed against various epitopes of RSV for the detection of RSV in nasopharyngeal aspirates. The monoclonal antibodies were used singly and in pools of different specificities which in turn were tested in both direct and indirect IF. In a preliminary study, aspirates from 227 infants were examined for RSV by standard methods. The results were compared with the detection of RSV in these aspirates using nine separate monoclonal antibodies and a pool consisting of five monoclonal antibodies. Respiratory syncytial virus was detected in 64 (28%) by cell culture, in 68 (30%) by indirect IF using bovine polyclonal antibody (BPA), and in 75 (33%) by indirect IF using the monoclonal antibody pool. The nine individual monoclonal antibodies when tested separately were less sensitive, detecting between 8 and 77% of all aspirates found to be positive by culture. After statistical analysis of the results obtained in the preliminary study, a refined monoclonal antibody pool was prepared and in a further study was tested by both direct and indirect IF in parallel with our two standard methods. Slides prepared from 303 nasopharyngeal aspirates collected between 1981 and 1984 and either tested the same day or stored at -20 degrees C were used to evaluate these reagents. Overall agreement between the four tests was found in 274 (90%) specimens. Cell culture detected RSV in 68 (22%) specimens, indirect IF with BPA in 67 (22%), indirect IF with monoclonal antibody in 72 (24%), and direct IF with monoclonal antibody in 79 (26%). The pool of monoclonal antibodies used in direct or indirect IF was thus more sensitive than our standard methods for the detection of RSV in nasopharyngeal aspirates, and direct IF tests could be completed in 40 minutes.     

Evaluation of serum rosette inhibitory factors in chronic liver disease.

Serum rosette inhibitory factors (IF) were detected in 61% of 18 sera from HBsAg negative and in 78% of 14 sera from HBsAg positive chronic active liver disease (CALD) patients, who were not under immunosuppressive treatment. In CALD patients, who were under treatment for at least 6 months, IF were detected in 66% of the HBsAg positive patients and only in 18% of the HBsAg negative ones. After precipitation of the serum gamma-globulins by ammonium sulphate, IF were found in the supernatant only in HBsAg positive sera, while completely disappeared in HBsAg negative ones. A significant correlation between serum IF and factors reacting in immunofluorescence (IFL) with a T enriched preparation of lymphocytes was documented only in HBsAg negative cases. No correlation was found between serum IF and circulating immune complexes (IC) or lymphocytotoxins in any of the sera tested. From all these data it is concluded that serum IF detectable in HBsAg negative CALD cases are probably immunoglobulins. Our data would favour the hypothesis that they are anti-T lymphocytes antibodies, since no correlation was found between serum IF and circulating IC. Similar factors, detectable in HBsAg positive sera, are, at least in part, different. The role of such factors in the modulation of the immune response in CALD patients is discussed.     

猪小肠黏膜下层脱细胞支架的制备及组织学评价

目的评价不同浓度的十二烷基磺酸钠(SDS)处理猪小肠黏膜下层(SIS)的脱细胞效果,筛选最佳脱细胞浓度,为组织工程化角膜上皮的制备提供支架材料。方法配制0.1%、0.2%、0.3%、0.5%SDS随机分成A、B、C、D 4组,分别脱细胞处理SIS 15min、30min、1h、2h(n=20),同时观察SIS的大体形态变化,HE和4,6-二脒基-2-苯基吲哚(DAPI)染色观察脱细胞前后SIS的生物学特性,并对脱细胞支架进行基因组DNA分析(n=5)。结果脱细胞SIS肉眼观察呈乳白色、半透明的膜状物,具有一定的弹性、韧性及透光性;HE和DAPI染色光学显微镜观察结果显示,A组及B组处理30min时SIS生物支架无细胞及DNA残留,组织结构保留完整,胶原纤维间孔隙率增加;A组及B组处理30min脱细胞率可达90%以上。结论 0.1%及0.2%SDS处理30min条件下脱细胞效果较好,能更好地降低SIS的免疫原性,是SDS脱细胞处理SIS比较理想的浓度时间条件,为组织工程化角膜上皮的构建奠定理论基础。     

Acknowledgments

We examined the impact of father involvement on treatment. Participants were 107 families enrolled in parent-child interaction therapy (PCIT), including 56 involved-father (IF) families, 16 uninvolved-father (UF) families, and 35 absent-father (AF) families. All groups showed improvements during treatment to within the average range on the Eyberg Child Behavior Inventory (ECBI), although mothers from AF families reported better treatment outcome than mothers from IF families. Improvements occurred on the Beck Depression Inventory (BDI) and the Parenting Stress Index (PSI) as well, but there were no group differences. At a 4-month follow-up, mothers in IF families maintained treatment gains on the ECBI. In contrast, mothers in AF families reported significant decline at follow-up, although their scores remained within the normal range. Results suggest that father participation in treatment may not affect immediate treatment outcome but may help to maintain the beneficial effects of PCIT.     

