骨髓间充质干细胞定向分化肝细胞及肝内移植研究

目的 观察体外诱导骨髓问充质干细胞分化及肝纤维化形成环境中移植情况。方法 首先行骨髓间充质干细胞提取、分离和培养，加入肝细胞生长因子（HGF，20μg／L）和表皮生长因子（EGF，1．5mg／L）诱导定向分化。肝纤维化形成的大鼠随机分成2组，每组10只。使用5-溴脱氧尿苷（BrdU）标记诱导的骨髓间充质干细胞，经门静脉向肝纤维化形成的SD大鼠肝脏移植，对照组用BrdU标记未经诱导的骨髓间充质干细胞。2周后通过免疫组织化学方法检测大鼠肝脏标记细胞的分布及BrdU^＋／ALB^＋细胞数量。结果 体外诱导骨髓间充质干细胞定向分化的细胞CK8及ALB表达阳性。移植2周后大鼠肝脏均可检测到BrdU标记细胞，与对照组相比诱导后骨髓问充质干细胞组BrdU^＋／ALB^＋细胞数较多，差异有统计学意义（P〈0．05）。结论 经体外诱导骨髓间充质干细胞能分化为肝细胞，移植在大鼠肝纤维化形成环境中，白蛋白表达细胞数更多。     

人骨髓基质干细胞向肝细胞分化过程中白蛋白的表达研究

目的观察在体外诱导人骨髓基质干细胞向肝细胞分化过程中自蛋白的表达特征。方法从手术切除弃置的人肋骨骨髓中分离基质细胞,在含有肝细胞生长因子(HGF)、淋巴细胞抑制因子(UF)、纤维母细胞生长因子(FGF)等条件培养基中培养细胞。应用免疫荧光方法在共聚焦显微镜下观察肝细胞特异性白蛋白染色;同时,于诱导培养后多时间点测定培养细胞产生的白蛋白水平。结果人骨髓来源的基质干细胞在条件培养基中经诱导后,分化为成熟附壁细胞;细胞胞浆内肝细胞特异性标志物白蛋白呈阳性染色;已分化细胞合成并分泌白蛋白,其表达水平随细胞分化显示时间依赖性变化特征。结论人骨髓基质干细胞经诱导培养后可向成熟肝细胞分化并具有合成和分泌白蛋白功能,可以作为临床肝细胞移植及生物人工肝等治疗终末期肝病的重要肝细胞来源。     

骨髓源性肝干细胞的确认及定向分化的实验研究

目的 确定骨髓源性肝干细胞的表面标记，体外培养诱导其分化为肝细胞。方法 采用流式细胞仪检测多种大鼠肝损伤模型骨髓中干细胞标记的细胞群体的数量变化，寻找可能的肝干细胞，免疫磁珠分离各骨髓干细胞标记的细胞群体，进行体外培养诱导分化，观察细胞形态的变化，免疫组化技术检测清蛋白(白蛋白)，AFP，CK8／18等肝细胞标记的表达。结果 各模型大鼠骨髓内β2微球蛋白阴性(β2m^-)细胞显著增高，体外培养经诱导后呈多角形细胞表现，清蛋白，AFP，CK8／18表达阳性。其他干细胞类型细胞数量变化小，体外培养未见肝细胞样变化。结论 β2m^-细胞的数量随肝损伤而变化，在体外具有向肝细胞分化的能力，可能成为肝干细胞的标记物。     

骨髓源性肝干细胞定向分化及脾内移植研究

目的　寻找骨髓源性肝干细胞的表面标记 ,进行定向分化及脾内移植研究。方法　流式细胞仪检测淤胆大鼠骨髓中干细胞群体的数量变化 ,寻找肝干细胞标记 ,免疫磁珠分离 ,进行体外诱导分化和肝再生模型的脾内移植 ,观察细胞形态的变化 ,免疫组织化学和免疫荧光技术检测白蛋白、AFP、CK8/18等肝细胞标记的表达。结果　淤胆鼠 β2微球蛋白阴性 (β2 m-)细胞数量较对照组数量明显增高 ,分别为 (6.17± 2 .70 ) %和 (0 .79± 0 .61) % (P <0 .0 1) ,β2 m-细胞体外培养及脾内移植均可出现肝细胞样细胞 ,白蛋白、AFP、CK8/18表达阳性。结论　β2m 细胞在体内外具有向肝细胞分化的能力 ,脾内移植是肝干细胞移植可供选择的部位之一。     

