组织工程学研究中偏光显微镜的应用

偏光显微镜是地质界用来观察晶体矿物的重要仪器，其理论基础是结晶光学，特点是即可观察结构，又可根据光学性质（折光率值不同）来判断成分，通过在研究ＮＥＣＭ和注射性胶原制作的过程中，用偏光显微镜和光镜观察同一部位的组织片，加以对比分析。因为胶原是生物体内的特殊“晶体”，故认为偏光显微镜应用在组织工程研究中，不仅开阔了观察领域，而且是重要的观测手段和方法。用偏光显微镜观察了猪骨ＥＣＭ胶原框架，以及注射性胶     

从NECM制取的可注射性胶原的生物学评价

从ＮＥＣＭ制取可注射胶原是从大分子降解为胶原分子，因此不需要交联剂和稳定剂。溶液中仅有胶原分子、无交联剂。本实验是将可注射胶原液注入大鼠尾部皮下组织内，共注射３０只，分三批杀死（１．５个月，３．０个月和半年），取注射部位的皮下组织作组织切片，染色（ＨＥ）、光镜观察（包括偏光显微镜）。     

动物皮肤、肌腱、软骨和骨的ECM制作

细胞外基质（ＥＣＭ）研究是组织工程学的重要部分，即是重要基础研究内容，又是研究细胞外基质的替代物课题。针对目前临床上大量应用人工合成的ＥＣＭ存在的问题（炎性反应、排斥反应、相容性等），本研究试图用动物的组织（自然的）ＥＣＭ替代人工合成的ＥＣＭ。用组织工程学方法处理动物组织，获得的产物经ＨＥ、奥新蓝染色、光镜观察和不染色切片偏光显微镜观察，证实经处理后的动物组织ＥＣＭ仅剩下肌腱、皮肤、软骨和骨的胶原框架。     

动物皮肤,肌腱,软骨和骨的ECM制作

细胞外基质研究是组织工程学的重要部分，即是重要基础研究内容，又是研究细胞外基质的替代物课题。针对目前临床上大量应用人工合成的ＥＣＭ存在的问题（炎性反应，排斥反应、相容性等），本研究试图用动物的组织（自然的）ＥＣＭ替代人工合成的ＥＣＭ。用组织工程学方法处理动物组织，获得的产物经ＨＥ、奥新蓝染色、光镜观察和不染色切片偏光显微镜观察，证实经处理后的动物ＥＣＭ仅剩下肌腱、皮肤、软骨和骨的胶原框架。     

从NECM制取的可注射性胶原的生物学评价

从ＮＥＣＭ制取可注射胶原是从大分子降解为胶原分子，因此不需要交联剂和稳定剂。溶液中仅有胶原分子，无交联剂。本实验是将可注射胶原液（１２０ｍｇ／ｍｌ）１ｍｌ注入大鼠尾部皮下组织内，共注射３０只，分三批杀死（１．５个月，３．０个月和半年），取注射部位的皮下组织作组织切片、染色（ＨＥ）、光镜观察（包括偏光显微镜）。发现三组在注射部位皮下组织内均有胶原分散存在。半年时胶原量少而且排列有序，未见包裹现象。除１．５个月少数动物皮下组织内有轻度炎性反应外，其余均未见到炎性反应。无排斥反应。局部组织无坏死现象。实验认为：本法所制可注射胶原液在半年内未发现特殊的生物学反应，胶原未完全吸收。     

动物NECM的稳定性评价

“动物ＮＥＣＭ的制作”文章发表近半年，已制作好的动物皮肤、肌腱、软骨和骨的ＮＥＣＭ已在保存液中保存了１３个月。为了观察ＮＥＣＭ和保存液的稳定性，本文做了如下几个方面的研究：①保存液：未放置ＮＥＣＭ新配制的和已放置１年的保存液；放置ＮＥＣＭ的保存液（１９９７年２月）。上述保存液均做细菌培养、镜下观察和成分分析。②电镜和组织学光镜观察：１９９７年２月制作的ＮＥＣＭ的光镜观察；放置１３个月后的ＮＥＣＭ电镜和光镜观察（包括偏光显微镜）；新鲜的正常相应组织的电镜和光镜观察。实验结果证明：猪皮肤和牛肌腱的ＮＥＣＭ已无细胞成分，基质为平行排列的胶原纤维丝或松散的胶原纤维网。而正常组织中细胞完整，胶原纤维呈致密交叉的网状编织结构。保存液清晰，无细菌生长，成分未变。     

