恒河猴外周血pDC1/pDC2的分离和细胞表现型的研究

目的 研究恒河猴外周血树突状细胞（DC）的亚群pDC1和pDC2的分离方法。并探讨其细胞表现型。方法 采集健康，SIV阴性的恒河猴外周血，用Ficoll-Hypaque梯度离心法提取外周血单核细胞（PBMC），利用与恒河猴有交叉反应的人单克隆抗体以及三色流式细胞仪分离pDC1和pDC2，并对其进行细胞表现型的鉴定。结果 首次分离出恒河猴pDC1和pDC2细胞；并保持其活性。pDC1细胞表现型为Lineage^-,HLA-DR^ 和BDCA1^ ;pDC2的细胞表现型为Lineage^-,HLA-DR^ 和IL-3Rα^ 。结论 成功分离和鉴定了与人相似的恒河猴pDC1和pDC2细胞，对今后异种移植排斥反应的研究具有一定的意义。     

恒河猴外周血pDC1/pDC2的形态学研究

目的 研究恒河猴外周血pDC1和pDC2的形态学特征。方法 采集健康、SIV阴性的恒河猴外周血，用Ficoll—Hypaque梯度离心法提取外周血单核细胞(PBMC)。利用与恒河猴有交叉反应的人单克隆抗体以及三色流式细胞仪分离pDC1和pDC2。新鲜分离和经CD40L培养的pDC1进行形态学观察。新鲜分离和经CD40L、rhIL—3和rhGM—CSF培养的pDC2进行形态学观察。结果 恒河猴外周血树突状细胞(DC)具有较大不规则偏心的细胞核，其细胞表面有大量典型的树突状突起。结论 本研究为pDC1和pDC2作为抗原递呈细胞(APC)在排斥反应中的免疫调整功能奠定基础。     

pDC1抑制T细胞增殖的实验研究

目的　研究 pDC1体外对T细胞增殖的抑制作用。方法　采集健康恒河猴外周血 ,用Ficoll Hypaque梯度离心法和三色流式细胞仪分离 pDC1,经混合淋巴细胞培养 (MLR)研究其免疫调整功能。结果　新鲜分离的 pDC1具有较弱的刺激T细胞增殖能力 ;在经CD40L培养后 pDC1即成熟DC1成为有效的T细胞刺激增殖者。结论　本实验用MLR方法成功地研究了恒河猴外周血pDC1体外抑制T细胞增殖的作用 ,为阐明pDC1诱导免疫耐受的作用机制奠定了基础。     

体外扩增恒河猴自体内皮细胞人工血管移植研究（一）

目的:为恒河猴人工血管移植研究提供内皮化人工血管.方法;将恒河猴表浅静脉内皮细胞在体外培养13.89±1.36天;扩增培养的内皮细胞衬里于用纤维蛋白胶预衬的ePTEE人工血管,继续培养9天.结果:细胞数增加147.93±88.68倍.所培养的细胞为二倍体细胞,纯度99%.原代及传代细胞培养上清液中6-keto-PGFla和vWF含量无显著性差异.细胞种植后2小时和9天、人工血管腔面见一层均匀的基质,基质表面有一层连续的内皮细胞单层.内皮细胞紧密排列,呈梭状,形态饱满.细胞种植后9天,内皮细胞胞浆内微丝、微管数量明显增加,表明细胞骨骼成熟.结论:内皮化人工血管可用于置换恒河猴动脉.     

肾移植受者术后早期外周血树突状细胞亚群的动态变化及意义

目的 分析肾移植受者手术前后外周血中树突状细胞(DC)及其亚群骨髓源性DC(mDC)和浆细胞源性DC(pDC)的动态变化,探讨其与排斥反应的关系.方法 检测28例肾移植受者术前,术后1、7和28 d外周血中白细胞总数和单个核细胞数(PBMNC);应用流式细胞术测定DC及其亚群的数量和pDC/mDC.应用酶联免疫吸附试验法测定手术前后血清白细胞介素(IL)-10和IL-12水平.15名健康志愿者作为正常对照.结果 移植组术前外周血DC总数、pDC和mDC数量均低于对照组(P＜0.05),但两组pDC/mDC的差异无统计学意义(P＞0.05).移植组受者术后第1天外周血DC数量骤然降低,然后缓慢上升,第28天恢复至手术前的73.7％;mDC和pDC术后也降低,但mDC恢复较快,pDC恢复缓慢,至术后28 d分别达到术前水平的80.1％和50.1％(P＜0.05).术后第7天,移植组发生排斥反应者mDC数量高于未发生排斥反应者(P＜0.01).受者手术前后IL-10和IL-12的水平变化不明显.结论 DC及其亚群的变化与肾移植受者免疫状态有关,其变化异常提示受者免疫状态不稳定,在受者发生急性排斥反应时,可以作为诊断的参考指标.     

