一氧化氮及去内皮对颈动脉窦压力感受器活动的影响

本实验应用自制的颈动脉窦窦内劝灌流－窥上表面灌流的双重灌流装置,研究了外源性一氧化氮及内皮对家名义离体ＣＳ压力感受器活动的影响,并进一步探讨其作用机制。     

一种高精度离体心肌等长收缩灌流装置

乳头肌是心脏学科常用的研究标本,将乳头肌放在灌流装置中进行人工灌流,通过换能器即可记录其机械活动。研究乳头肌等长收缩力学性能和顺应性的灌流记录装置,必须具备下述两个条件: 1.准确记录乳头肌的静息张力和主动张力:静息张力(RT)是研究顺应性的基本参数;     

一种简易的大鼠离体肝脏灌流装置

参照Miller等人n 0]的大鼠离体肝脏灌流方法,我们设计制作了一套灌流装置,此装置由三部分组成:超级恒温器(维持灌流系统恒温用)、气体交换系统及灌流仪(用有机玻璃制作:上部有加热弯管,肝门静脉插管,温度计及恒温水浴夹层;下部有放置肝脏的漏斗和平板、灌流室、胆汁引流管、通气口及恒温水浴夹层)。用含O.15%葡萄糖的Krebs-Ringor碳酸氢盐缓冲液为灌流介质,流速2—3毫升/分钟/克肝,灌流4小时,在灌流期间持续通入95%Or-5?20灌流后肝脏无坏死灶出现,肝切片显微观察,结构基本正常。通过胆汁分泌量及耗氧量测定,证明本法可维持肝脏正常功能至少4小时。我们将灌流液浓缩后,作脂质测定及脂蛋白电泳分析:总胆固醇为0.062±0.008毫克/克肝(n=7,p=95%);有一条相当于大鼠血清极低密度脂蛋白(VLDL)及两条相当于高密度脂蛋白(HDL)的色带。本装置构造简单,保证恒温,流速较稳定,适用于物质代谢及其调节与药物代谢的研究。目前,我们已将此法用于大鼠肝脏脂质代谢、脂蛋白代谢及肝脏肝素可释放脂酶活性的研究(均见另文)。     

一种简易的大鼠离体肝脏灌流装置

参照Miller等人的大鼠离体肝脏灌流方法,我们设计制作了一套灌流装置,此装置由三部分组成:超级恒温器(维持灌流系统恒温用)、气体交换系统及灌流仪(用有机玻璃制作:上部有加热弯管,肝门静脉插管,温度计及恒温水浴夹层;下部有放置肝脏的漏斗和平板、灌流室、胆汁引流管、通气口及恒温水浴夹层)。用含0.15%葡萄糖的Krebs-Ringer碳酸氢盐缓冲液为灌流介质,流速2～3毫升/分钟/克肝,灌流4小时,在灌流期间持续通入95%O_2-5%CO_2。灌流后肝脏无坏死灶出现,肝切片显微观察,结构基本正常。通过胆汁分泌量及耗氧量测定,证明本法可维持肝脏正常功能至少4小时。我们将灌流液浓缩后,作脂质测定及脂蛋白电泳分析:总胆固醇为0.062±0.008毫克/克肝(n=7,p=95%);有一条相当于大鼠血清极低密度脂蛋白(VLDL)及两条相当于高密度脂蛋白(HDL)的色带。本装置构造简单,保证恒温,流速较稳定,适用于物质代谢及其调节与药物代谢的研究。目前,我们已将此法用于大鼠肝脏脂质代谢、脂蛋白代谢及肝脏肝素可释放脂酶活性的研究(均见另文)。     

离体心脏灌流系统今昔谈

17世纪初,英国医生哈维发表<心血运动论>一书,创立"血液循环学说",明确了心脏在血液循环中的重要作用.然而,心脏是如何工作的,哪些因素影响和调节心脏功能,仍然未知,这也是生理学家着重探讨的科学问题,但一直没有突破性进展.直到一百多年前,离体心脏灌流系统的出现,才使心脏生理学的发展进入一个新时代.利用该装置,生理学家获得的新发现如雨后春笋、层出不穷,从而奠定了心脏生理学的基础.本文对这种经典的生理学实验装置--离体心脏灌流系统的发展历程作一回顾,期望对今天的生理学研究有所裨益.     