从皮肤的细胞外基质（NECM）中制取可注射胶原

常规制取注射性胶原的方法是用酶降解法，经反复酸提、盐析、离心、交联等步骤，进行纯化，该法将胶原降解，酶提取分子片断，用交联剂组成可注射胶原。本法是从ＮＥＣＭ开始制取，ＮＥＣＭ已为胶原框架，因此步骤简化很多，仅需解裂胶原网状结构，酶解末端蛋白以消除特异性，最后降解成３００ｎｍ左右胶原。本法不用交联剂和稳定剂。生物原材料来源广泛，有利临床推广应用。经半年动物实验证明，本法制取的可注射胶原无排斥反应和炎     

从皮肤的细胞外基质(NECM)中制取可注射胶原

常规制取注射性胶原的方法是用酶降解法，经反复酸提、盐析、离心、交联等步骤，进行纯化，该法将胶原降解，酸提取分子片断，用交联剂组成可注射胶原。本法是从ＮＥＣＭ开始制取，ＮＥＣＭ已为胶原框架，因此步骤简化很多，仅需解裂胶原网状结构，酶解末端蛋白以消除特异性，最后降解成３００ｎｍ左右胶原。本法不用交联剂和稳定剂。生物原材料来源广泛，有利临床推广应用。经半年动物实验证明，本法制取的可注射胶原无排斥反应和炎性反应，也无包裹现象，半年时仍残留有部分胶原     

肾小球硬化及老年肾细胞外基质的变化研究

本文通过一侧肾切加重复阿霉素注射制作加速肾小球硬化大鼠模型，应用酶联免疫吸附试验（ＥＬＩＳＡ）对分离的肾小球内细胞外基质［胶原Ⅲ、胶原Ⅳ、层粘连蛋白（ＬＮ）和纤维连结蛋白（ＦＮ）］进行了硬化过程动态的定量研究，并将正常鼠（３月）和老年鼠（２６月）作了比较。结果显示，胶原Ⅲ在正常鼠肾小球内用ＥＬＪＳＡ方法未检测到；疾病所致的肾小球硬化过程胶原Ⅲ持续增加，弥漫性肾小球硬化期，较正常肾增加１１．８倍。胶原Ⅳ、ＬＮ和ＦＮ为正常肾小球内存在的细胞外基质（ＥＣＭ），在肾小球硬化的早、中期明显增加；弥漫性肾小球硬化期分别为正常肾的１２．３、１６．３和５．１倍。老年鼠与正常鼠相比，胶原Ⅲ无显著性变化（Ｐ＞０．０５），胶原Ⅳ、ＬＮ和ＦＮ分别为正常鼠的９．１、１０．０和２．８倍。本研究证实，ＥＣＭ的积聚是肾小球硬化过程中重要的病理特征，胶原Ⅲ（一种间质胶原）似与疾病所致的肾小球硬化发展有更密切和直接的关系。     

肾病患儿免疫细胞对肾小球上皮细胞合成基质的影响

目的为了明确免疫细胞对肾小球上皮细胞（ｇｌｏｍｅｒｕｌａｒｅｐｉｔｈｅｌｉａｌｃｅｌＧＥＣ）合成功能的直接作用。方法应用肾小球细胞体外培养，同位素掺入及放射免疫技术，以总胶原，层粘连蛋白，Ⅲ型前胶原及Ⅳ型胶原的合成为观察指标，动态研究了不同病理类型原发性肾病综合征（ＩＮＳ）患儿外周血单个核细胞（ｐｅｒｉｐｈｅｒａｌｂｌｏｏｄｍｏｎｏｎｕｃｌｅａｒｃｅｌＰＢＭＣ）对ＧＥＣ生物功能的影响。结果（１）肾病极期未经激素治疗组（未治组）ＰＢＭＣ上清明显促进了ＧＥＣ合成层粘连蛋白；（２）未治ＰＢＭＣ上清抑制了ＧＥＣ合成总胶原；（３）未治组ＰＢＭＣ上清促进了Ⅲ型前胶原的合成，而对Ⅳ型胶原的合成无明显影响；（４）肾病患儿ＰＢＭＣ的上述作用与是否足量激素治疗有关，而与尿蛋白能否阴转、肾组织病理类型、肾病临床类型等无直线相关关系。结论原发性肾病患儿循环免疫细胞可影响ＧＥＣ合成细胞外基质的功能。免疫细胞的这种活性可被激素治疗改变。     