腺相关病毒载体介导结缔组织生长因子对体外转染恒河猴和人腰椎间盘细胞蛋白多糖和Ⅱ型胶原影响的比较

目的比较腺相关病毒载体介导结缔组织生长因子（connective tissue growth factor,CTGF）对体外培养的恒河猴和人腰椎间盘细胞转染后蛋白多糖含量和Ⅱ型胶原的影响。方法将恒河猴及成人腰椎间盘髓核细胞进行体外培养,应用rAAV2-CTGF体外转染细胞,分别通过^35S标记蛋白多糖的方法和Ⅱ型胶原的SP-ABC免疫组化法检测腺相关病毒载体介导生长因子对椎间盘细胞蛋白多糖合成和Ⅱ型胶原的影响。结果恒河猴椎间盘细胞能进行体外培养并传代,其形态学特性与培养的成人椎间盘细胞相似,rAAV2-CTGF与对照组相比可促进恒河猴和人椎间盘髓核细胞的蛋白多糖生物合成（P〈0．01）,恒河猴和成人相比生长因子对于蛋白多糖的促进作用无明显差异（P〉0．05）。结论成功培养恒河猴及成人椎间盘细胞,腺相关病毒载体介导CTGF能显著促进髓核细胞蛋白多糖的合成,生长因子对恒河猴和人椎间盘细胞蛋白多糖含量的影响具有很大的相似性。     

电磁脉冲对恒河猴淋巴细胞的损伤效应及其机制

目的:研究电磁脉冲对恒河猴淋巴细胞的损伤作用及其机理,为其防护提供参考依据。方法:用本实验室建立的电磁脉冲发生源进行照射,照射场强为6×10~4V/m,在5min 内重复照射30次。脉宽为25～30 ns,单次脉冲前沿为20ns。用原位末端标记技术和麦格-姬姆萨染色检测凋亡细胞,用碱磷酶免疫组化技术检测 Bax和 Bcl-2凋亡相关蛋白的表达。结果:(1)上述场强照后1d,淋巴细胞凋亡即开始增多,照后3d 接近峰值,一直持续到照后14d,与对照(照前)值比较,在照后3、7、14d 淋巴细胞凋亡指数分别为对照值的2.2、2.5和2.2倍。(2)照射后外周血淋巴细胞数量也迅速减少,相同场强照射后3、7、14d 约为对照值的74%,77%79%。(3)照射后3d,淋巴细胞 Bax 蛋白即出现增加,7d 达到峰值,Bax 阳性的淋巴细胞约为对照值的3.1倍。而 Bcl-2蛋白照射后出现减少,于照后7d 降至最低,Bcl-2阳性的淋巴细胞约为对照值的36%。这些结果提示 Bax 能促进 EMP 诱发的淋巴细胞凋亡,而 Bcl-2则起抑制作用。结论:EMP 能够诱发恒河猴外周血淋巴细胞的大量凋亡,细胞凋亡可能是导致 EMP 照射后淋巴细胞数量迅速降低和免疫功能出现抑制的主要原因之一。Bax 和 Bcl-2在 EMP 诱发的淋巴细胞凋亡调控中起重要作用。     

西罗莫司对异种动脉补片移植后的免疫调节作用研究

目的探讨西罗莫司在异种动脉补片移植中的免疫调节作用。方法选择野生型巴马猪至食蟹猴异种动脉补片移植手术后14 d受体猴的外周血单核细胞(POD14)为研究对象。设置二甲基亚砜(DMSO)对照组(体积比为1︰1 000)和西罗莫司实验组(终浓度为0.1μmol/L和0.5μmol/L),分别培养1.0 d和5.5 d,检测POD14细胞活性;设置DMSO对照组和西罗莫司实验组(终浓度为0.1μmol/L),培养5.5 d,检测POD14细胞中T、B细胞的数量并检测细胞因子含量和信使核糖核酸(mRNA)表达水平。结果与DMSO对照组比较,终浓度为0.1μmol/L和0.5μmol/L的西罗莫司处理1.0 d后,POD14细胞活性降低(P0.01～0.001);终浓度为0.1μmol/L和0.5μmol/L的西罗莫司处理POD14细胞5.5 d后,POD14细胞活性均明显降低(均为P0.001)。与DMSO对照组比较,西罗莫司(终浓度0.1μmol/L)降低POD14细胞中CD3+CD4+T细胞和CD3+CD8+T细胞的数量(P0.05～0.01),而CD3-CD20+B细胞数量略有升高(P0.01)。与DMSO对照组比较,西罗莫司实验组的细胞因子干扰素(IFN)-γ、白细胞介素(IL)-2、IL-4、IL-5和IL-6含量均明显降低(P0.05～0.001);西罗莫司降低细胞因子IFN-γ、肿瘤坏死因子(TNF)-α、IL-2、IL-4、IL-5和IL-6的mRNA表达水平(P0.05～0.001)。结论西罗莫司抑制异种动脉补片移植术后受体猴POD14细胞的增殖,主要机制是降低T细胞数量和抑制免疫排斥相关细胞因子的表达和分泌。     