电针足三里穴对肠缺血再灌注损伤大鼠小肠黏膜上皮紧密连接蛋白ZO-1的调节作用

目的探讨电针足三里穴对肠缺血再灌注损伤大鼠小肠黏膜上皮紧密连接蛋白闭锁小带蛋白(ZO)-1的调节作用。 方法按随机数字表法将30只SD大鼠分为模型组、电针足三里穴组、电针非经非穴组,每组10只。每组大鼠均于麻醉后沿腹正中线切口,分离肠系膜上动脉,用无损伤血管夹夹闭其起始部位,30 min后松夹恢复血流60 min,造成小肠缺血/再灌注损伤。电针足三里穴组于大鼠缺血后即刻行电针双侧足三里穴30 min,强度为2～3 mA,频率2～100 Hz;电针非经非穴组采用相同频率和强度于缺血后即刻刺激大鼠非经非穴(足三里穴外侧旁开0.5 cm)30 min;模型组不做任何治疗。于缺血再灌注损伤60 min时,以腹主动脉放血法处死各组大鼠,留取每只大鼠的血液和10 cm远端回肠组织。将留取的大鼠血液经10 000×g,4 ℃离心10 min后取上清得到血浆20 μL,全自动生化分析仪检测大鼠血浆脏器功能指标,包括谷丙转氨酶(GPT)、肌酸激酶同工酶(CK-MB)、尿素氮、肌酐;免疫荧光染色及蛋白质印迹法检测各组大鼠小肠黏膜上皮紧密连接蛋白ZO-1分布情况与表达水平。数据比较采用单因素方差分析和LSD-t检验。 结果缺血再灌注损伤60 min时,模型组大鼠GPT、CK-MB、尿素氮和肌酐水平分别为(88.1±11.6) μ/L、(482.3±69.7) μ/L、(11.6±3.7) mmol/L、(52.3±13.2) μmol/L,电针足三里穴组大鼠分别为(37.3±6.4) μ/L、(213.7±44.3) μ/L、(5.2±1.4) mmol/L、(30.1±5.7) μmol/L,电针非经非穴组大鼠分别为(85.7±13.4) μ/L、(460.2±72.3) μ/L、(10.8±4.2) mmol/L、(53.4±12.1) μmol/L,3组大鼠各指标比较,差异均有统计学意义(F＝69.4063、55.3611、10.9582、14.6817,P<0.05)。与模型组比较,电针足三里穴组大鼠血浆中GPT、CK-MB、尿素氮和肌酐水平与均降低,2组比较差异均有统计学意义(t＝12.126、10.285、5.116、4.883, P<0.05),而电针非经非穴组大鼠血浆中GPT、CK-MB、尿素氮和肌酐水平与模型组相近,差异均无统计学意义(P>0.05);电针足三里穴组大鼠血浆中GPT、CK-MB、尿素氮和肌酐水平均低于电针非经非穴组,比较差异均有统计学意义(t＝－10.307、－9.193、－4.000、－5.509, P<0.05)。免疫荧光染色结果显示,缺血再灌注损伤60 min时,模型组大鼠小肠黏膜上皮紧密连接蛋白ZO-1分布异常,连续性破坏,表达减少;电针足三里穴组大鼠小肠黏膜上皮紧密连接蛋白ZO-1分布密度较大、完整、连续;电针非经非穴组大鼠小肠黏膜上皮紧密连接蛋白ZO-1断裂,部分缺失,肠黏膜上皮紧密连接蛋白ZO-1荧光较弱、连续性消失。蛋白质印迹法检测结果可得,缺血再灌注损伤60 min时,电针足三里穴组大鼠小肠黏膜上皮紧密连接蛋白ZO-1蛋白表达(1.67±0.43)显著高于模型组(0.86±0.31)和电针非经非穴组(0.89±0.29),比较差异均有统计学意义(t＝4.517、3.965,P<0.05);电针非经非穴组大鼠小肠黏膜上皮紧密连接蛋白ZO-1蛋白表达与模型组比较,差异无统计学意义(P>0.05)。 结论电针足三里穴可有效调节肠黏膜上皮紧密连接蛋白ZO-1的正常分布与表达,能有效保护肠缺血再灌注损伤后肠黏膜屏障功能,减轻肠缺血再灌注后脏器功能损害。     

Effect of interferon on exogenous murine leukemia virus infection.