Vasopressor hormone response following mesenteric traction during major abdominal surgery

Background : We investigated the vasopressor hormone response following mesenteric traction (MT) with hypotension due to prostacyclin (PGI₂) release in patients undergoing abdominal surgery with a combined general and epidural anesthesia. Methods : In a prospective, randomized, placebo-controlled study we administered 400 mg ibuprofen (i.v.) in 42 patients scheduled for abdominal surgery. General anesthesia was combined with epidural anesthesia (T4-L1). Before as well as 5, 15, 30, 45, and 90 min after MT we recorded plasma osmolality, hemodynamics and measured 6-keto-PGFlα (stabile metabolite of PGI₂), TXB₂ (stabile metabolite of thromboxane A₂) active renin, and arginine vasopressin (AVP) plasma concentrations by radioimmunoassay. Catecholamine levels were assessed by high-pressure liquid chromatography (HPLC) with electrochemical detection. Results : Following MT, arterial hypotension occurred along with a substantial PGI₂ release. This was completely abolished by ibuprofen administration. Although plasma levels of 6-keto-PGF_1α (1133 (708) vs. 60 (3) ng/L, median (median absolute deviation), P=0.0001, placebo vs. ibuprofen) remained significantly elevated, blood pressure was restored within 30 min after MT in the placebo group. At the same point in time plasma concentrations of TXB² (164 (87) vs. 58 (1) ng/L, P=0.0001), epinephrine (46 (33) vs. 14 (6) ng/L, P=0.001), AVP (41 ± (18) vs. 12 (7) ng/L, P=0.0004), and active renin (27 (12) vs. 12 (4) ng/L, P = 0.001) were significantly higher in placebo-treated patients. Conclusion : Under combined general and epidural anesthesia arterial hypotension following MT due to endogenous PGI₂ release is associated with enhanced release of AVP, active renin, epinephrine and thromboxane A₂, presumably contributing to hemodynamic stability within 30 min after MT.     

A Teaspoon Pump for Pumping Blood with High Hydraulic Efficiency and Low Hemolysis Potential

Abstract: Virtually all blood pumps contain some kind of rubbing, sliding, closely moving machinery surfaces that are exposed to the blood being pumped. These valves, internal bearings, magnetic bearing position sensors, and shaft seals cause most of the problems with blood pumps. The original teaspoon pump design prevented the rubbing, sliding machinery surfaces from contacting the blood. However, the hydraulic efficiency was low because the blood was able to "slip around" the rotating impeller so that the blood itself never rotated fast enough to develop adequate pressure. An improved teaspoon blood pump has been designed and tested and has shown acceptable hydraulic performance and low hemolysis potential. The new pump uses a nonrotating "swinging" hose as the pump impeller. The fluid enters the pump through the center of the swinging hose; therefore, there can be no fluid slip between the revolving blood and the revolving impeller. The new pump uses an impeller that is comparable to a flexible garden hose. If the free end of the hose were swung around in a circle like half of a jump rope, the fluid inside the hose would rotate and develop pressure even though the hose impeller itself did not "rotate"; therefore, no rotating shaft seal or internal bearings are required.     

Microspheres Based Detoxification System: A New Method in Convective Blood Purification

Abstract: A variety of protein-bound or hydrophobic substances, accumulating as a result of pathologic conditions such as exogenous or endogenous intoxications, are removed poorly by conventional detoxification methods because of low accessibility (hemodialysis), insufficient adsorption capabilities (hemosorption), low efficiency (peritoneal dialysis), or economic limitations (high-volume plasmapheresis). Combining advantages of existing methods with microspheric technology, a module-based system was designed. Major operating parameters of the latter can be modified to allow for adjustment to individual clinical situations. An extracorporeal blood circuit including a plasmafilter is combined with a secondary high-velocity plasma circuit driven by a centrifugal pump. Different microspheric adsorbers can be combined in one circuit or applied in sequence. Thus, a prolonged treatment can be tailored using specially designed selective adsorber materials. Comparing this system with existing methods (high-flux hemodialysis, molecular adsorbent recycling system), results from our in vitro studies and animal experiments demonstrate the superior efficiency of substance removal.     

