大鼠心肌梗塞后梗塞区胆碱能神经支配的变化

目的：探讨大鼠心肌梗塞区胆碱能神经支配的变化。方法：实验用20只大鼠，以组织化学Karnovsky-Roots法显示胆碱能神经纤维。应用多功能图像分析仪测定梗塞区胆碱能神经纤维密度。结果：大鼠心肌梗塞后4天，梗塞区胆碱能神经纤维密度显著下降；梗塞后14天，梗塞区胆碱能神经纤维完全消失；梗塞后120天，心肌梗塞区的部分区域出现胆碱能神经纤维，结论：大鼠心肌梗塞后14天，梗塞区发生完全的去胆碱能神经支配，120天后部分区域出现胆碱能神经再支配。     

同种异体骨髓单个核细胞移植治疗大鼠急性心肌梗死实验研究

目的探讨大鼠同种异体骨髓单个核细胞（bone marrow mononuclear cells，BM—MNCs）在急性心肌梗死区分化增殖潜能及其修复重建心肌作用。方法健康雄性Wistar大鼠24只，用结扎冠状动脉左前降支的方法建立大鼠心肌梗死模型，随机分为对照组（AMI＋培养基，12只），移植组（AMI＋BM—MNCs，12只）；分别将制备的培养基和BM—MNCs悬液心外膜下植入梗死心肌周围。移植术后4周，观察心肌梗死区及其周边区组织形态学特点、心肌梗死面积变化。结果实验组与对照组相比心肌梗死面积明显缩小（P〈0．01）；实验组心肌梗死区内有BrdU标记阳性的BM—MNCs移植细胞存活，向心肌源性细胞分化并且诱导了大量的新生毛细血管。结论同种异体BM—MNCs移植在细胞水平完成了对心肌梗死区再心肌化和再血管化过程，可以改善急性心肌梗死后的心脏功能。     

同种异体脂肪组织源性间质干细胞移植治疗大鼠急性心肌梗死

目的：建立体外分离扩增脂肪组织源性间质干细胞方法，并探讨同种异体脂肪干细胞移植治疗大鼠心肌梗死的效果及可行性。方法: 18只雄性SD大鼠随机分为假手术组、急性心肌梗死对照组（AMI组）及AMI+细胞移植组。分离大鼠腹部脂肪组织干细胞，体外扩增，BrdU标记后于结扎左冠状动脉前降支后1h移植入梗死心肌，移植后4周进行血流动力学检测心功能并取出心脏进行病理切片观察和免疫组织化学染色检测移植细胞在梗死心脏中的定居、存活情况。结果: 大鼠腹部脂肪组织可分离培养出大量间质干细胞。细胞移植治疗组左心室收缩压高于AMI对照组（P＜0.01），舒张末压显著降低（P＜0.01），左心室内压最大上升、下降速率明显加快（P＜0.05）；病理组织切片显示梗死边缘区心肌面毛细血管计数明显增加，梗死区心肌组织内及毛细血管壁中均可见移植标记细胞。结论: 脂肪组织可作为干细胞又一新的来源，同种异体脂肪干细胞移植治疗AMI有效、可行。     

