脂质过氧化在肾缺血和缺血再灌注损伤中的变化

本实验观察大鼠肾缺血75min及缺血60min后再灌注15min时肾组织脂质过氧化(LPO)和有关酶类变化。结果显示,肾脏缺血和缺血/再灌注后肾皮质和髓质中脂质过氧化产物丙二醛(MDA)含量均显著增高。超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性无明显变化。缺血/再灌注后黄嘌呤氧化酶(XO)活性显著升高。上述结果提示,肾缺血和缺血/再灌注时的LPO增强与氧自由基(OFR)产生增多有关。但上述二种情况时OFR产生机制不尽相同。     

肾神经在缺血再灌注性肾损伤室旁核电活动变化中的作用

目的:观察保留和去除大鼠肾神经后,肾缺血再灌注性肾损伤过程中室旁核电活动的变化,了解肾神经在缺血再灌注性肾损伤时室旁核电活动变化中的作用。方法:20只SD大鼠(250±30g)随机分为二组(n=10):①神经组:分离出左侧肾神经,夹闭肾动脉缺血30min,再灌注30min。②去肾神经组:分离并剪断左侧肾神经,夹闭肾动脉缺血30min,再灌注30min。参照大鼠脑立体定位图谱(George Paxinos,Charles Watson),用微电极全程记录各组肾缺血30min,再灌注30min室旁核放电活动。结果:保留肾神经组大鼠缺血和再灌注瞬间室旁核电活动明显减少(P<0.05)。随着缺血和再灌注时间的延长,室旁核放电活动逐渐增强(P<0.05)。去肾神经组大鼠缺血和再灌注瞬间时室旁核放电未出现快速性变化。在缺血和再灌注过程中,室旁核放电进行性增加,与保留肾神经组比较,去神经组缺血和再灌注期间各观察时间点室旁核放电均显著性增强(P<0.05)。结论:急性肾缺血再灌注可引起室旁核放电活动增强;肾缺血再灌注过程中室旁核放电活动的快速性变化与肾神经有关;肾神经具有抑制肾缺血再灌注时室旁核紧张性的作用。     

有色明胶血管灌注切片标本制作

有色明胶血管灌注法在国内外出版的有关组织学技术的书中均有介绍。因难于掌握灌注速度、温度和时间等,常致充盈不良或失败。我们采用在动物麻醉的情况下直接往血管内灌注有色明胶,不仅充盈良好,而且组织变化较小。常规石蜡包埋     

氯氮平对局灶性脑缺血再灌注大鼠脑内单胺类神经递质含量的影响

观察氯氮平对大鼠局灶性脑缺血再灌注单胺类神经递质变化与组织病理学改变的影响，探讨氯氮平对缺血再灌注损伤的保护作用及其机制。采用线栓法制作大鼠大脑中动脉局灶性脑缺血再灌注模型，用荧光分光光度计测定缺血2h再灌注lh时大脑皮层及纹状体NE、DA、5-HT含量；脑组织切片HE染色，光镜下观察组织病理学变化。结果可见；氯氮平12mg/kg、24mg/kg组能显著提高缺血2h再灌lh时大鼠大脑皮层及纹状体NE、DA、5-HT含量，并能显著改善间质脑水肿。表明氯氮平对脑缺血再灌注损伤有保护作用，其作用机制可能与减少缺血再灌注期间单胺类神经递质的释放有关。     

肢体缺血预处理大鼠肝损伤保护效应中内皮素1的变化和意义

背景：研究体内最强的缩血管物质内皮素1在肢体缺血预处理保护大鼠肢体缺血再灌注后肝损伤中的变化和意义,有助于从肝脏微循环角度探讨肢体缺血预处理的保护作用。 目的：探讨内皮素1在肢体缺血预处理保护大鼠肢体缺血再灌注后肝损伤中的变化和意义。 方法：雄性Wistar大鼠随机分为对照组、肢体缺血再灌注组和肢体缺血预处理组。肢体缺血预处理组以橡皮带预先阻断双后肢血流5 min,然后恢复血流灌注5 min,反复4次进行缺血预处理。然后肢体缺血再灌注组和肢体缺血预处理组以橡皮带环绕结扎大鼠双后肢根部,阻断血流4 h后松解,恢复血流灌注4 h制备肢体缺血再灌注模型,并于再灌注前20 min于左侧颈外静脉插管滴注生理盐水。对照组双后肢松弛环绕橡皮带但不阻断血流,其后操作同肢体缺血再灌注组。 结果与结论：大鼠肢体缺血再灌注后血浆内皮素1、透明质酸酶、丙二醛、谷丙转氨酶、谷草转氨酶水平和肝组织内皮素1、丙二醛、髓过氧化物酶水平均明显升高(P < 0.05),肢体缺血预处理干预后上述指标均明显降低(P < 0.05)。光镜下可见肢体缺血再灌注组肝细胞肿胀,肝索排列不规则;肢体缺血预处理组上述损伤表现减轻。结果可见大鼠肢体缺血预处理对肢体缺血再灌注后肝损伤的保护作用可能与抑制了内皮素1的缩血管作用从而改善肝脏的微循环有关,也可能与内皮素1含量的降低减少了白细胞过度聚集活化和减弱脂质过氧化有关。     

