化学萃取同种异体神经种植许旺细胞的体外实验

目的：通过自体许旺细胞与化学萃取同种去细胞异体神经桥接物体外结合培养的研究，寻找一种在结构功能上与自体神经相似而抗原性少或无的物质来修复较长距离的神经缺损。方法：使用近交系F344乳鼠和双差速贴壁及Arab-C抑制成纤维细胞生长的细胞培养方法来获得大量纯度的许旺细胞。成年SD大鼠作为异体神经来源，利用化学萃取去除其中细胞成分降低其抗原性，制备去细胞神经桥接物，再把前者通过微注射方法注入到后者内部继续培养观察细胞生长情况。用抗S-100标记许旺细胞计算纯度，用HE染色、电镜观察神经萃取及细胞种植其内后生长情况。结果：许旺细胞培养纯度达92%以上。Triton-X-100及脱氧胆酸钠具有完全萃取掉神经纤维中的许旺细胞及髓鞘，萃取后所得到的桥接物适合于许旺细胞体外生长，并且细胞在其内有迁移排成行的特性。结论：通过化学萃取方法所得到的异体去细胞神经桥接物种植自体许旺细胞后可能为一种理想的神经缺损修复材料。     

绿色荧光蛋白标记神经干细胞的体外研究

目的　构建携带绿色荧光蛋白 (GFP)基因的逆转录病毒载体pLNCX2 GFP ,用GFP对神经干细胞进行标记示踪。方法　应用基因克隆的方法 ,制备 pLNCX2 GFP ,借助阳离子脂质体转染包装细胞PA3 17,G418筛选阳性克隆 ,获取病毒上清 ;从胚胎大鼠脑中解剖分离和培养神经干细胞 ,用病毒上清感染大鼠胚胎神经干细胞。结果　经酶切电泳和DNA测序表明成功构建了重组GFP逆转录病毒 ,pLNCX2 GFP转染包装细胞后可以产生GFP逆转录病毒 ,病毒感染大鼠胚胎神经干细胞可以长期表达绿色荧光。结论　逆转录病毒能够快速、稳定、长期地将GFP基因转入神经干细胞 ,这种标记方法非常有利于神经干细胞移植后结构和功能的研究     

绿色荧光蛋白标记的犬脂肪干细胞体外复合珊瑚材料的生长特性

目的研究犬脂肪干细胞(AdiposeDividedStromalCells,ADSCs)作为种子细复合珊瑚材料体外培养的生长特性。方法分离犬ADSCs,成骨诱导并转染绿色荧光蛋白(GFP)后,复合珊瑚材料培养。检测细胞总数,激光共聚焦观察细胞形态。结果ADSCs-珊瑚复合物体外增殖良好。激光共聚焦显示培养2周后细胞呈老化形态。结论GFP转染不影响细胞传代、增殖。细胞-材料复合物体外培养7天后回植较为适宜。     

绿色荧光标记共培养睾丸体细胞体内、外培养观察

目的：对Wistar大鼠睾丸间质体细胞转染携带绿色荧光蛋白基因的慢病毒,为进一步标记睾丸间质体细胞移植组织工程实验奠定基础。方法：获取Wistar大鼠睾丸间质体细胞,用慢病毒转载绿色荧光对其转染标记,体外培养,在共聚焦荧光显微镜下动态观察细胞生长、增殖等情况。回植于去势鼠体内,培养一定时期后取心脏血进行雄激素的检测。结果：睾丸间质体细胞于90h后,在荧光显微镜蓝光激发波长下,可发出明亮的绿色荧光,转染率达80%。传代后的细胞仍能发出明亮的绿色荧光。回植后血清学检测有明显高于对照组的激素检出。结论：经GFP转染后的睾丸间质体细胞仍具有睾丸间质体细胞的特性。采用携带绿色荧光蛋白基因的慢病毒标记睾丸体细胞中梭形及不规则形细胞（SLCs等）转染细胞效率高且持久、简便有效,且能保持睾丸间质体细胞原有生物学特性。     