大鼠急性心肌梗死后的心肌细胞凋亡和凋亡相关基因表达的变化

目的： 观察大鼠急性心肌梗死（AMI）后的心肌细胞凋亡和caspase-3、Bcl-2和Bax表达的变化。 方法: 105只雌性SD大鼠，随机取78只结扎左冠状动脉建立AMI模型，24 h存活43只作为心肌梗死组（MI组）；另27只设为假手术组（S组）；两组再按观察时点随机分为48 h和4周两亚组，即：MI 48 h（n=11）和MI 4周（n=13）组，S48 h（n=10）和S4周（n=10）组。末端脱氧核糖核苷酸转移酶介导的dUTP切口末端标记技术（TUNEL）和DNA凝胶电泳检测心肌细胞凋亡。免疫组化方法和Western blotting检测caspase-3、Bcl-2和Bax的表达。 结果： MI 48 h组动脉收缩压（SBP）、舒张压（DBP）、平均压（MAP）、左心室收缩压（LVSP）和左心室内压最大上升下降速率（±dp/dt）均显著低于S组（P<0.05, P<0.01），左心室舒张末压（LVEDP）显著高于S组（P<0.05）；MI 4周组除SBP、DBP和MAP无显著差异（均P>0.05）外，上述其它指标的变化与MI 48 h组相同，且LVEDP升高更为显著（P<0.01）；MI 48 h和4周两组梗死/疤痕区、梗死边缘区和非梗死区的心肌细胞凋亡指数均显著升高P<0.05,P<0.01），心肌细胞中“凋亡执行因子”caspase-3和“凋亡促进基因”Bax的表达亦均显著增高（P<0.05,P<0.01），而“凋亡抑制基因”Bcl-2仅在MI 48 h组梗死区心肌细胞中表达增加，“抑制凋亡复合基因”Bcl-2/Bax的比值仅在MI 48 h组降低。 结论： 大鼠AMI后，梗死区及其边缘区和非梗死区均有心肌细胞凋亡发生，伴“凋亡执行因子”caspase-3和“凋亡促进基因”Bax的表达增加；AMI早期，“凋亡抑制基因”Bcl-2在梗死区表达增加，但“抑制凋亡复合基因”Bcl-2/Bax的比值下降。     

缺氧对急性心肌梗死后3周大鼠心室肌细胞持续性钠电流的影响

目的：研究缺氧对急性心肌梗死（AMI）大鼠心室肌细胞持续性钠电流的影响，以期更进一步探讨急性心肌梗死后心律失常的发生机制。 方法： 采用结扎大鼠冠状动脉左前降支建立AMI动物模型,应用膜片钳全细胞记录方法,观察AMI 3周心室肌外膜梗死区细胞持续性电流（INap）的变化。 结果： 常氧条件下，假手术组（n=9）的INap电流密度为0.144±0.022（pA/pF）,心梗组（n=9）的INap电流密度为0.121±0.013（pA/pF）,明显低于假手术组(P<0.01), 以上两组的INap 均可被河豚毒素（TTX）阻断。缺氧条件下，假手术组和心梗组的INap均随着缺氧时间的延续而增大，但假手术组INap的增大明显大于心梗组，两组的INap均可被谷胱甘肽（1 mmol/L）所阻断。 结论： 急性心肌梗死后，无论是在常氧或是再次缺氧情况下，心肌梗死区与非梗死区细胞INap的大小均存在差异，造成心肌复极离散度增大，可能是导致AMI后出现折返性室性心律失常的原因之一。     

中西药结合促进心梗后大鼠胆碱能神经的恢复

目的：观察葛根素与神经生长因子联合应用对大鼠心肌梗死后去胆碱能神经支配的影响，以证实二者联合应用对心梗后大鼠胆碱能神经功能恢复的作用。方法：实验用SD大鼠32只，分为正常组、心肌梗死组、神经生长因子组及联合用药组。术后2d取材，以Karnovsky-Roots法，显示胆碱能神经纤维，应用多功能真彩色病理图像分析系统分析胆碱能神经纤维密度。结果：心肌梗死组存活心肌中胆碱能神经纤维密度最低，联合用药组胆碱能神经纤维密度最高。结论：葛根素与神经生长因子联合应用能够明显减缓大鼠心肌梗死后的去胆碱能神经支配，促进神经功能的恢复。     

葛根素对大鼠心肌梗死后梗死部位肾上腺素能神经纤维的影响

目的观察葛根素对大鼠心肌梗死后梗死部位。肾上腺素能神经纤维分布的影响，以求证实葛根索的神经保护作用。方法Wister大鼠120只，分为假手术组、心肌梗死模型组和葛根素组。分别于术后6h、2、4、7、14时取材，应用免疫组织化学方法，显示肾上腺素能神经纤维，应用多功能真彩色病理图像分析系统分析。肾上腺素能神经纤维的密度。结果心肌梗死模型组大鼠在术后6h、2、4、7、14时，梗死区肾上腺素能神经纤维密度均比假手术组明显降低（P〈0．01）。葛根素组大鼠在术后7、14d时，梗死区。肾上腺素能神经纤维密度较心肌梗死模型组明显增多（P〈0．01）；与假手术组相比无显著差异（P〉0．05）。结论葛根素能干预大鼠心肌梗死后的肾上腺素能神经纤维分布减少，具有神经保护作用。     