骨髓基质细胞体外分化移植治疗大鼠脊髓损伤的初步研究

[目的]探讨大鼠骨髓基质细胞体外分化为神经干细胞后移植治疗大鼠脊髓损伤的可行性。[方法]骨髓基质细胞经培养及定向分化为神经干细胞，后者由5-溴脱氧尿嘧啶核苷法标记，制备大鼠脊髓损伤模型，伤后第9d移植神经干细胞，实验分组：细胞移植组、PBS填充组、正常对照组。应用组化法观察移植细胞是否存活，取材前24h显露坐骨神经，行辣根过氧化物酶逆行示踪法观察脊髓损伤处的修复重建。[结果]骨髓基质细胞在定向分化为神经干细胞后标记并移植于脊髓损伤区，标记的阳性细胞可在受体脊髓内检测到，辣根示踪技术显示细胞移植组较PBS填充组阳性细胞明显增多，差别有统计学意义。[结论]大鼠骨髓基质细胞在体外分化为神经干细胞后移植于脊髓损伤区，移植细胞可以存活，并参与脊髓损伤处神经传导通路的结构重建。     

骨髓基质细胞移植于缺血心肌的研究

国家自然科学基金资助项目 ( 30 170 935)目的　研究骨髓基质细胞移植于缺血心肌后的分化增殖情况和对心功能的保护作用。方法　以同基因背景的Lewis大鼠为研究对象 ,将溴氮胞苷 (BrdU)标记的骨髓基质细胞移植于结扎冠状动脉的大鼠缺血心肌内 ,4周后观察移植细胞的分化增殖情况和促血管生长作用 ,并用超声检测心功能改变。结果　细胞移植 4周后 ,可以在缺血心肌内找到BrdU标记的移植细胞 ,其中相当一部分细胞已分化为肌浆球蛋白重链 (MHC)阳性的心肌细胞。另外IIIV因子染色可见实验组移植区内有大量的血管新生 (2 3± 4/HPF) ,而对照组中却很少发现 (2± 1 /HPF) ,差异有非常显著性 (P <0 .0 1 )。超声检查显示移植骨髓基质细胞后可显著改善缺血心脏功能 ,其中实验组动物射血分数 (EF)升高了 0 .2 51± 0 .1 0 3 ,而对照组却降低了 0 .0 94± 0 .0 0 3 ,差异有非常显著性 (P<0 .0 1 )。结论　骨髓基质细胞移植于缺血心肌后可以向心肌细胞转化 ,并可明显改善心肌功能     

骨髓间充质干细胞定向肝细胞样分化的研究

目的探讨骨髓间充质干细胞定向肝细胞样分化的诱导条件，为肝组织工程提供新的种子细胞来源。方法分离小鼠骨髓间充质干细胞，在特定肝细胞培养液中用肝细胞生长因子(HGF)和表皮细胞生长因子(EGF)定向诱导。在倒置显微镜下观察细胞形态。应用免疫荧光法鉴定细胞性质。结果骨髓间充质干细胞经HGF和EGF诱导7d后变为肝细胞样圆形；2周后免疫荧光染色可见分化后细胞表达肝细胞标志物CK18和白蛋白。结论骨髓间充质干细胞在特定条件诱导下可以向肝细胞方向分化。有可能作为肝组织工程的种子细胞来源。     

5-BrdU标记的骨髓间充质干细胞对大鼠创面愈合的作用研究

目的探讨以5-溴脱氧尿嘧啶（5-BrdU）标记的骨髓间充质干细胞（Bone marrow mesenchymal stem cells，BMSCs）在大鼠创面愈合中的作用。方法分离培养大鼠BMSCs并用流式细胞仪技术鉴定。在大鼠背部脊柱一侧制作全层皮肤缺损模型，实验组经尾静脉注入经5-BrdU标记的BMSCs，移植细胞数为2×10^7。对照组为5-BrdU标记的BMSC移植的正常大鼠。术后3d、7d、14d、21d、28d分批处死动物，观察移植细胞参与创面愈合的情况。结果流式细胞仪鉴定细胞表达CD29、CD90；CD45表达呈阴性。实验组：经5-BrdU体外标记的BMSCs进行细胞移植14d后，大鼠创面局部及边缘可见5-BrdU标记的阳性细胞聚集，免疫组化染色可见基底层、真皮层、新生血管周围有散在红棕色标记细胞；对照组中未发现明显的聚集现象；抗BrdU／FⅧ、BrdU／波形蛋白免疫双标技术检测结果显示：在创伤部位有部分BMSCs分化为血管内皮细胞和成纤维细胞参与创面愈合。大鼠正常皮肤中未见明显的BMSCs聚集和增殖分化情况。结论经尾静脉注射移植的BMSCs参与皮肤创面愈合。     