cAMP产生的兔股动脉舒张部分由NO介导

目的:研究cAMP产生的周围血管扩张作用是否与激活一氧化氮合酶(NOS)有关。方法:利用家兔股动脉恒流灌注模型,观察股动脉内灌注不同浓度的dbcAMP(膜渗透性cAMP)或腺苷酸环化酶的直接激动剂forskolin后灌注压的变化及NOS抑制剂L-NAME对其作用的影响。结果:dbcAMP和forskolin均产生浓度依赖的周围血管舒张,NOS抑制剂L-NAME部分阻断这种作用。结论:cAMP产生的周围血管扩张作用部分通过激活NOS实现。     

体外循环缺血-再灌注对人心房肌细胞一氧化氮合成酶的影响

目的：探讨体外循环心脏缺血一再灌注对人心房肌一氧化氮合成酶的影响。方法：用免疫组织化学结合计算机图像分析技术，检测在心脏缺血一再灌注前后１１例病人心房肌肉一氧化氮合成酶的三种同功异构酶一神经性一氧化氮合成酶、诱导性一氧化氮合成酶和内应性一氧化氮合成酶含量的变化。结果：心脏缺血一再灌注后神经性一氧化氮合成酶反应产物的平均灰度值明显减少，内皮性一氧化氮合成酶者明显增加，而诱导性一氧化氮合成酶者无明显改变。结论：心脏缺血一再灌注引起人心房肌神经性一氧化氮合成酶表达增加、内皮性一氧化氮合成酶表达减少，而对诱导性一氧化氮合成酶无明显影响。心脏缺血一再灌注后心脏功能失调可能与心房肌以上变化有关。     

大鼠短暂性局灶性脑缺血早期HMGB1在半暗带区的表达

目的:探讨调节炎症反应的细胞因子HMGB1在脑缺血/再灌注损伤中的时程变化特点。方法:采用大鼠MCAO模型,利用ELISA及免疫组织化学染色方法观察缺血/再灌注后不同时间点脑内HMGB1蛋白的表达变化。结果:ELISA检测提示再灌注后大鼠缺血侧大脑HMGB1表达显著增高,在10 h和70 h形成两次高峰(P<0.05);免疫组织化学染色提示再灌注1 h后部分大鼠(1/5)和再灌注后4,10,22和70 h后全部大鼠梗死区周边半暗带区的细胞外间隙有HMGB1阳性染色颗粒,后期并在部分细胞内出现HMGB1高表达。结论:结果提示,HMGB1不仅与脑缺血/再灌注的晚期损伤有关,亦可能参与了早期损伤过程。     

心脏α1受体与灌注性心律失常关系的研究

本实验结扎日本大耳白兔冠状动脉左室支２５ｍｉｎ，再灌注２０ｍｉｎ，观察缺血及再灌注对缺血区心肌α１受体密度和亲和力的影响，并于结扎后５ｍｉｎ给予α１受体阻断剂哌唑嗪，观察其对灌注后α１受体变化及再灌注性心律失常发生的影响。结果表明，缺血性和再灌注性心律失常可伴随α１受体密度下降；再灌注注性心律失常的发生亿与α１受体的亚型构成比有关；非亚型选择性α１受体阻断剂的抗再灌注性心律失常作用不稳定，在一定条     