介绍一种大鼠大脑中动脉阻塞／再灌流模型

介绍一种大鼠大脑中动脉阻塞／再灌流模型郑健董为伟１（第三军医大学新桥医院，重庆６３００３７；重庆医科大学神经病学研究所）局灶脑缺血／再灌流动物模型的建立是研究“再灌流”损害的基础。我们对Ｎａｇａｓａｗａ等建立的大鼠大脑中动脉线栓阻塞法局灶脑缺血模型做...     

家兔离体心脏缺血再灌注模型制作方法的改进及其效果

目的:通过对家兔离体心脏缺血再灌注模型制作方法的改进,延长家兔离体心脏存活时间并提高其存活状态,为研究离体心脏缺血再灌注提供更为稳定的模型.方法:健康新西兰家兔20只随机分为对照组与实验组,每组10只;通过改进实验方法与优化剖取家兔心脏术式操作,记录两组离体心脏复跳和从复跳到稳定跳动时间、比较两组离体心脏的存活时间及状态.结果:改进组优化手术操作完全暴露家兔心脏、减少了心肌机械性损伤,用改进台氏液冲洗离体心脏,缩短了从心脏取出到主动脉挂于灌流装置上该过程的时间,从而缩短了心脏从离体到再灌注此段的缺血时间;灌流液的温度从30℃缓慢升至37℃,此过程避免了离体心脏从4℃环境直升为37℃的初期供氧与灌流液温度条件变化而带来的心肌内环境紊乱,加之在灌流装置中增加了心脏保温罩、不断更换以95 ％O2+5％CO2氧饱和气化的新鲜台氏液,以共同维持离体心肌的正常生理功能.结果两组对比,改进组家兔离体心脏复跳时间和从复跳到稳定跳动时间比对照组缩短、明显显示其稳定有节律跳动时间延长(P＜0.01).结论:本改进法可使家兔离体心脏复苏快、且能持续有节律稳定跳动7h左右,达到延长家兔离体心脏存活时间的目的,该模型可适用于专业实验教学和科学研究.     

清醒山羊颈动脉体灌流模型及其评估

本文对清醒山羊颈动脉体灌流模型作了详细介绍。手术步骤包括:(1)在颈部任一侧结扎枕动脉、切断窦神经及切除颈动脉体;(2)完成对供应另一侧(灌流侧)脑和颈部血管的改道手术,以便从血液供应上游离颈动脉体;(3)在灌流侧安置颈静脉插管和颈动脉插管,建立灌流通路。实验中应用体外循环方法以动物自身血液灌流颈动脉体。该模型具有两个显著优点:(1)实验中可保持动物处于正常生理状态;(2)对脑组织和颈动脉体的血液血气及pH能分别加以人为控制。实验前动物均经过氰化钠试验检查颈动脉体和主动脉体的化学敏感性,用以评价灌流模型的有效性。文中还对这一模型的应用进行了讨论。     

电子顺磁共振法测定清栓酶在沙鼠脑缺血再灌流损伤中的抗自由基作用

本文设计了沙鼠脑缺血再灌流实验模型,以清拴酶作脑缺血后静脉注射再灌流模型和非药物再灌流模型组对照,用电子顺磁共振法(ESR)测定脑组织自由基波谱,结果清栓酶组出现自由基信号很弱,接近正常组,显著区别于未用药脑缺血再灌流30分钟和60分钟组(P<0.01),证明清栓酶有抗自由基和再灌注损伤的作用.     

