目的 探讨睡眠剥夺大鼠喉组织闭锁小带蛋白1(zonula occludens 1,Zo-1)及闭合蛋白3(claudin-3)表达变化,为揭示咽喉反流性疾病的发病机制提供参考.方法 健康成年雄性SD大鼠40只,随机分为实验组(30只),分为睡眠剥夺1月组、睡眠剥夺2月组和睡眠剥夺3月组,实验组使用改良多平台水环境法每天剥夺动物白天睡眠8 h,分别持续1、2和3月(分别为睡眠剥夺1、2、3月组,每组10只),对照组鼠笼饲养,正常睡眠.完成造模后各组分别处死取喉组织行Western blot和实时荧光定量RT-PCR分析,观察Zo-1及claudin-3蛋白和mRNA表达变化,比较四组的差异.结果 ①与对照组比较,睡眠剥夺1月组、3月组Zo-1及claudin-3蛋白表达均降低,2月组Zo-1及claudin-3蛋白表达均升高,但差异无统计学意义(P＞0.05);与2月组相比,3月组Zo-1蛋白表达下降(P＜0.05),1月组及3月组claudin-3蛋白下降(P＜0.05);②与对照组比较,1月组及2月组Zo-1 mRNA表达均上升,3月组Zo-1 mRNA表达下降,1月组claudin-3 mRNA表达升高,2月组及3月组claudin-3 mRNA表达降低,但差异无统计学意义(P＞0.05);与1月组相比,3月组Zo-1 mRNA表达下降(P＜0.05),2月组及3月组claudin-3 mRNA表达下降(P＜0.05).睡眠剥夺1、2、3月组之间比较,随着睡眠剥夺时间的延长,Zo-1和claudin-3蛋白表达先降低、后升高、然后再降低(P＜0.05);Zo-1和claudin-3 mRNA表达逐渐降低(P＜0.05).结论 睡眠剥夺可能通过氧化应激干扰大鼠喉粘膜上皮主要紧密连接蛋白的表达,导致喉上皮细胞屏障功能障碍,使反流物更容易进入细胞间隙,与咽喉反流的发生和发展密切相关.

1 前 言 数十年以来的医疗美容基础及临床研究实践表明,面部衰老包含皮肤质地的改变、软组织松垂与容量的减少、韧带老化及骨组织的吸收等多重因素.因此,面部年轻化的合理治疗必然是多种方法、多维度序贯性的联合治疗方案. 随着注射美容技术的兴起,注射用填充剂在面部年轻化非手术治疗方案里占有越来越重要的地位.注射材料的研发经历了高分子化学阶段、高分子生物化学阶段和生物组织工程阶段.通过组织工程化的安全同种异体及异种软组织材料或者是通过干细胞研究的相关辅助方法来实现更为理想的组织营养和再生修复功能,可能是实现可注射材料抗衰的更佳联合方案的重要发展方向.异种脱细胞真皮基质(acellular dermal matrix,ADM)作为生物组织工程支架的代表性材料,因其具有很好的临床应用前景而受到整形美容领域的关注[1-2].

[目的]比较三种肌腱移植物重建前交叉韧带(ACL)的组织学早期愈合过程.[方法]新西兰兔60只,随机分为三组,分别用自体肌腱(自体组)、深低温冷冻同种异体肌腱(深冻组)、冷冻干燥同种异体肌腱(冻干组)重建ACL,每组20只.于术后3、8、12、24周时取材,行组织学观察.[结果]移植物关节内段:术后3周各组移植物内细胞结构消失;术后8周移植物浅层有成纤维细胞增殖,但深层为无细胞纤维;术后12周各组移植物浅层细胞数增多,由浅入深逐渐长入移植物;术后24周各组移植物浅层与深层细胞密度均匀一致,深层无细胞区域消失.移植物腱骨界面:术后3周腱骨界面成纤维细胞增殖;术后8周各组腱骨界面软骨细胞出现,出现散在斜形sharpey纤维;术后12周各组软骨细胞增多,各组斜形sharpey纤维数量增多,自体组出现垂直形sharpey纤维,有连续性“潮线”结构;术后24周垂直形sharpey纤维比例增多,连续性“潮线”结构范围扩大.随时间推移,三组移植物关节内段和腱骨界面内的细胞数均显著增加(P＜0.05).细胞数排序为自体组＞深冻组＞冻干组,术后8周和12周时,三组间差异有统计学意义(P＜0.05),而术后24周时,三组间差异已无统计学意义(P＞0.05).[结论]冻干异体肌腱和深冻异体肌腱早期组织学愈合过程较自体肌腱延迟,但24周后三种肌腱移植物的愈合程度趋同.