纳洛酮对脑缺血大鼠海马神经元及氧自由基代谢的影响

目的探讨纳洛酮对脑缺血/再灌注损伤的防治作用及其机理。方法大鼠随机分为假手术组、脑缺血/再灌注损伤模型组、纳洛酮组和丹参组。采用四动脉结扎法建立脑缺血/再灌注损伤大鼠模型。测定脑缺血10分钟,再灌注30分钟脑组织丙二醛(MDA)浓度、超氧化物歧化酶(SOD)活性和脑组织水含量,观察海马CA1区神经元记数及病理变化。结果再灌注30分钟脑组织的MDA浓度、脑组织水含量显著增高,脑组织SOD活性及海马CA1区正常神经元记数显著降低(P<0.05和P<0.01)。使用纳洛酮后上述各指标的异常变化减轻,与再灌注组比有显著性差异(P<0.05和P<0.01)。结论纳洛酮可减轻全脑缺血/再灌注损伤,其机制与降低中枢神经元的氧自由基水平有关。     

振荡流动对组织工程用灌注式生物 反应器中剪切力分布影响

目的 考察振荡流动以及三维支架孔径和孔隙率对生物反应器内流速和剪切力分布的影响,并根据理论计算结果为脱细胞骨三维支架和灌注式生物反应器制备提出优化方法。方法 针对实验室前期制备的骨组织工程用脱细胞骨三维支架和灌注式生物反应器,将脱细胞骨三维支架简化为各向同性的多孔介质,对生物反应器内的流速和剪切力分布进行理论建模。结果 振荡流作用时,多孔支架材料内速度和达西剪切力呈现一致的变化规律,不同半径处流速和达西剪切力差异减小,有利于在骨组织工程中对种子细胞进行均匀三维培养。提高入口灌流速度可提高平均达西剪切力;增加多孔支架孔径或孔隙率对支架内流速峰值影响不大,但会显著降低平均达西剪切力;提高入口振荡流动振荡频率可降低支架内流速最大峰值,显著减小不同半径处流速的差异。结论 适宜的振荡流易产生利于骨组织工程干细胞所需剪切力,研究结果有望为优化骨组织工程中种子细胞的三维培养方法提供理论指导。     

川芎嗪对失血性休克家兔肠系膜毛细血管血流动力学的影响

家兔１６只，随机分为生理盐水对照组和川芎嗪治疗组，麻醉后造成失血性休克，用显微电视录像静像步进技术测定川芎嗪对小肠系膜毛细血管内径、血流速度及血流量的作用。结果提示，川芎嗪比对照组可加快失血性休克家兔小肠系膜毛细血管血流速度、改善肠系膜微循环血流灌注，但对毛细血管口径无明显作用。     

Computational modelling of the mechanical environment of osteogenesis within a polylactic acid–calcium phosphate glass scaffold

A computational model based on finite element method (FEM) and computational fluid dynamics (CFD) is developed to analyse the mechanical stimuli in a composite scaffold made of polylactic acid (PLA) matrix with calcium phosphate glass (Glass) particles. Different bioreactor loading conditions were simulated within the scaffold. In vitro perfusion conditions were reproduced in the model. Dynamic compression was also reproduced in an uncoupled fluid-structure scheme: deformation level was studied analyzing the mechanical response of scaffold alone under static compression while strain rate was studied considering the fluid flow induced by compression through fixed scaffold. Results of the model show that during perfusion test an inlet velocity of 25 μm/s generates on scaffold surface a fluid flow shear stress which may stimulate osteogenesis. Dynamic compression of 5% applied on the PLA–Glass scaffold with a strain rate of 0.005 s^?1 has the benefit to generate mechanical stimuli based on both solid shear strain and fluid flow shear stress on large scaffold surface area. Values of perfusion inlet velocity or compression strain rate one order of magnitude lower may promote cell proliferation while values one order of magnitude higher may be detrimental for cells. FEM–CFD scaffold models may help to determine loading conditions promoting bone formation and to interpret experimental results from a mechanical point of view.     

Three-dimensional modeling of transport of nutrients for multicellular tumor spheroid culture in a microchannel

The growth dynamics of avascular tumors in a microchannel bioreactor is investigated. A three-dimensional flow and nutrient transport model, incorporating the multicellular tumor spheroid (MTS) growth model, has been developed to study the influence of nutrients (oxygen and glucose) supply and distribution on the MTS growth. Numerical simulations based on the EMT6/Ro tumor cells show that the continuous-flow perfusion is more efficient to deliver nutrients to the MTS than the diffusion-only static culture. It is further demonstrated that as long as there is bulk flow, the growth of a single tumor spheroid at the early stage is insensitive to the flow velocity and the channel size. For multiple tumor spheroids in the same microchannel, however, increasing the perfusion velocity can improve the nutrient environment for the disadvantageous downstream tumor spheroid. The flow shear stress exerting on the MTSs in the current microchannel bioreactor is estimated to be far below the critical value to affect the MTS growth, which means that there is still much room for increasing perfusion velocity to satisfy the higher nutrient requirement by the growing tumor spheroids.     