Tissue perfusion in relation to cardiac output during continuous positive-pressure ventilation and administration of propranolol or verapamil

Background : Our objective was to determine whether administration of propranolol or verapamil modifies the hemodynamic adaptation to continuous positive-pressure ventilation (CPPV), in particular the regional distribution of cardiac output (CO).
Methods : General hemodynamics and regional blood flows assessed by microsphere technique (15 (μm) were recorded in 16 anesthetized pigs during spontaneous breathing (SB) and CPPV with 8 cm H₂O end-expiratory pressure (CPPV8) before and after intravenous administration of propranolol (0.3 mg · kg⁻¹ followed by 0.15 mg · kg⁻¹ · h⁻¹, n=8) or verapamil (0.1 mg · kg⁻¹ followed by 0.3 mg · kg⁻¹ · h⁻¹, n=8).
Results : CPPV8 depressed CO by 25% without shifts in its relative distribution with the exception of a noteworthy increase in adrenal perfusion. Propranolol increased arterial blood pressure, and due to a fall in heart rate, CO dropped by 25%. The kidneys and, to a lesser extent, the splanchic region and central nervous system received increased fractions of the remaining CO at the expense of skeletal muscle flow. Similar patterns were seen during SB and CPPV8 such that the combination of propranolol and CPPV8 depressed CO by 50%. The circulatory effects of verapamil were less evident but myocardial perfusion tended to increase.
Conclusions : The combination of propranolol or verapamil with CPPV does not result in any specific hemodynamic interaction in anesthetized pigs, except that the combined effect of propranolol and CPPV may severely reduce CO.     

Volatile anaesthetics reduce adhesion of blood platelets under low-flow conditions in the coronary system of isolated guinea pig hearts

Background : Inhibitory effects of volatile anaesthetics on platelet aggregation have been demonstrated in several studies. However, the influence of volatile anaesthetics on intracoronary platelet adhesion has not been elucidated so far.
Methods : Isolated hearts of guinea pigs were perfused with buffer in the absence or presence of volatile anaesthetics (0.5 and 1 MAC) at constant coronary flow rates of 5 ml/min for 25 min, then 1 ml/min for 30 min and again 5 ml/min for 10 min. Before, during and after low-flow perfusion, a bolus of human platelets was applied into the coronary system. To simulate thrombogenic conditions, 0.3 U/ml human thrombin was infused during low-flow perfusion and reperfusion. The number of platelets sequestered to the endothelium was calculated from the difference between coronary in- and output of platelets. The myocardial production of lactate and consumption of pyruvate and coronary perfusion pressure were also determined.
Results : At a flow rate of 5 ml/min only about 3% of the applied platelets did not emerge from the coronary system, in any group. In contrast, 13.1±1.2% (mean±SEM) of infused platelets became adherent in low-flow perfusion in the control group without anaesthetic. The adherence was reduced with each 1 MAC isoflurane (to 6.2±1.2%), sevoflurane (to 4.4±0.9%) or halothane (to 3.2±1.5%) (each P <0.05 vs. control). Volatile anaesthetic, 0.5 MAC, did not inhibit platelet adhesion to a statistically significant extent in any case. Perfusion pressure and metabolic parameters were not statistically different between the control and the hearts exposed to anaesthetics.
Conclusion : Volatile anaesthetics in a concentration of 1 MAC can reduce the adhesion of platelets in the coronary system under reduced flow conditions. This action does not arise from vasodilation or inhibition of ischaemic stress.     

Bariatric Surgery in Some "Central and East-European (former Eastern bloc)"Countries - Current Status and Prediction for the Next Millennium

Background: Obesity is increasing globallly, including in the formerly "Eastern Bloc" countries. Methods: A survey was made of obesity and bariatric surgery. Results: In the 8 East and Central European countries studied, with total population 300 million, roughly 43% of the population was overweight (BMI 25-30), 23% obese (BMI > 30), with about 15 million people morbidly obese (BMI > 40). From 0-10 morbidly obese individuals/100,000/year undergo bariatric surgery. Conclusion: Most countries were found to provide inadequate treatment for obesity.The majority of the morbidly obese are not treated effectively. However, health-care awareness of obesity and bariatric surgeons are slowly increasing.     