人骨髓基质细胞培养及向成骨细胞的诱导分化

目的研究人骨髓基质细胞体外培养及向成骨细胞诱导分化的实验方法。方法采用梯度离心法获得人骨髓基质细胞，细胞纯化后使用分化培养液将骨髓基质细胞向成骨细胞方向诱导分化。通过形态学观察、生化指标检测、细胞染色和矿化结节测定等方法，确定细胞的功能状态和分化程度。结果显微镜观察显示获得的人骨髓基质细胞生长状况良好，生化指标稳定；经分化培养液培养的细胞增殖速度明显减慢，生长状态平稳。细胞在分化培养过程中，上清液中碱性磷酸酶分泌量明显增加，细胞碱性磷酸酶染色明显浓染，随时间呈显著增强趋势；采用常规培养液培养的骨髓基质细胞，在汇合后不能形成明显的矿化结节。用分化培养液培养的人骨髓基质细胞，在14d时开始出现矿化结节，在21d时呈现密集的茜素红染色矿化结节。结论梯度离心法获得的人骨髓基质细胞生长情况良好，功能状态稳定；体外培养的人骨髓基质细胞在一定条件下可以向成骨细胞方向诱导分化，并具有良好的成骨细胞功能特征，可以满足进一步研究的需要。     

磁标记猪骨髓间充质干细胞自体肝内移植后MR活体示踪研究

目的探讨利用磁共振成像对移植肝脏的磁标记猪骨髓间充质干细胞进行活体示踪的可行性。方法获取猪自体骨髓间充质干细胞，分离、培养、扩增。应用菲立磁（Feridex）标记细胞，普鲁士蓝染色鉴定，标记细胞组（n=6）和未标记细胞组（n=4）行经门静脉行肝内移植，分别于移植前，移植后6h、3d、7d行磁共振T1WI，T2WI，GRE序列成像，7d后行组织切片普鲁士蓝染色。结果：普鲁士蓝染色表明MSCs的标记率达接近100％，磁标记MSCs肝内移植后行磁共振T2＊WI序列呈明显低信号改变，并持续至细胞移植后7d，组织切片普鲁士蓝染色显示7d后肝内仍有移植的磁标记细胞存在于肝实质及肝窦中。结论利用SPIO可以在体外成功标记猪骨髓间充质干细胞，磁共振成像可以对经门静脉移植到肝内的磁标记MSCs进行活体示踪。     

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 comparison of the inhibitory effects of four volatile anaesthetics on the metabolism of chlorzoxazone, a substrate for CYP2E1, in rabbits

Background: Halothane inhibits in vitro and in vivo activity of cytochrome P-450 (CYP) 2E1. There are several fluorinated volatile anaesthetics besides halothane, and most of them are defluorinated by CYP2E1. It is unclear whether other fluorinated anaesthetics inhibit the in vivo activity of CYP2E1.
Methods: We compared the inhibitory effects of therapeutic concentrations of four inhalational anaesthetics, halothane, enflurane, isoflurane, and sevoflurane, on chlorzoxazone metabolism in rabbits receiving artificial ventilation.
Results: All four inhalational anaesthetics decreased arterial blood pressure and increased plasma chlorzoxazone concentration. However, no significant differences in the plasma chlorzoxazone concentration were found between the four anaesthetics. The estimated chlorzoxazone clearance increased after beginning inhalation with all four agents, but no significant difference in clearance was noted between agents.
Conclusions: At therapeutic concentrations, the in vivo inhibitory effect on chlorzoxazone metabolism was similar for all four inhalational anaesthetics examined, even though their chemical characteristics and extent of hepatic metabolism differ considerably.     

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.     

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.     

Membranes in Artificial Organs

Abstract: Membrane processes play a pivotal and enabling role in modern replacement therapy for acute and chronic organ failure and in the management of immunologic diseases. In fact, virtually all contemporary extracorporeal blood purification methods employ membrane devices, and the next generation of artificial organs and tissue engineering therapies are almost certain to be similarly grounded in membrane technology. In this short essay, we comment on the similarities and differences among synthetic membranes and their natural counterparts and also provide a critical overview of the demographics and technology of hemodialysis, hemofiltration, apheresis, oxygenation, and emerging membrane technologies and applications.     

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.     

Dilemma of Membrane Biocompatibility and Reuse

Abstract: Numerous articles have been published on the multiple use of dialyzers and on the effect of different reprocessing chemicals and techniques on the dialyzer biocompatibility and performance. The results often appear contradictory, especially those comparing standard biocompatibility parameters. Despite this confusion, a discerning review of the published works allows certain limited conclusions to be drawn. Reprocessing of used hemodialyzers changes the biocompatibility profile of a dialyzer as defined by the parameters complement activation. leukopenia, and cytokine release. The effect of reprocessing depends on the chemicals and reprocessing technique applied and also on the type of membrane polymer being subjected to the reprocessing procedure. Reports of pyrogenic reactions indicate that the flux of the membrane also influences how suitable it is for safe reuse. An increased risk of allergic and pyrogenic reactions appears to be associated with dialyzer reuse. Furthermore, there has been a lack of investigations into the immunologic effect of the layer of adsorbed and chemically altered proteins that remains on the inner surface of reprocessed dialyzers. We conclude that the clinical benefit of dialyzer reuse cannot be generally accepted from a biocompatibility point of view.