Numerical simulation of blood flow in femoral perfusion: comparison between side-armed femoral artery perfusion and direct femoral artery perfusion

Computational numerical analysis was performed to elucidate the flow dynamics of femoral artery perfusion. Numerical simulation of blood flow was performed from the right femoral artery in an aortic model. An incompressible Navier–Stokes equation and continuity equation were solved using computed flow dynamics software. Three different perfusion models were analyzed: a 4.0-mm cannula (outer diameter 15 French size), a 5.2-mm cannula (18 French size) and an 8-mm prosthetic graft. The cannula was inserted parallel to the femoral artery, while the graft was anastomosed perpendicular to the femoral artery. Shear stress was highest with the 4-mm cannula (172 Pa) followed by the graft (127 Pa) and the 5.2-mm cannula (99 Pa). The cannula exit velocity was high, even when the 5.2-mm cannula was used. Although side-armed perfusion with an 8-mm graft generated a high shear stress area near the point of anastomosis, flow velocity at the external iliac artery was decreased. The jet speed decreased due to the Coanda effect caused by the recirculation behind sudden expansion of diameter, and the flow velocity maintains a constant speed after the reattachment length of the flow. This study showed that iliac artery shear stress was lower with the 5.2-mm cannula than with the 4-mm cannula when used for femoral perfusion. Side-armed graft perfusion generates a high shear stress area around the anastomotic site, but flow velocity in the iliac artery is slower in the graft model than in the 5.2-mm cannula model.     

Growth of primary embryo cells in a microculture system

We present optimal perfusion conditions for the growth of primary mouse embryonic fibroblasts (mEFs) and mouse embryonic stem cells (mESCs) using a microfluidic perfusion culture system. In an effort to balance nutrient renewal while ensuring the presence of cell secreted factors, we found that the optimal perfusion rate for culturing primary embryonic fibroblasts (mEFs) in our experimental setting is 10 nL/min with an average flow velocity 0.55 μm/s in the microchannel. Primary mEFs may have a greater dependence on cell secreted factors when compared to their immortalized counterpart 3T3 fibroblasts cultured under similar conditions. Both the seeding density and the perfusion rate are critical for the proliferation of primary cells. A week long cultivation of mEFs and mESCs using the microculture system exhibited similar morphology and viability to those grown in a petri dish. Both mEFs and mESCs were analyzed using fluorescence immunoassays to determine their proliferative status and protein expression. Our results demonstrate that a perfusion-based microculture environment is capable of supporting the highly proliferative status of pluripotent embryonic stem cells.     

Development of a new selective coronary perfusion cannula

The two most common types of coronary perfusion cannulae currently being used are the “balloon type”, with a balloon at the tip, and the “fenestrated type”, which has holes along the side near the tip. However, on occasion an unusually high perfusion pressure or a considerable amount of leakage is encountered during infusion of the cardioplegic solution. We have examined the properties of a newly developed Kochi Medical School (KMS)-type cannula and compared these to the properties of the balloon-type and fenestrated-type cannulae in an ex vivo experimental model that contains ostia of 4, 3, or 2 mm in diameter. Ejected flow velocity, circuit pressure, and the amount of leakage were measured at an infusion rate of 100 and 200 mL/min, with the latter two parameters measured under the counterpressure of 0 and 50 cmH₂O to examine the influence of coronary vascular resistance. Without counterpressure, the balloon type presented with the highest flow velocity (263 cm/s at 200 mL/min) and perfusion pressure (64 mmHg at 200 mL/min) but without leakage. The fenestrated type yielded a considerable amount of leakage (40 % at an ostium size of 2 mm). The KMS type showed a lower flow velocity and circuit pressure with less leakage. Under 50 cmH₂O counterpressure, however, only the KMS-type cannula could inject the water to any ostium size at both flow rates. These results suggest that the concept of the KMS-type cannula may be advantageous to achieving a secure infusion to a diseased coronary ostium.     