高渗胁迫和灌流培养策略提高HEK 293细胞生产重组腺病毒载体产量

由于各种疾病在全球范围内的肆虐,国际市场对重组腺病毒载体(adenoviral vector,Adv)疫苗的需求量急剧增加,而工艺研究是解决这一问题的有效手段之一。在细胞接毒前施加高渗胁迫可以提高分批培养模式下的Adv产量,新兴的灌流培养也可以显著提高Adv的产量。将高渗胁迫工艺与灌流培养相结合,有望进一步提升高细胞密度生产过程中的Adv产量。本研究利用摇瓶结合拟灌流培养作为生物反应器灌流培养的缩小模型,使用渗透压为300–405 mOsm的培养基研究了高渗胁迫对细胞生长和Adv生产的影响。结果显示,在细胞生长阶段使用370 mOsm的高渗透压培养基,在病毒生产阶段使用300 mOsm的等渗透压培养基的灌流培养工艺有效地提高了Adv的产量。进一步研究发现这可能归因于病毒复制后期HSP70蛋白的表达量增加。将这种工艺放大至生物反应器中,Adv的产量达到3.2×10¹⁰ IFU/mL,是传统灌流培养工艺的3倍。本研究首次将高渗胁迫工艺与灌流培养相结合的策略应用于HEK 293细胞生产Adv,同时揭示了高渗胁迫工艺增产Adv的可能原因,为HEK 293细胞生产其他类型Adv的工艺优化提供了借鉴。     

CHO细胞无血清结团灌流培养：超声-沉降柱二合一灌流系统

利用CHO细胞能在培养过程中自然结团的特性,采用超声—沉降柱二合一灌流系统能促进细胞结团和加强截留的特性,我们用无血清培养基连续灌流培养基因重组CHO细胞MK3-A2株,分泌表达rhTNK-tPA获得了成功。培养周期为77-110天,细胞结团率为90%左右,直径在285～570μm之间,细胞截留率保持在95%左右,成活率为85%以上,细胞密度达到2×107/ml左右,rhTNK-tPA生产率平均为89 mg/L/d,最高时达216mg/L/d。 结果表明,使用该灌流系统进行细胞结团培养可以取代微载体培养用于动物细胞制药的规模化生产。     

梗阻性低位直肠癌保肛手术可行性探讨

目的:探讨梗阻性低位直肠癌保肛治疗(直肠癌前切除术(dixon手术))的可行性及术后肠瘘的防治。方法:回顾性分析我科2009年1月-2012年1月梗阻性低位直肠癌的保肛治疗(dixon)24例手术患者(梗阻性保肛组)临床资料及非梗阻性低位直肠癌保肛治疗(dixon)的24例患者(非梗阻性保肛组)临床资料,比较梗阻性与非梗阻性低位肠梗阻保肛治疗的临床疗效,分析梗阻性低位肠梗阻保肛治疗的可行性。结果:梗阻性保肛组住院天数:11.9天,非梗阻性肠梗阻保肛组8.7天P0.05;梗阻性保肛组发生肠瘘:4例(16.7%),非梗阻性肠梗阻保肛组发生肠瘘:1例(4.2%)P0.05,经充分引流后肠瘘愈合,无1人死亡,两组术后至出院期间死亡人数:0例;梗阻性保肛组肠功能恢复(以排气排便为指标):5.1天,非梗阻性保肛组肠功能恢复:3.8天,P0.05;术后6个月腹泻便秘患者两组相同为24人;术后6个月梗阻性保肛组肿瘤复发6人(25%),非梗阻性保肛组肿瘤复发5人(20.8%),P0.05。结论:梗阻性低位肠梗阻保肛治疗住院期疗效较非梗阻性保肛组差,中远期疗效无明显差异。梗阻性低位直肠癌可行保肛治疗。     

Alternating flow filtration as an alternative to internal spin filter based perfusion process: Impact on productivity and product quality

This article reports the results obtained from comparison of internal spin filter (ISF) and alternating flow filtration (ATF) as cell retention systems, regarding cell growth, volumetric perfusion rate, cell specific perfusion rate and cell productivity in the fermentation process. As expected we were able to reach higher cell densities and to achieve longer runs since ATF systems are known to be less affected by fouling. Volumetric production of the reactor using the ATF system was 50‐70% higher than the production achieved using the ISF due to higher cell density and a two‐fold increase in the perfusion rate. On the other hand, downstream processing performances were evaluated regarding chromatographic steps yields and productivity and quality attributes of the purified materials. Similar results were obtained for all evaluated systems. The fact that we were able to achieve a 2 working volumes (WV)/day perfusion rate using an ATF system as cell retention device allowed us to virtually double the WV of a 25 L reactor. These results constitute valuable data for the optimization of recombinant protein production in perfusion processes since a two‐fold increase in the average production of a manufacturing facility could be easily achieved as long as downstream scale up is possible. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1010–1014, 2017     