Lipofectamine介导转染神经干细胞的研究

目的观察阳离子脂质体法转染体外培养神经干细胞的转染效率和外源基因的表达。方法从1d龄新生鼠大脑皮层组织培养神经干细胞，带有报告基因GFP的穿梭质粒pAd．Tract-CMV经Lipofectamine介导转染神经干细胞后观察GFP表达，用流式细胞仪测定转染率。并观察阳离子脂质体对神经干细胞的毒性作用。结果荧光显微镜观察到被转染的神经干细胞长期表达绿色荧光蛋白。流式细胞仪结果显示转染率最高可达到39．99％。转染时，阳离子脂质体浓度超过24ml／L时表现出细胞毒性。结论阳离子脂质体hpofectamine介导转染神经干细胞效率较高，外源基因表达时间长。     

携带肝细胞生长因子基因慢病毒转染人脂肪来源干细胞复合纤维蛋白胶支架构建可注射型组织工程脂肪的实验研究

目的探讨构建可注射型组织工程脂肪的可行性,为临床软组织缺损修复及美容填充提供简便易行的方法。方法取脂肪抽吸术获取的脂肪组织,采用酶消化法分离培养获得人脂肪来源干细胞(human adipose-derived stem cells,h ADSCs),进行细胞形态观察、流式细胞术及成脂诱导鉴定。用携带肝细胞生长因子(hepatocyte growth factor,HGF)和绿色荧光蛋白(green fluorescent protein,GFP)的慢病毒HGF-GFP-LVs,以不同感染复数(multiplicity of infection,MOI)(分别为10、30、50、100)转染h ADSCs,计算转染效率以确定最适MOI。取SPF级6周龄单纯T细胞缺陷BALB/c雌性裸鼠10只,将经过最适MOI HGF-GFP-LVs转染并成脂诱导的h ADSCs与纤维蛋白胶混合注入裸鼠背部右侧皮下(转染组),单纯成脂诱导的h ADSCs与纤维蛋白胶混合注入背部左侧皮下(未转染组),单纯纤维蛋白胶注入颈后正中皮下(空白对照组),每点注射0.4 m L。移植后12周处死裸鼠,取出移植物,行大体观察、湿重测量、HE染色、GFP荧光标记检测及免疫荧光染色观察,评价种子细胞的体内成脂能力以及移植物血管新生情况。结果经鉴定所培养细胞为h ADSCs。MOI为10和30时HGFGFP-LVs转染率较低,MOI为50和100时转染率较高(均80%),确定最适MOI为50。移植12周,转染组和未转染组均获得外观类似脂肪组织的新生物,湿重分别为(32.30±4.06)mg和(25.27±3.94)mg,差异有统计学意义(t=3.929,P=0.001);空白对照组未见新生物。转染组新生脂肪细胞数为(126.93±5.36)个/视野,显著高于未转染组的(71.36±4.52)个/视野(t=30.700,P=0.000)。荧光显微镜下可见部分单房脂肪细胞发出网状绿色荧光。免疫荧光染色示转染组血管密度为(16.37±2.76)个/视野,显著高于未转染组的(9.13±1.68)个/视野(t=8.678,P=0.000)。结论以h ADSCs为种子细胞,经慢病毒转染HGF基因并成脂诱导后与纤维蛋白胶支架复合可在体内成功构建成熟脂肪,能促进移植后的血管新生,从而提高移植物的存活率。     

绿色荧光蛋白转基因神经干细胞脑出血大鼠脑内移植后血管生成与神经形态学的改变

目的 观察绿色荧光蛋白转基因神经干细胞(NSCs/GFP)脑出血大鼠脑内移植后血管生成与神经形态学改变.方法 分离、培养及纯化NSCs/GFP,并进行体外诱导分化.注射动脉血制作大鼠脑出血模型,3d后将NSCs/GFP注入血肿同侧尾状核,观察NSCs/GFP在脑内分化情况以及大鼠不同时间点行为学的变化.结果 成功分离、培养及鉴定NSCs/GFP,体外诱导分化为内皮细胞和平滑肌细胞；移植入脑出血大鼠脑内后,能促进其肢体功能恢复(P＜0.05),且能分化为神经元、星型胶质细胞、内皮细胞及平滑肌细胞.结论 NSCs/GFP移植入脑出血大鼠脑内能改善其运动功能,并有生成血管与神经的能力.     