Cell distribution in a scaffold with random architectures under the influence of fluid dynamics

Fluid dynamic environment and scaffold architectures have an important influence on cell growth and distribution inside the scaffold. A porous cylindrical scaffold with a central channel is seeded with the sheep mesenchymal stem cells (MSCs) in this study. Then the cell seeded scaffold is continuously perfused with alpha-MEM medium by a peristaltic pump for 7, 14, and 28 days. Histological study shows that the cell proliferation rates are different throughout the whole scaffolds. The different cell coverage is shown in various positions of the scaffold. A computational fluid dynamics (CFD) modeling is used to simulate the flow conditions within perfused cell-seeded scaffolds to give insight into the mechanisms of these cell growth phenomena. Relating the simulation results to perfusion experiments, the even fluid velocity (approximately 0.26-0.64 mm/s) and shear stress (approximately 0.0029-0.027 Pa) are found to correspond to increased cell proliferation within the cell-scaffold constructs. This method exhibits novel capabilities to compare results obtained for different perfusion rates or different scaffold microarchitectures and may allow specific fluid velocities and shear stresses to be determined that optimize the perfusion flow rate, porous scaffold architecture, and distribution of in vitro tissue growth.     

Optimization of dynamic culture conditions: effects on biosynthetic activities of chondrocytes grown in collagen sponges

Application of mechanical stimulation, using dynamic bioreactors, is considered an effective strategy to enhance cellular behavior in load-bearing tissues. In this study, two types of perfusion mode (direct and free flow) are investigated in terms of the biosynthetic activities of chondrocytes grown in collagen sponges by assessment of cell proliferation rate, matrix production, and tissue morphology. Effects of the duration of preculture and dynamic conditioning are further determined. Our results have demonstrated that both bovine and human-derived chondrocytes demonstrate a dose-dependent response to flow rate (0-1 mL/min) in terms of cell number and glycosaminoglycan (GAG) content. This may reflect the weak adhesion of cells to the sponge scaffolds and the immature state of the constructs even after 3 weeks of proliferative culture. Our studies define an optimal flow rate between 0.1 and 0.3 mL/min for direct perfusion and free flow bioreactors. Using fresh bovine chondrocytes and a lower flow rate of 0.1 mL/min, a comparison was made between free flow system and direct perfusion system. In the free flow bioreactor, no cell loss was observed and higher GAG production was measured compared with static cultured controls. However, as with direct perfusion, the enhancement effect of free flow perfusion was strongly dependent on the maturation and organization of the constructs before the stimulation. To address the maturation of the matrix, preculture periods were varied before mechanical conditioning. An increase in culture duration of 18 days before mechanical conditioning resulted in enhanced GAG production compared with controls. Interestingly, additional enhancement was found in specimens that were further subjected to a prolonged duration of perfusion (63% increase after an additional 4 days of perfusion) after prematuration. The free flow system has an advantage over the direct perfusion system, especially when using sponge scaffolds, which have lower mechanical properties; however, mass transfer of nutrients is still more optimal throughout the scaffolds in a direct perfusion system as demonstrated by histological analysis.     

基于体外实验和数值仿真的冠脉介入手术中球囊去充盈瞬间的血流动力学研究

目的研究冠脉介入手术中血流参数在球囊去充盈瞬间发生的动态变化,探讨球囊去充盈对术后无复流发生风险的潜在影响。方法搭建体外实验装置,利用高速摄像机拍摄球囊变形过程和由染色剂标记的流场(流体介质为水),使用图像分析技术提取球囊变形参数,并估测球囊下游的流体流动速度;构建计算机仿真模型,导入实测的球囊变形数据,在多种灌注压和流体介质条件下模拟球囊去充盈过程。结果球囊在去充盈过程中呈现显著的非线性变形特征。球囊下游流速的数值计算结果与实测数据吻合良好,两者均显示流速随去充盈后的时间增长和灌注压升高而增大。数值计算进一步揭示,球囊下游流速在接近冠脉血流速度生理值时,球囊-管壁间隙流速和壁面剪应力分别达到其生理值的8~10倍和60~70倍。结论球囊去充盈引起球囊-管壁间隙流体急剧加速和壁面剪应力异常升高,从而增大斑块、血栓碎屑剥离的风险。鉴于壁面剪应力的升高程度随灌注压升高而增大,在冠脉介入手术中采取术前降压或选择舒张期去充盈等措施可能有助于降低无复流的发生风险。