Flt3配体重组腺病毒治疗小鼠胃癌的实验研究

Flt3配体（FL）是新的早期造血生长因子。研究表明。它能显著增加树突状细胞（DC）、自然杀伤细胞（NK）的数量和功能。DC和NK细胞在机体抗肿瘤免疫反应中起着关键作用，因此如何增加DC、NK细胞的数量和功能，一直是我们研究的热点。虽然Flt3有此作用。但其在体内表达的水平很低，时间短。不能达到抗肿瘤的需要，     

传代种植密度对人间充质干细胞增殖及成骨分化的影响

目的 :研究人间充质干细胞 (mesenchymalstemcells ,MSCs)传代培养时 ,细胞种植密度对MSCs增殖及成骨诱导分化影响。方法 :将第 2代MSCs以 8× 10 3 /cm2 、 3× 10 3/cm2 、 8× 10 2 /cm2 密度接种 ,分析其生长曲线 ,并扩增培养 18d ,记录细胞扩增数量 ,再分析扩增后细胞的生长曲线和成骨诱导能力。结果 :人骨髓MSCs阴性表达ALP、CD3 4;在 8× 10 3/cm2 种植密度下 ,细胞倍增时间 40h ,18d后细胞数量扩增 (5 1± 13 )倍 ;在 3× 10 3 /cm2 种植密度下 ,细胞扩增 (2 8± 6)倍 ;在 8× 10 2 /cm2 种植密度下 ,细胞总数仅增加 (5± 3 )倍。在低密度下获得的细胞增殖能力强于高密度组 ,两组细胞均具有成骨诱导能力。结论 :种植密度对MSCs增殖有明显影响 ,快速扩增MSCs作为骨组织工程种子细胞 ,宜选择 8× 10 3 /cm2 种植密度。     

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.     

Is the recovery profile of mivacurium independent of the rate of decay of its plasma concentration in patients with normal plasma cholinesterase activity?

Background: The duration of action of muscle relaxants is poorly correlated to the rate of decay of their plasma concentration. The plasma concentration of mivacurium may rapidly decrease below its active concentration because of the extensive hydrolysis of mivacurium. By inflating a tourniquet on one upper limb for 3 min after the administration of atracurium, mivacurium or vecuronium, we studied the influence of the initial decline of their plasma concentration on their effect. Methods: In 50 patients anaesthetised with thiopental, isoflurane and fentanyl, the effect of bolus doses of 0.15 or 0.25 mg . kg^?1 mivacurium (MIV 15, MIV 25), 0.3 or 0.5 mg . kg^?1 atracurium (ATR 30, ATR 50) and 0.06 or 0.1 mg . kg^?1 vecuronium (VEC 06, VEC 10) were measured on both arms (evoked response of the adductor pollicis to train-of-four stimulation every 12 s), a tourniquet being applied on one arm just before and during 3 min after the muscle relaxant bolus. Results: Tourniquet inflation of 3 min almost abolished the neuromuscular effect of mivacurium. In the vecuronium groups and in the ATR 50 group, tourniquet inflation did not modify the maximum degree of depression of the twitch response. Also, the duration of action of vecuronium was unaffected by the tourniquet. In the ATR 30 group, times to return of the twitch response to 25% (duration 25%) and 75% (duration 75%) of control response were significantly shorter in the cuffed arm, 23 min vs 27 min, and 41 min vs 45 min, respectively. In the ATR 50 group, only duration 25% was significantly shorter in the cuffed arm (41 min vs 45 min). Conclusion: The results suggest that the rate of decline of the plasma concentration of mivacurium is so rapid, that a very low and almost clinically ineffective concentration is present as soon as 3 min after its administration. The results also indicate that the recovery from a mivacurium-induced neuromuscular blockade is not influenced by the rate of decay of its plasma concentration in patients with genotypically normal plasma cholinesterase.