目的 探讨睡眠缺乏和高脂饮食对大鼠喉组织的影响.方法 32只雄性SD大鼠分为四组,分别为睡眠剥夺组、高脂饮食组、睡眠剥夺加高脂饮食组、对照组,每组8只,采用改良多平台水平面法建立大鼠部分睡眠剥夺动物模型(睡眠剥夺组),高脂饮食组喂食含有35％ 脂肪的饲料,睡眠剥夺加高脂饮食组剥夺部分睡眠同时喂食高脂饲料,对照组以维持饲料笼养,各组处理3个月后处死动物,取大鼠舌、牙龈、软腭、咽、喉、食管组织黏膜以福尔马林固定、石蜡包埋,切片、HE染色,光镜下观察组织病理学变化,比较四组之间的差异.结果 与对照组比较,除喉部声门下组织外,四组大鼠其他部位未见明显的形态学改变.在三组实验动物中,声门下粘膜明显变厚,粘膜上皮或出现纤毛结构消失,或出现细胞数量减少或破坏消失,粘膜下层大量炎症细胞浸润,腺体呈肿瘤样增生,其病变程度以睡眠剥夺组最为严重,另两组间无明显差异.结论 长期睡眠缺乏和高脂饮食可导致大鼠喉部声门下区组织发生明显病理改变,其中睡眠缺乏造成的影响最为严重,具体机制有待进一步探讨.

目的 观察大鼠腰椎小关节突在不同振动频率下关节软骨退变程度及腰痛相关因子的表达情况,同时探究振动对骨密度的影响.方法 收集对照组和振动组(振动组接受15 min/d,频率5～60 Hz、振幅3 mm的垂直振动12 w)大鼠腰椎小关节软骨,采用HE染色和AB-PAS染色,进行Mankin评分评价软骨退变程度;免疫组化染色观察凋亡软骨细胞、COX-2、PGE2、CⅡ在小关节突软骨表达;双能X线吸收法测量振动前后股骨颈骨密度.结果 对照组、5 Hz振动组、20 Hz振动组、60 Hz振动组小关节Mankin评分依次增加,有显著性差异(P＜0.05);组间凋亡软骨细胞、COX-2、PGE2表达随着振动频率增加而显著性增强(P＜0.05);对照组与5Hz振动组CⅡ表达差异不明显(P＞0.05),其余各组间CⅡ表达差异明显(P＜0.05);组间骨密度增加值无显著性差异.结论 长时间垂直振动可导致软骨退变,振动频率越大,小关节退变越重;振动与致痛因子表达具有相关性;5～60 Hz、3 mm振幅振动不能促进幼龄大鼠BMD增加.

Objective To explore the effects of ischemic postconditioning on the reperfusion injury sal-vage kinase and the opening of mitochondrial permeability transition pore(mPTP) at different time phases of myocardial ischemia-reperfusion injury in rats.Methods Isogenic adult Lewis rat models of cervical hetero-topic and working-heart transplantation were established.The 120 recipients were divided into ischemia-reperfusion group,ischemic postconditioning group and sham operation group ,8 rats in the sham operation group, 56 in the other two groups respectively,8 for each time phase point.Myocardial infarction size,expres-sion of p-AKT and p-ERK1/2 protein and the opening of mPTP were measured respectively at 3, 6, 12,24-hour and 2,4, 7-day after reperfusion.Results The expression of p-AKT and p-ERK1/2 protein and the opening of mPTP were statistically significant different between the ischemia-reperfusion group and ischemic postconditioning group at 7 time points after reperfusion(P<0.05),while both of which were higher than the sham operation group.The myocardial infarction range of the postconditioning group was less than the ische-mia-reperfusion group(P <0.05).Conclusion Ischemic postconditioning can increase the expression of p-AKT and p-ERK1/2 protein,decrease the opening of mPTP and myocardial infarction range between 3 hours and 7 days after the reperfusion after myocardial ischemia in rats .