Recombinant antibody production by perfusion cultures of rCHO cells in a depth filter perfusion system

Recombinant Chinese hamster ovary cells, producing recombinant antibody against the human platelet, were cultivated in a depth filter perfusion system (DFPS). When perfusion cultures with working volume of 1 L were operated at perfusion rates of 5/d and 6/d, volumetric antibody productivities reached values 28 and 34 times higher than that of batch suspension culture in Erlenmeyer flasks and 43 and 53 times higher than that of batch culture in a controlled stirred tank reactor, respectively. Perfusion cultures in the DFPS showed stable antibody production over the whole culture period of up to 20 days. In the DFPS, inoculated cells in suspension were entrapped in a few hours within the depth filter matrix by medium circulation and retained there until the void space of the filter matrix was saturated by the cultured cells. After cells in the depth filter matrix reached saturation, overgrown viable cells at a perfusion rate of 5/d or 6/d were continuously collected into waste medium at a density of 2-4 x 10(5) cells/mL, which resulted in stable operation at high perfusion rates, maintaining values of process parameters such as glucose/lactate concentration, pH, and dissolved oxygen concentration. Because the DFPS overcomes most drawbacks observed with conventional perfusion systems, it is preferable to be used as a key culture system to produce monoclonal antibody stably for a long culture period.     

梗阻性低位直肠癌保肛手术可行性探讨

目的：探讨梗阻性低位直肠癌保肛治疗（直肠癌前切除术（dixon手术））的可行性及术后肠瘘的防治。方法：回顾性分析我科2009年1月．2012年1月梗阻性低位直肠癌的保肛治疗（dixon）24例手术患者（梗阻性保肛组）临床资料及非梗阻性低位直肠癌保肛治疗（dixon）的24例患者（非梗阻性保肛组）临床资料,比较梗阻性与非梗阻性低位肠梗阻保肛治疗的临床疗效,分析梗阻性低位肠梗阻保肛治疗的可行性。结果：梗阻性保肛组住院天数：11．9天,非梗阻性肠梗阻保肛组8．7天P〈0．05;梗阻性保肛纽发生肠瘘：4例（16．7％）,非梗阻性肠梗阻保肛组发生肠瘘：1例（4.2％）P〈0．05,经充分引流后肠痿愈合,无1人死亡,两组术后至出院期间死亡人数：0例;梗阻性保肛组肠功能恢复（以排气排便为指标）：5．1天,非梗阻性保肛组肠功能恢复：3．8天,P〈0．05;术后6个月腹泻便秘患者两组相同为24人;术后6个月梗阻性保肛组肿瘤复发6人（25％）,非梗阻性保肛组肿瘤复发5人（20．8％）,P〉0．05。结论：梗阻性低位肠梗阻保肛治疗住院期疗效较非梗阻性保肛组差,中远期疗效无明显差异。梗阻性低位直肠癌可行保肛治疗。     

Hybridoma perfusion system using a sedimentation device

Summary A novel sedimentation method with a spiral decanter was utilized with a bioreactor for propagation of hybridoma cells at high densities. The live cell concentration was increased and cell lysis was greatly reduced in this system compared to a tangential flow hollow fiber perfusion system. The specific monoclonal antibody productivity was higher than that obtained using a hollow fiber perfusion system or in a batch culture. Cell specific productivity usually declined over time in long term experiments. The use of the sedimentation device eliminated progressive deterioration of reactor performance usually associated with a perfusion device.     