免疫应答中小鼠脾GAP-43神经支配的可塑性--神经纤维数量和分布的变化

为了解脾交感神经在免疫功能增强时的形态变化，用免疫组织化学方法观察了ＢＡＬＢ／ｃ小鼠脾内生长相关蛋白样免疫反应（ＧＡＰ－４３－ＬＩ）性神经在结核菌素衍生蛋白（ＰＰＤ）诱发的免疫反应高峰期数量和分布的变化。结果显示，动物在接受两次ＰＰＤ注射（２１ｄ＋７ｄ）后，脾ＧＡＰ－４３－ＬＩ神经纤维密度明显增加并伴有分布和形态的变化。于对照动物，脾ＧＡＰ－４３－ＬＩ神经纤维主要分布于血管周围，少量伸入动脉周围淋巴鞘的脾基质中，而在免疫刺激的动物，不仅血管周围的神经纤维明显增多，动脉周围淋巴鞘外层、边缘区和红髓等免疫应答活跃的部位也出现许多神经纤维。神经纤维分支和纤维上膨体明显增多。以上结果提示：免疫应答中脾神经成分发生活跃的结构重塑，这些变化有可能与神经的免疫调节功能有关。     

糖尿病性勃起功能障碍大鼠阴茎和脊髓nNOS及AchE表达的变化

目的：观察大鼠糖尿病性勃起功能障碍（ED）与腰骶段脊髓和阴茎神经源性一氧化氮合酶（nNOS）和乙酰胆碱酯酶（AchE）阳性神经元或神经纤维变化的相关性。方法：注射链脲佐菌素建立糖尿病大鼠模型,4周和12周后注射阿扑吗啡（APO）进行大鼠阴茎勃起功能实验,取大鼠阴茎和腰骶段脊髓,用ABC免疫组织化学法和组织化学法分别显示nNOS和AchE阳性神经元或神经纤维。结果：与对照组相比,糖尿病4周时,大鼠阴茎勃起次数无显著性差异;12周时显著性减少;糖尿病4周时,脊髓和阴茎nNOS和AchE阳性神经元或神经纤维均无显著变化,而12周时均显著减少。结论：糖尿病性ED的出现伴随脊髓和阴茎内NO和乙酰胆碱的减少。     

急性心肌梗死后心脏TGF-β1的表达和心室重塑(英文)

目的：探讨大鼠急性心肌梗死（AMI）后心肌纤维化和TGF-β1表达及炎症反应的分子机制。 方法： 建立大鼠AMI模型及假手术组，于术后第1、4和8周末测血流动力学后取心脏。RT-PCR和免疫组化SABC法检测TGF-β1基因和蛋白的表达。用氯胺T法测心肌组织羟脯氨酸的含量。心肌病检观察心肌组织炎症细胞浸润情况。 结果： 与假手术组相比，AMI后第1、4和8周末血流动力学有明显改变(P<0.01)。在梗死区、交界区和非梗死区羟脯氨酸和TGF-β1基因和蛋白表达均增高（P<0.05），第1周的表达高于第4和8周。羟脯氨酸和TGF-β1蛋白表达呈现明显正相关（r＝0.75－0.99，P<0.05）。AMI后第1周梗死区和交界区炎症细胞浸润明显，第4和8周心肌炎症细胞减少。TGF-β1在梗死后1周时主要见于心肌细胞、中性粒细胞、巨噬细胞和部分淋巴细胞的胞浆内表达，在4周和8周见于成纤维母细胞及间质。 结论： AMI后心肌细胞、炎症细胞、成纤维母细胞胞浆及基质中TGF-β1表达增高，与羟脯氨酸的变化及炎症过程存在联系，可能在AMI后心室重塑和心肌炎性修复过程中起重要作用。     

Surviving acute myocardial infarction: survivin expression in viable cardiomyocytes after infarction