目的 建立猪皮肤软组织海水浸泡爆炸伤创面动物模型.方法 选取8头(3头在预实验中死亡,5头进入实验)小型猪作为实验动物,在每头猪的双侧肩胛部、臀部制造4个损伤程度相当的海水浸泡爆炸伤创面,创面累及全层皮肤至脂肪层,肌肉、骨骼无明显损伤.结果 当雷管距皮肤0 cm时,创面较大,出血较多,伴邻近肢体骨折和局部肌肉缺损,于海水浸泡后6h死于全身多脏器损伤合并失血性休克;当雷管距皮肤1.0 cm时,肩胛部创面在范围、深度上均符合要求,但合并肺损伤,该猪于海水浸泡后32 h死于大面积肺梗死;当雷管距皮肤1.5 cm时,肩胛部和臀部的创面在范围、深度上均符合要求,创面可重复性好,但该猪于海水浸泡后10 h死于持续性低体温.立即改变海水浸泡方式,将炸后实验小型猪放置海水中改为海水持续浸泡爆炸创面1h,模型可重复性好,动物存活好.结论 雷管距猪皮肤为1.5 cm时,爆炸创面经海水浸泡1h,创面可重复性好,动物存活好,适合实验研究.

Objective To investigate the protective effect of ischemic postconditioning on myocardial ischemia-reperfusion in rats with cardiac transplantation at different phases. Methods The model of cervical heterotopic heart transplantation (left heart work) was established in inbred line Lewis rats. After survived for 2 d, the rats were divided into ischemia-reperfusion group (each n=8 at 7 phases:3 h, 6 h, 12 h, 24 h, 2 d, 4 d and 7 d), ischemic postconditioning group (each n=8 at the same 7 phases as above mentioned phases) and sham-operation group (n=8). The value of serum CK-MB, and index of myocardial apoptosis and area of myocardial infarction of transplanted heart were compared after reperfusion. Results At 6 phases of 3 h, 6 h, 12 h, 24 h, 2 d and 4 d, the difference in average value of CK-MB had statistical significance between ischemia-reperfusion group and ischemic postconditioning group (all P<0.05). The value of CK-MB was significantly higher in ischemia-reperfusion group and ischemic postconditioning group than that in sham-operation group (all P<0.05). At phase of 6 h, the peak value of CK-MB was significantly lower in ischemic postconditioning group than that in ischemia-reperfusion group [(34.73±8.83) U/L vs. (52.58±10.05) U/L, P<0.01]. At 7 phases of 3 h, 6 h, 12 h, 24 h, 2 d, 4 d and 7 d, the difference in the index of myocardial apoptosis and area of myocardial infarction had statistical significance between ischemia-reperfusion group and ischemic postconditioning group (all P<0.05). The index of myocardial apoptosis was higher in ischemia-reperfusion group and ischemic postconditioning group than that in sham-operation group (all P<0.05). Conclusion Ischemic postconditioning can reduce the peak value of serum CK-MB, myocardial apoptosis and area of myocardial infarction in rats with heart transplantation within 3 h to 7 d after myocardial ischemia-reperfusion.

Objective To investigate the influence of different vibration frequency on the degeneration of the lumbar intervertebral disc and the bone mineral density ( BMD) of the femur, and to explore the effect of protective equipment on the degeneration of the lumbar intervertebral disc in rats.Methods BMD of rats before and after the vibration training was detected .After the vibration training, nucleus pulposus of the intervertebral disc was collected , and HE staining and the immunohistochemical study of the nucleus pulposus tissue were performed .Results Along with the increase of the vibration frequency, the apoptosis of the intervertebral disc cells increased obviously.Compared with that in protective equipment group, the percentage of positive cells and optical density value of the positive particles detected using TUNEL method in non-protective equipment group increased significantly (P<0.05). BMD of the femur showed no significant difference before and after the vibration ( P>0.05 ) .Conclusion Vibration has no obvious influence on BMD .But along with the increase of the vibration frequency , the degeneration of the intervertebral disc increases . And the protective equipment has protective effect on the degeneration of the intervertebral disc caused by vibration .