Isoproterenol potentiates atrial fibrillation induced by acetylcholine]

A perfusion with normal Tirode solution containing isoproterenol, acetylcholine and their combination into the sinus node artery of the anesthetized open-chest dogs was used to induce atrial fibrillation. The perfusion of isoproterenol, alone, was unable to induce atrial fibrillation, though significantly increased atrial rate. Meanwhile the perfusion of acetylcholine, alone, did induce atrial fibrillation in all animals. The mixed perfusion of isoproterenol and acetylcholine led to decreasing the threshold (minimum) concentration of acetylcholine to induce atrial fibrillation. Herewith, atrial fibrillation appeared at later time from a perfusion start and lasted for more long time. No significant slowing down of sinus rhythm was registered before the initiation of atrial fibrillation. The data suggest that initiation of paroxysmal atrial fibrillation may only be mediated by parasympathetic activity and dependents on a level of adrenergic activity.     

VCTDSA联合CT灌注成像在急性缺血性卒中诊断运用探究

目的：探讨64层容积CT数字减影血管造影（Volume computed tomography digital subtraction angiography, VCTDSA）联合CT灌注成像在急性缺血性卒中诊断价值。方法：回顾性分析45例临床确诊为急性缺血性卒中患者的临床资料,分别给予VCTDSA与CT灌注成像处理,分析这两种技术对急性缺血性卒中患者的诊断敏感性和特异性。结果：45例患者检查有颅内有不同部位出血灶,多见于脑室,均伴有不同程度的异常血管网形成,其中VCTDSA图像质量优于CT灌注成像,VCTDSA较DSA可准确显示和测量动静脉瘘口大小,VCTDSA与MRA在瘤体长轴、瘤颈的比较中无显著性差异。结论：与其他减影CTA比较,VCTDSA联合CT灌注成像在急性缺血性卒中患者的诊断中具有优势。     

In vivo and in vitro intervention with L-carnitine prevents abnormal energy metabolism in isolated diabetic rat heart: chemical and phosphorus-31 NMR evidence

The beneficial effects of in vivo injections (200 mg/kg, twice daily) or in vitro perfusion (5.0 mM) of L-carnitine on an intrinsic abnormality in energy metabolism was investigated in isolated, perfused diabetic rat heart. Hearts were aerobically perfused for 60 min with elevated fatty acid substrate to simulate diabetic conditions. Phosphorus-31 nuclear magnetic resonance spectroscopy revealed a temporal decline in myocardial ATP levels (to approx 82%) during perfusion of diabetic hearts, but not in control hearts. This reduction was prevented by prior treatment in vivo with L-carnitine or by providing L-carnitine acutely in the perfusion medium. Chemical analysis of tissue extracts indicated that L-carnitine injections were effective in replenishing the decrease in total myocardial carnitine content which was present in diabetic hearts and in preventing the accumulation of long chain fatty acyl CoA. Perfusion with L-carnitine also attenuated the elevation of long chain fatty acyl CoA in diabetic hearts. This study gives additional support to the hypothesis that decreases in ATP which occur in the isolated, perfused diabetic heart are correlated with a concomitant elevation in long chain fatty acyl CoA, a known inhibitor of adenine nucleotide translocase. In the presence of elevated exogenous fatty acids, a primary deficiency in the total myocardial carnitine pool would result in elevations in tissue concentrations of long chain fatty acyl CoA since carnitine is a required carrier for transport of fatty acids into mitochondria. Replenishment of the carnitine in vivo was shown to be sufficient to prevent subsequent alteration in long chain fatty acyl CoA and ATP in isolated perfused diabetic hearts despite the burden of elevated fatty acid substrates.     

Significance of Perfusate Lactic Acidosis in Cadaveric Renal Transplantation

Thirty-two human cadaver kidneys were assessed by continuous perfusion before transplantation. Altogether, 26 were transplanted. Ten kidneys functioned immediately, of which nine had excellent renal function at three months. Sixteen had delayed onset of function, of these only five had excellent function at three months. There was no difference in warm ischaemic interval between the two groups or in ice storage time. The immediate function group, however, were perfused for twice as long as the delayed function group. The two groups could not be separated on donor history, but they could be precisely separated during perfusion by measuring lactic acidosis and lactic acid dehydrogenase (LDH). The biggest single difference between the groups was in terms of donor preparation.