BACKGROUND: Apoptosis is a key feature in postinfarction remodelling leading to progressive myocyte loss. Both proapoptotic and antiapoptotic factors contribute to the delicate balance between death and survival. The survivin pathway has emerged as essential in the control of apoptosis, although its role in heart disease is unknown.AIM: To evaluate survivin expression after acute myocardial infarction (AMI). METHODS: Survivin expression was assessed immunohistochemically in the peri-infarct and remote viable myocardium in 17 consecutive patients who died 1-30 weeks after AMI and in four control hearts. RESULTS: Survivin was expressed by myocytes in the peri-infarct area in eight patients and in the remote region in 13 patients. The rate of survivin expression after AMI was significantly higher in the remote versus peri-infarct regions and compared with control hearts. Its expression was inversely associated with the presence of dilated cardiopathy and of apoptosis, independently from the gross pathology infarct size. CONCLUSIONS: Survivin myocardial expression after AMI may be associated with the survival of at risk myocardium and may be indicative of more favourable remodelling after AMI. These findings identify a potential new target for the treatment of postinfarction remodelling.     

抑制AChE后对神经再生的影响——AChE可能有神经营养因子样作用

乙酰胆硷酯酶(ACHE)在神经系统的分布模式多年来一直令人困惑。近年对AChE的非胆硷能递质传递功能的研究已引起了相当的重视。本文研究了大鼠坐骨神经压榨后,肌肉注射不可逆的胆硷酯酶抑制剂DFP,通过Bodian染色和透射电镜观察神经损伤后溃变和再生的过程。结果显示DFP注射动物损伤神经的再生能力明显减弱,表现为(1)再生纤维生长速度较对照组减慢(p<0.01),(2)新生纤维数量减少(3)溃变物质清除缓慢,(4)雪旺氏细胞增生欠活跃。上述结果表明AChE在其参与胆硷能递质传递之外还和神经组织的维持和再生有一定的关系。结合AChE在神经发育和生长中的一些间接证据,本文讨论了AChE很可能有神经营养因子样作用的问题。     

乳鼠心肌细胞移植对兔急性心肌梗死区微血管密度的影响

目的 观察乳鼠心肌细胞移植对兔急性心肌梗死区微血管密度及微血管再生的影响. 方法 新西兰大白兔16只,新生乳鼠40只.建立兔急性心肌梗死模型后,将培养的SD乳鼠心肌细胞移植到兔心肌梗死区,分别于移植后1、2、3、4周取材,用碱性磷酸酶组织化学法和免疫荧光法进行染色,光镜观察,LEICA Qwin V3图像分析系统分析兔心肌梗死边缘区毛细血管密度及其再生情况. 结果 乳鼠心肌细胞移植后的1、2、3、4周不同的时间段,兔心肌梗死区的毛细血管密度及新生毛细血管数量均明显高于对照组(P<0.05).定量分析显示,毛细血管密度呈现先升后降的趋势. 结论异体异种心肌细胞移植能够促进兔心肌梗死区的毛细血管新生,有利于改善心肌的血液供应.     

大脑皮质损伤后Ⅲ层和Ⅵ层内胆碱能纤维再生的研究

实验从2月龄SD大鼠64只，于顶叶皮质切一深3mm、宽2mm的冠状切口，用染AChE纤维的组织化学方法分析术后1～8周切口吻、尾侧区皮质Ⅱ层和Ⅵ层内胆碱能纤维的变化．结果表明：正常组皮质Ⅱ层纤维密度高于Ⅵ层（P＜0.01）；损伤组和损伤后胰岛素脑室内注射组（0．6IU／次／3d）在损伤后l～2周，皮质Ⅱ层内存留的纤维密度高于Ⅵ层，3周后Ⅵ层纤维再生幅度超过Ⅱ层（P＜0．05）；而在链尿菌素所致胰岛素缺乏的皮质损伤组，损伤后4周时Ⅵ层纤维密度仍低于Ⅱ层（P＜0．05）。提示皮质Ⅵ层内胆碱能纤维损伤后再生幅度大于Ⅱ层，推测可能与脑内胰岛素及其受体在皮质深部分布密度较高有关．     