目的 研究振动对大鼠椎间盘细胞凋亡及股骨骨密度的影响.方法 选用40只6周龄雄性SD大鼠,随机分成4组:正常对照组10只;振动实验组30只(分别为振动频率5 Hz 10只,振动频率20 Hz 10只,振动频率60 Hz 10只).对实验组动物进行垂直振动训练,正常对照组不进行振动干预,置于与实验组相同环境下,相同面积围栏内自由活动,时间同实验组.12周后对各组大鼠股骨进行 BMD检查,然后将大鼠处死,取出(L5-L6)椎间盘组织,固定、切片.对各组大鼠椎间盘标本组织进行形态学观察(HE染色);TUNEL检测凋亡(FITC+POD).结果 振动实验组大鼠与正常组大鼠比较椎间盘髓核细胞数减少;TUNEL检测阳性细胞的百分率和阳性颗粒的平均光密度值增高,差异均有统计学意义(P＜0.05).而各组大鼠股骨BMD无显著性差异(P＞0.05).结论 一定频率的垂直机械振动可以对大鼠椎间盘髓核细胞凋亡产生影响;且在一定的范围内,随着振动频率的增加,相应振动实验组大鼠椎间盘髓核细胞凋亡增多,椎间盘组织退变加速.而一定频率的垂直振动对健康幼龄大鼠股骨BMD影响不显著.

在北京市科委科研项目的支持下,作者制定了北京市地方标准一《实验用小型猪病理学诊断规范》.本文介绍了这一地方标准的制定原则,对主要内容的编制作了说明,并对其它实验动物病理学诊断标准的制定进行了讨论.

目的 建立椎间盘内破裂的动物模型,观察其病理学及行为学表现.方法 实验用雄性SD大鼠96只,随机分为3组,每组32只.假手术组(对照组),全麻后取仰卧位,经腹腔正中入路,钝性撕开后腹膜,暴露L6～7椎间盘,逐层缝合.椎间盘轻度破坏组(手术1组),全麻后取仰卧位,经腹腔正中入路,暴露L6～7椎间盘.于椎间盘前正中点以直径0.2mm的特制针头刺入L6～7椎间盘至后侧纤维环下,深度控制为3mm.少许明胶海绵堵塞针口,逐层缝合.关腹前滴入约1ml左右生理盐水.椎间盘重度破坏组(手术2组),全麻后取仰卧位,经腹腔正中入路,暴露L6～7椎间盘.于椎间盘前正中点以直径0.2mm的特制针头刺入L6～7椎间盘至后侧纤维环下,深度控制为3mm.仍以前正中针口为刺入点,与第一针道各成30°角向左右分别穿刺至后外侧纤维环下.结果 手术椎间盘内可见由前方至后方的裂隙,后侧的外层纤维环未破裂.术后髓核内纤维组织样细胞透明样变性,与胶冻状物交织在一起,内层纤维软骨细胞变性.椎间盘内破裂模型手术2组的大鼠术后静止不动时间延长、运动时间减少,同假手术组相比,有统计学意义.结论 建立了一种椎间盘内破裂的动物模型,并观察了手术间盘的病理及行为学改变过程,该模型可模拟临床上的椎间盘源性下腰痛.