When interferon (IF)-treated NIH/3T3 cells were exogenously infected with the Moloney strain of murine leukemia virus (M-MLV), no viral progeny release was detected as long as IF remained in the medium. This was evidently an inhibition of the virus replication and not a consequence of interfering with the establishment of the infection since, when IF was removed either before infection or later after infection, virus release gradually approached the rate of untreated control after a temporary delay. Furthermore, a direct examination revealed that IF treatment had no effect on the formation of infectious centers. IF treatment led to a reduced viral RNA synthesis. A similar inhibition of viral RNA synthesis was observed when the potent protein synthesis inhibitor cycloheximide (CH) was added early after infection. However, when added at a late stage, the drug had no effect on viral RNA synthesis. It appears, therefore, that IF-induced inhibition of viral RNA synthesis is not a feedback consequence of an inhibition of a later step but, rather, an inhibition of some early step. This conclusion was substantiated by the finding that when IF was eliminated from pretreated cultures up to 10 hr before infection, progeny release was still affected.     

Binding of Escherichia coli heat-stable enterotoxin to rat intestinal cells and brush border membranes

The association of heat-stable enterotoxin (STa) produced by enterotoxigenic Escherichia coli 431 with isolated rat intestinal epithelial cells and brush border membranes was characterized. Specific binding of strain 431 125I-STa to a single class of specific high-affinity receptors was saturable and temperature dependent and reached a maximum between 5 and 10 min. A 1,000-fold excess of unlabeled 431 STa competitively displaced 90 to 95% of radiolabeled enterotoxin bound to brush border membranes. In contrast, specific binding of 431 125I-STa to intestinal cells ranged from 40 to 65%. The number of STa-specific receptors on rat intestinal cells determined by Scatchard analysis was 47,520 +/- 14,352 (mean +/- standard error of the mean) per cell, with affinity constants (KaS) of 2.55 X 10(11)and 4.32 x 10(11) liters/mol determined for intestinal cells and brush border membranes, respectively. Villus intestinal cells appeared to possess about twice as many STa receptors as did crypt cells. Dissociation of specifically bound 431 125I-STa from intestinal cells and brush border membranes was minimal (2 to 5%). In addition, neither the rate nor the extent of dissociation was increased by a 1,000-fold excess of unlabeled homologous 431 Sta. Binding experiments with 431 125I-STa and brush border membranes showed that purified unlabeled STas from enterotoxigenic E. coli strains 667 (class 1 porcine enteropathogen), B-41 (bovine enteropathogen), and human strains 213C2 (Mexico) and 153961-2 (Dacca, Bangledesh) exhibited patterns of competitive inhibition similar to those of homologous unlabeled 431 STa (class 2 enteropathogen). A lipid extract which contained gangliosides and glycolipids exhibited dose-dependent competitive inhibition of heat-labile enterotoxin binding to brush border membranes but did not inhibit binding of 431 125I-STa. Purified heat-labile enterotoxin from strain 286C2 did not inhibit binding of 431 STa to brush border membranes. Pronase treatment of brush border membranes reduced binding of 431 125I-STa by about 30%, suggesting that the STa receptor was a protein or a glycoprotein. The putative STa receptor was radiolabeled with 431 125I-STa and solubilized with sodium deoxycholate. One major radioactive band was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by radioautography. These data suggested that STas bind essentially irreversibly to a specific receptor on the cell surface of intestinal cells before activation of guanylate cyclase.     

Cholera toxin protects against action by Clostridium difficile toxin A. The role of antisecretory factor in intestinal secretion and inflammation in rat

The protein antisecretory factor (AF) inhibits intestinal fluid secretion induced by the cholera toxin (CT) and Clostridium difficile toxin A (CDA). The present work investigated whether CT-induced AF protects against the enterotoxin action by CDA. Rats were pretreated perorally with CT or buffer as control, whereafter CDA-induced fluid secretion and cytotoxicity was tested in vivo in ligated intestinal loops; the mucosal level of AF was estimated using the Western blot technique. Rats given repeated peroral doses of CT became tolerant to CDA, the inhibition of fluid secretion and of cytotoxicity being 79% in eight out of nine animals. The repeated CT-treatment also induced long-lasting rise of AF in the mucosal epithelium. Recombinant AF given either perorally or intravenously inhibited both fluid secretion and cytotoxicity by CDA; similar results were obtained with a truncated 16-mer AF peptide. In conclusion: peroral CT-treatment induced tolerance to CDA in rat small intestine. The tolerance was probably mediated by AF induced via action of cholera toxin on the enteric nervous and immune system.