经脂肪源性干细胞诱导的施万样细胞用于组织工程化人工神经的实验研究

目的研究经大鼠脂肪源性干细胞（ADSCs）体外诱导分化的施万样细胞应用于组织工程化外周神经,修复大鼠坐骨神经缺损的效果。方法Wistar大鼠48只,体重200-250g,随机分成3组,每纽i6只,分别用下面3种不同的方法修复15mm坐骨神经缺损：DMEM组（支架内注射培养基）、诱导组（支架内注射施万样细胞）和自体神经移植组．通过足迹实验（坐骨神经功能指数测定）、神经电生理检测、胫前肌湿重比率测定进行功能检测;应用透射电镜、图像分析系统进行组织学观察。结果术后12周诱导组的SFI指数、神经传导速度、潜伏期、波幅以及胫前肌重量恢复、神经纤维数目、轴突直径、髓鞘厚厦好于DMEM组（P〈0．05）,接近自体移植组。再生神经中标记细胞观察显示PKH-26标记的ADSCs,依然呈红色荧光。结论ADSCs诱导分化后的施万样细胞与去细胞同种异体神经两者构建的组织工程化神经能有效地修复大鼠坐骨神经缺损,其效果与自体神经移植相似：ADSCs经诱导后的细胞可以作为组织工程种子细胞新的来源。     

大鼠PnNOS基因修饰脂肪源性干细胞的构建

目的 构建过表达大鼠阴茎神经源性一氧化氮合酶（PnNOS）基因大鼠脂肪源性干细胞（ADSCs）,为基因修饰ADSCs移植治疗勃起功能障碍（ED）大鼠模型提供种子细胞.方法 获取大鼠PnNOS基因,并与线性化的腺病毒真核表达载体pDC315-EGFP定向克隆连接.构建正确的pDC315-PnNOS-EGFP穿梭质粒转染293 T细胞,采用Western Blot检测PnNOS基因在293 T细胞中的表达情况.利用AdMax腺病毒包装系统包装产生重组腺病毒,包装后扩增纯化并测定病毒滴度.PnNOS基因重组腺病毒转染大鼠ADSCs,观察GFP表达情况和Western Blot检测PnNOS基因表达情况.结果 经PCR鉴定、限制性酶切分析、测序鉴定和目的质粒Western Blot表达检测鉴定,证实pDC315-PnNOS-EGFP重组腺病毒载体构建成功.重组腺病毒包装成功且病毒滴度为5.0×109 PFU/ml.Western Blot于大鼠ADSCs中检测到约161 KDa大小条带,其与PnNOS蛋白分子量大小基本相一致.结论 大鼠PnNOS基因修饰的ADSCs构建成功,从而为基因修饰ADSCs移植治疗ED提供了良好的基因工程细胞.     

化学去细胞异体神经促神经再生的体外实验研究

[目的]对大鼠化学去细胞异体神经蛋白成分进行电泳分析,观察去细胞异体神经中的蛋白组分;制备去细胞神经薄膜,接种背根神经节,观察其结构对神经生长的影响。[方法]按Sondell法制备大鼠的去细胞异体神经,将制备好的神经进行电泳分析,观察28～30 kDa区域的髓鞘蛋白的去除程度;将制备的神经进行冰冻切片,接种鸡胚背根神经,进行神经纤维荧光染色,观察神经纤维在神经切片上的生长方向。[结果]去细胞异体神经电泳结果显示:28～30 kDa区域髓鞘蛋白完全消失,化学萃取的去细胞神经可以完全去除髓鞘蛋白;背根神经节发出大量的神经纤维沿着去细胞神经基底膜管方向生长。[结论]去细胞异体神经中无残留引起免疫反应的髓鞘蛋白,保留的基底膜管结构对促神经纤维再生具有引导性作用。     

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.