目的对海藻酸钙和卡波姆凝胶经支气管镜介入兔肺支气管内的安全性进行评价,为含抗结核药物凝胶肺内应用提供依据。方法 45只日本长耳兔随机分成对照组、海藻酸钙组和卡波姆组,每组15只,实验观测4周。检测体温、体重、血常规、T细胞亚群及肺病理学改变。结果各组间体温、体重、血常规及外周血T细胞亚群的比较均无显著性差异(P>0.05)。海藻酸钙组和卡波姆组凝胶介入前与介入后CD3+、CD4+、CD8+百分比比较亦无显著性差异(P>0.05)。海藻酸钙组和卡波姆组的受试动物左肺底细支气管无凝胶成分残留,肺大体及光镜病理观察与对照组相比均未产生明显变化。光镜病理观察可见海藻酸钙组的右肺底有局灶性肺泡破裂,肺泡间隔无炎性细胞浸润。卡波姆凝胶组的左肺底偶见局灶性肺不张。结论海藻酸钙和卡波姆凝胶肺内应用均具有较好的安全性,可作为含抗结核药物凝胶的良好基质。

Objective To evaluate the effect of carbomer gel and e-gum bronchoscopy intervention in the rabbit lung and provide evidence for using antituberculosis medicament gel administered in lung.Methods Forty-five Japanese long-eared rabbits were randomly divided into control group,carbomer group and e-gum group.Animal temperature,avoirdupois,hemic routine,T lymphocyte subsets,and pathology were tested.Results There were no statistical difference in animal temperature,avoirdupois,hemic routine and T lymphocyte subsets of each group.There were no statistical difference in percent of CD3+,CD4+,CD8+between before and after injection in carbomer group and e-gum group.Lung tissue pathology of carbomer group was no abnormality.There was no carbomer gel in left lung.In e-gum group,a small amount of gel was found in left bronchia of the rabbit.One of rabbits got pneumonia and lymphocyte was found in Spacing of alveolus. Conclusions Carbomer gel and e-gum can be used in lung for security and be good medium to develop antituberculosis medicament gel.

Objective To investigate the effect of recipient derived dendritic cells(DC) loaded with 8-methoxypsoralen plus UVA irradiation(PUVA)-treated donor splenic lymphocytes(PUVA-SP) on Th1/Th2 balance and cardiac allograft rejection in recipient rats.Methods Heterotopic vascularized heart transplantation model was established in inbred DA and LEW rats.Spleen lymphocytes of DA rat were treated with PUVA.Bone marrow-derived DCs of LEW rat were co-cultured with PUVA-treated or untreated DA spleen lymphocytes(PUVA-SP or SP),and the productions of IFN-γ and IL-10 in the culture supernatant were determined by Luminex method.Recipient LEW rats were given infusion of PUVA-SP DC,SP DC or PBS(Control group) through the peripheral vein 7 days before heart transplantation.The survival of cardiac allografts was evaluated by palpation every day.The concentrations of Th1(IFN-γ,IL-2) and Th2(IL-10) cytokines in serum of recipients were determined by Luminex method,and the samples of cardiac allograft were collected for pathological evaluation 6 days after transplantation.Results After being co-cultured with donor PUVA-SP,singnificantly more IL-10 and IFN-γ were secreted by recipient DC compared with that by DC cultured with donor SP(P<0.01).Comparing with the infusion of SP DC or PBS,the infusion of PUVA-SP DC significantly decreased the serum levels of IL-2 and IFN-γ in cardiac allograft recipients,but it increased the serum level of IL-10.The allograft of PUVA-SP DC group survived much longer than those of control group and SP DC group(27.2d vs 6.7d and 5.5d,P<0.01) with lower rejection grade.Conclusion PUVA-SP DC is a novel negative immunoregulatory tool,which is able to mediate Th2 immune deviation in vivo and down-regulate the reaction of allograft rejection.

Objective: To make magnetic compression anastomosis between the small intestines as a novel treatment, using the advances in interventional radiology. Methods: One magnet was put into the ileum of an adult dog, another magnet was put into the colon of the dog, the ileum between the two magnets was ligatured causing a complete obstruction of the small intestine. Two magnets were immediately attracted towards each other transmurally, so that the walls of intestine were compressed together. Results: Two magnets compressed the stricture transmurally, causing gradual ischemic necrosis of the stricture. The ischemic necrosis created an anastomosis between the two magnets. The magnets of 2 300～2 770 gauss were discharged from the anal 6～14 days after the procedure. There was a complete fistula between the ileum and the colon. The magnets of 3 200 gauss were discharged 3- 4 days post the procedure, and two dogs were died of perforation on colon caused by the magnets of 3 200 gauss. Conclusion: Magnetic compression anastomosis(MCA)is a novel interventional method that creates an anastomosis between the small intestine and the colon. The magnets from 2 300～2 770 gauss are safe. MCA could be applied in patients.