颅脑损伤促进的坐骨神经再生

BACKGROUND:Studies have shown that craniocerebral injury can promote the repair of sciatic nerve injury in rats, but its precise mechanism remains unclear. OBJECTIVE:To further explore the action mechanism of craniocerebral injury on the repair of sciatic nerve injury using morphology and histology. METHODS:Sixty specific-pathogen-free healthy male Sprague-Dawley rats were randomly divided into two groups. Rats with craniocerebral injury and sciatic nerve injury were considered as the experimental group. Rats with simple sciatic nerve injury were considered as the control group. Classical Feeney method was used in models of craniocerebral injury and SunderlandV sciatic nerve injury. At 8 and 12 weeks after modeling, sciatic nerve index was detected. Masson staining and NF200 immunofluorescence staining were used to observe the nerve regeneration at the anstomotic site. Transmission electron microscope was used to observe the number of regenerative axons. RESULTS AND CONCLUSION:At 8 and 12 weeks after modeling, compared with the control group, gait and sciatic nerve index recovered better in the experimental group. In the experimental group, Masson staining showed fewer nerve membrane collagen fibers, and the axon arranged neatly. NF200 immunohistochemistry showed that in the experimental group, the density of regenerated nerves was high, and nerves were regularly distributed. Transmission electron microscopy showed that in the experimental group, regenerative axons were regularly arranged, collagen scar was less, and myelin layer arranged regularly. Results suggested that the craniocerebral injury in rats may promote the repair of peripheral nerve injury by reducing scar collagen in nerve endings.     

Cell viability and inflammatory response in hydrogel sponges implanted in the rabbit cornea

In the quest for the development of a functional keratoprosthesis, the biocompatibility of the porous skirt material in the Chirila keratoprosthesis (KPro) was investigated. The population of live and dead cells within, and the inflammatory response to, a tissue-integrating poly(2-hydroxyethyl methacrylate) (PHEMA) sponge were studied. Samples of the hydrogel sponge were implanted in rabbit corneas and explanted at predetermined time points up to 12 weeks. The explanted sponges were subjected to cell viability assay using two types of fluoroprobes, 5-chloromethylfluorescein diacetate and ethidium homodimer-1. A semiquantitative analysis was performed to assess the number of dead cells within the sponge and in the area of corneal stroma proximal to the sponge. Five rabbits were used for each end point (2, 4 and 12 weeks). To investigate the inflammatory response to the sponge, immunocytochemistry, using specific antibodies to rabbit macrophages, enzyme histochemistry of chloroacetate esterase (to detect neutrophils) and transmission electron microscopy (TEM) were also employed at 24 h, 2, 4 and 12 weeks after implantation. Four weeks after implantation, fewer viable cells were observed in the sponge when compared to the 2-week implant. However, the proportion of viable cells increased dramatically by 12 weeks. The proportion of nonviable cells decreased gradually with time; central sponge contained 34±11% dead cells after 2 weeks, and 15±4.3% after 12 weeks. The staining of inflammatory cells demonstrated the presence of macrophages and neutrophils up to 12 weeks after implantation. TEM confirmed the presence of these cell types and others, including eosinophils and myofibroblasts, as well as blood capillaries. The presence of a significant number of viable cells at each time point and the uniform reduction of the nonviable cell proportion with time suggests that the sponge is a conducive environment supporting a prolific, viable cellular colonization. Dead cells observed in the first instance indicate a normal injury pattern. However, the presence of a small but significant proportion of invading inflammatory cells 12 weeks after implantation confirms a characteristic pattern of wound healing within the sponges.     

大鼠周围神经损伤后肌肉组织乙酰胆碱酯酶的变化

目的：探讨周围神经损伤后，在不同时间点受神经支配的肌肉组织中乙酰胆碱酯酶(AChE)的变化。方法：建立大鼠外周神经损伤的动物模型；制备特异识别神经系统AChE的单克隆抗体，以该抗体作为一抗，酶标羊抗鼠IgG作为二抗建立酶联免疫检测方法；测定正常对照组、坐骨神经神经损伤后0．5h、3h、12h组，坐骨神经断点处、胚神经及腓总神经所支配的肌肉组织AChE的含量变化。结果：坐骨神经损伤0．5h后，断点处肌肉组织中AChE含量最高，随时间延长逐渐降低；胫神经、腓总神经支配的肌肉组织中AChE含量0．5h时最低，3h最高，12h时降低。结论：坐骨神经损伤后，断点处及其远端神经支配的肌肉组织中AChE的变化呈时间依赖性，但变化不一。     