Synergistic interaction between the effects of propofol and midazolam with fentanyl on phrenic nerve activity in rabbits

Background: It has been shown that the depressive effects of both propofol and midazolam on consciousness are synergistic with opioids, but the nature of their interactions on other physiological systems, e. g. respiration, has not been fully investigated. The present study examined the effect of propofol and midazolam alone and in combination with fentanyl on phrenic nerve activity (PNA) and whether such interactions are additive or synergistic. Methods: PNA was recorded in 27 anaesthetised and artificially ventilated rabbits. In three groups, propofol, fentanyl and midazolam were administered intravenously in incremental doses to construct dose-response curves for the depressant effects of each one on PNA. In another two groups, the effect of pretreatment with either fentanyl 1 μg · kg^?1 i. v. or midazolam 0.05 mg · kg^?1 i. v. on the effects of propofol and fentanyl respectively on PNA were studied. Results: Propofol and fentanyl caused a dose-dependent depression of PNA with complete abolition at the highest total doses of 16 mg · kg^?1 i. v. and 32 μg · kg^?1 i. v., respectively. In contrast, midazolam in incremental doses to a total of 0.8 mg · kg^?1 reduced mean PNA by 63%, but approximately 12% of PNA remained at a total dose as high as 6.4 mg · kg^?1. The mean ED₅₀s, calculated from dose-response curves, were 5.4 mg · kg^?1, 3.9 μg · kg^?1 and 0.4 mg · kg^?1 for propofol, fentanyl and midazolam, respectively. Initial doses of either fentanyl 1 μg · kg^?1 i. v. or midazolam 0.05 mg · kg^?1 i. v. acted synergistically with subsequent doses of either propofol or fentanyl to abolish PNA at total doses of 8 mg · kg^?1 and 8 μg · kg^?1, respectively. Conclusion: Fentanyl has a synergistic interaction with both propofol and midazolam on PNA and hence potentially on respiration.     

Influence of dopexamine hydrochloride on haemodynamics and regulators of circulation in patients undergoing major abdominal surgery

Background: Catecholaminergic support is often used to improve haemodynamics in patients undergoing major abdominal surgery. Dopexamine is a synthetic vasoactive catecholamine with beneficial microcirculatory properties. Methods: The influence of perioperative administration of dopexamine on cardiorespiratory data and important regulators of macro- and microcirculation were studied in 30 patients undergoing Whipple pancreaticduodenectomy. The patients received randomized and blinded either 2 μg · kg^?1 · min^?1 of dopexamine (n=15) or placebo (n=15, control group). The infusion was started after induction of anaesthesia and continued until the morning of the first postoperative day. Endothelin-1 (ET-1), vasopressin, atrial natriuretic peptide (ANP), and catecholamine plasma levels were measured from arterial blood samples. Measurements were carried out after induction of anaesthesia, 2 h after onset of surgery, at the end of surgery, 2 h after surgery, and on the morning of the first postoperative day. Results: Cardiac index (CI) increased significantly in the dopexamine group (from 2.61±0.41 to 4.57±0.78 1 · min^?1 · m^?2) and remained elevated until the morning of the first postoperative day. Oxygen delivery index (DO₂I) and oxygen consumption index (VO₂I) were also significantly increased in the dopexamine group (DO₂I: from 416±91 to 717±110 ml/m² · m²; VO₂I: from 98±25 to 157±22 ml/m² · m²), being significantly higher than in the control group. pHi remained stable only in the dopexamine patients, indicating adequate splanchnic perfusion. Vasopressive regulators of circulation increased significantly only in the untreated control patients (vasopressin: from 4.37±1.1 to 35.9±12.1 pg/ml; ET-1: from 2.88±0.91 to 6.91±1.20 pg/ml). Conclusion: Patients undergoing major abdominal surgery may profit from prophylactic perioperative administration of dopexamine hydrochloride in the form of improved haemodynamics and oxygenation as well as beneficial influence on important regulators of organ blood flow.     

Immunomodulation in Transplantation with Photopheresis

Abstract: Photopheresis is a technique in which peripheral blood mononuclear cells, in the presence of a photoacti-vatable compound, are exposed extracorporeally to ultraviolet A light and reinfused, inducing a host autoregula-tory immune response. Experimental work and ongoing clinical studies are helping to define the role of this novel, safe, and non-toxic immunomodulating technology in the field of transplantation.