Objective To investigate the effect of recipient derived dendritic ceils (DC) loaded with PUVA-treated donor splenic lymphocytes (PUVA-SP) on humoral and cellular immunity and allograft rejection in allograft recipients. Methods Heterotopic vascularized heart transplantation model was established from inbred DA to LEW rat. DA rat spleen lyrnphoeytes were treated with 8-methoxypsoralen plus UVA irradiation (PUVA). LEW rat bone marrow-derived DC were co-cultured with PUVA-treated or untreated DA spleen lymphocytes (PUVA-SP or SP), and the costimulatory molecules (CD86 and CD80) of the treated DC (PUVA-SP-DC or SP-DC) and untreated DC were detected by flow cytometry. Recipient LEW rats were subjected to infusion of PUVA-SP-DC, SP-DC or PBS through the peripheral vein at the 7th day before heart transplantation. Cardiac allgrafts survival was evaluated by daily palpation. Circulating alloantibody IgG levels of recipient rats were determined by flow cytometry, and proliferative responses of recipient T cells to donor or third party antigen were examined by mixed leukocyte reaction (MLR). The spleen was collected at the 6th day after transplantation. Results PUVA treatment effectively induced apoptosis of DA rat spleen lymphocytes. After co-cultured with donor PUVA-SP, recipient DC still maintained an immature phenotype with low levels of CD80 and CD86. Injection of PUVA-SP-DC significantly decreased serum anti-donor alloantibody levels of recipient rats, induced donor antigen specific hyporesponsiveness of recipient T cells,and prolonged allograft survival. Moreover, the spleens of the recipient rats, infused with PUVA-SP-DC,were the smallest compared to those of the recipients infused with PBS or SP-DC.Conclusion PUVA-SP-DC could effectively down-regulate anti-donor cellular and humorat adaptive immunity, leading to prolongation of cardiac allograft survival

Objective To compare the organ weight and relative weight between SPF and conventional minipigs.Methods Ten male and 10 female minipigs were divided into SPF and conventional groups.The weight and relative weight of seven organs were analyzed.Results The body weight of minipigs in the SPF group was significantly lower than that in conventional group.The gross findings of lung and spleen were changed in the Wuzhishan minipigs.The relative weight of liver,heart,brain and adrenal gland in the SPF minipigs was significantly higher than that in conventional group while its relative weight of the lung and spleen was significantly lower than that in the conventional group.Conclusion There are some differences of the organ form and organ to body weight between SPF and conventional minipigs,and the above described data may be used as physiological indexes in the laboratory animals.

我们为了在临床上开展磁铁压迫吻合术治疗不宜手术或拒绝手术的肠梗阻,进一步探讨磁铁压迫吻合治疗的作用原理,建立了磁铁压迫吻合术治疗肠梗阻的模型.

目的研究地塞米松与麻醉剂速眠新配伍的应用效果。方法大鼠72只随机分为3组,每组24只,A组和B组分别肌肉注射地塞米松0.1 mL/kg和1.0 mL/kg之后30 min,,对照组仅肌肉注射速眠新0.20 mL/kg。结果3组大鼠麻醉诱导时间相同,均不超过3 min;B组麻醉维持时间最长、苏醒时间最短;A组次之。A组和B组麻醉期心率和动脉平均血压均低于对照组,但差异无统计学意义(P>0.05)。呼吸监测显示,麻醉后A组和B组的大鼠呼吸数显著高于A组(P<0.05)。A组和B组麻醉后血浆ACHT和皮质酮水平未升高,而对照组显著升高。结论采用地塞米松配伍速眠新对大鼠进行复合麻醉,不仅能增强麻醉,而且能缓解麻醉过程中的心率加快和呼吸抑制症状,维持实验过程中各项指标较平稳,安全性高。