Autonomic innervation of the human cardiac conduction system: Changes from infancy to senility—An immunohistochemical and histochemical analysis

In order to study the changes in the pattern of autonomic innervation of the human cardiac conduction system in relation to age, the innervation of the conduction system of 24 human hearts (the age of the individuals ranged from newborn to 80 years), freshly obtained at autopsy, was evaluated by a combination of immunofluorescence and histochemical techniques. The pattern of distribution and density of nerves exhibiting immunoreactivity against protein gene product 9.5 (PGP), a general neural marker, dopamine β‐hydroxylase (DBH) and tyrosine hydroxylase (TH), indicators for presumptive sympathetic neural tissue, and those demonstrating positive acetylcholinesterase (AChE) activity, were studied. All these nerves showed a similar pattern of distribution and developmental changes. The density of innervation, assessed semiquantitatively, was highest in the sinus node, and exhibited a decreasing gradient through the atrioventricular node, penetrating and branching bundle, to the bundle branches. Other than a paucity of those showing AChE activity, nerves were present in substantial quantities in infancy. They then increased in density to a maximum in childhood, at which time the adult pattern was achieved and then gradually decreased in density in the elders to a level similar to or slightly less than that in infancy. In contrast, only scattered AChE‐positive nerves were found in the sinus and atrioventricular nodes, but were absent from the bundle branches of the infant heart, whereas these conduction tissues themselves possessing a substantial amount of pseudocholinesterase. During maturation into adulthood, however, the conduction tissues gradually lost their content of pseudocholinesterase but acquired a rich supply of AChE‐positive nerves, comparable in density to those of DBH and TH nerves. The decline in density of AChE‐positive nerves in the conduction tissues in the elders was also similar to those of DBH and TH nerves. Our findings of initial sympathetic dominance in the neural supply to the human cardiac conduction system in infancy, and its gradual transition into a sympathetic and parasympathetic codominance in adulthood, correlate well with the physiologic alterations known to occur in cardiac rate during postnatal development. The finding of reduction in density of innervation of the conduction tissue with ageing is also in agreement with clinical and electrophysiological findings such as age‐associated reduction in cardiac response to parasympathetic stimulation. Finally, our findings also support the hypothesis that, in addition to the para‐arterial route, the parafascicular route of extension along the conduction tissue constitutes another pathway for the innervation of the conduction system of the human heart during development. Anat Rec 264:169–182, 2001. © 2001 Wiley‐Liss, Inc.     

基于图像分析的周围神经Karnovsky-Roots法染色规律的效果观察

目的　基于图像分析探讨用于鉴别周围神经功能束的乙酰胆碱酯酶（AChE）染色Karnovsky-Roots法24小时内的染色规律,为基于乙酰胆碱酯酶染色的周围神经功能束三维重建确定标准化的孵育条件。 方法　新鲜人体截肢腓总神经标本,连续横断冰冻切片,片厚10 μm,共切取56 张,随机分为7组,每组8张切片,采用Karnovsky-Roots法进行乙酰胆碱酯酶孵育,其中1～6 组分别为1、2、4、8、12和24 h,第7组切片为阴性对照组。采用Image-Pro Plus 6.0 图像分析软件对不同时间染色组图像进行图像分析,指标为：(1) AChE 反应阳性区域的面积;(2) AChE 反应阳性区域的平均光密度;(3) 　AChE 反应阳性区域的累积光密度。组间结果采用SPSS13.0进行t-test 统计检验,以P<0.05为有统计学差异。 结果　8h 组的平均光密度值与12 h组有显著性差异,而12 h 与24 h 的平均光密度值无显著性差异;8 h、12 h 和24 h 间的染色面积、累计光密度值均存在显著性差异,但镜下可观察到24 h组存在过度染色。 结论　孵育12～24 h间的染色效果趋于一致,染色结果稳定可靠,神经束功能性质鉴别容易,神经束膜与染色的神经纤维间有清晰的分界,利于轮廓获取,可作为基于乙酰胆碱酯酶染色的周围神经功能束三维重建的标准化的育条件。