ATP对兔骨髓间充质干细胞向神经细胞分化的影响

摘要：目的 探讨骨髓间充质干细胞(MSCs)在ATP诱导下体外分化成神经细胞的可行性：方法抽取兔骨髓,淋巴细胞分离液密度梯度分离单个核细胞,体外培养传代,第3代时加入ATP,观察MSCs分化为神经细胞的形态学变化,并行诱导细胞的组织化学染色。结果MSCs在体外可以扩增,住ATP诱导下向神经样细胞分化,行巢蛋白(Nestin)、神经元特异性烯醇化酶(NSE)及胶质纤维酸性蛋白(cFAP)抗体免疫反应染色,阳性数分别约为Nestin30％、NsE：32％、GFAP15％。结论ATP能够诱导兔骨髓间充质干细胞向神经细胞分化。     

共培养条件下嗅鞘细胞对骨髓间充质干细胞向神经样细胞分化的影响

目的:观察体外骨髓间充质干细胞(BMSC)和嗅鞘细胞(OEC)非接触共培养时,OEC对BMSC向神经样细胞诱导分化的影响.方法:采用5～6周龄、体重100g左右的SPF级雄性Sprague Dawley (SD)品系大鼠20只,分离培养大鼠BMSC和OEC,BMSC取自双侧股骨,OEC取自嗅上皮.使用6孔0.4μm的Transwell共培养系统进行培养.将2×104/cm2的第3代OEC置于共培养系统上层,3×104/cm2的第3代BMSC置于下层.对共培养7d和14d后的BMSC行免疫组化染色,检测神经细胞特异性蛋白巢蛋白(Nestin)、神经元类型Ⅲβ-微管蛋白(Tuj-1)和神经胶质纤维酸性蛋白(GFAP)的表达情况.流式细胞仪检测Nestin、Tuj-1和GFAP阳性细胞的分布和所占的比例.结果:免疫组化检测显示,共培养7d和14d后BMSC均表达Nestin、Tuj-1和GFAP.流式细胞仪分析,共培养7d后Nestin、Tuj-1和GFAP阳性细胞比例分别为46.3％、76.5％和23.2％,共培养14d后分别为27.7％、89.1％和50％.结论:非接触共培养条件下,OEC能诱导BMSC向神经样细胞分化.     

脑脊液诱导人间充质干细胞定向分化为神经干细胞神经前体细胞的研究

目的 探索脑脊液诱导人间充质干细胞定向分化为神经干细胞的可行性.方法 分别采用同种异体脑脊液和细胞生长因子为诱导剂,体外诱导人脐血源和骨髓源间充质干细胞分化为神经干细胞.从形态学、免疫组化、荧光免疫绀化法、反转录聚合酶链反应(RT-PCR)方法对诱导后细胞进行鉴定.结果 两种方法诱导后间充质干细胞呈现神经干细胞改变,均可分化为神经元、星形胶质细胞和少突胶质细胞,并表现相应的特征形态结构、表型和生物学特性,细胞长势良好.而脑脊液诱导组,从形态上与细胞生长因子诱导组基本一致,但从时间上缩短神经干细胞、神经样细胞的生长周期.结论 两种诱导方法均可在体外定向诱导不间来源人间充质干细胞转化为神经样细胞,而脑脊液诱导组诱导时问和细胞周期均较短.     

不同诱导剂对骨髓间充质干细胞向神经元分化的影响

目的 寻找一种较为理想的诱导人骨髓间充质干细胞分化为神经元的诱导剂及方法.方法 采用人淋巴细胞分离液从骨髓中分离出骨髓间充质干细胞,体外培养、扩增后,以不同组合的复合神经诱导剂加以诱导,诱导期间观察细胞形态的变化,通过免疫细胞化学鉴定诱导细胞的分化情况,并用SPSS 12.0进行数据分析.结果 人骨髓间充质干细胞在加入不同组合的复合神经诱导剂后,均出现了胞体收缩、突起伸出的神经元样改变.免疫组化结果显示,兔抗人巢蛋白(nestin)、鼠抗人神经丝单克隆抗体(neurofilament,NF)出现了不同的阳性率,其中以E组阳性率最高,nestin(5.653±1.228)%,NF(74.724±3.651)%,兔抗人胶质纤维酸性蛋白(gliafiber acid protein,GFAP)免疫组化染色均呈阴性,SPSS 12.0分析显示各诱导组之间差异具有显著性意义(P＜0.05).结论 本实验探索出一种较为理想的诱导入骨髓间充质干细胞分化为神经元的复合神经诱导剂及方法.     

猕猴骨髓间充质干细胞与去细胞神经优化人工神经的体外构建

目的探讨猕猴骨髓间充质干细胞作为种子细胞与去细胞同种异体神经作为支架，体外构建人工神经的研究。方法采用密度梯度离心的方法分离并培养猕猴骨髓间充质干细胞，利用相差显微镜观察细胞生长情况并描绘其生长曲线；取猕猴3段4cm的周围神经，其中2段用4％triton—X-100及4％脱氧胆酸钠液分别萃取1次和2次，在新鲜神经和萃取神经中段取材，组织学检测及电镜学观察萃取前后的形态学变化；采用显微注射的方法把猕猴骨髓间充质干细胞种植到萃取两次的异体神经内，HE染色观察细胞在支架内的生长情况。结果猕猴骨髓间充质干细胞自我增殖能力强；萃取1次的异体神经细胞及髓鞘结构仍部分存留，而萃取2次的其细胞及髓鞘结构消失；HE染色可见细胞在萃取2次的异体神经内可爬行生长。结论猕猴骨髓间充质干细胞与化学萃取的去细胞同种异体神经适合体外构建人工神经。     

胚胎源脊髓组织诱导骨髓基质干细胞向神经元样细胞的分化

目的体外诱导大鼠骨髓基质干细胞（BMSC）分化为神经元样细胞。方法采用Percoll（密度：1．073g／ml）梯度分离液，离心分离鼠BMSC，体外培养，提取大鼠胚胎脊髓组织匀浆诱导BMSC分化为神经元样细胞。形态学观察及免疫组织化学鉴定神经元烯醇化酶（NSE）、神经丝蛋白（NF-200）、胶质纤维酸性蛋白（GFAP）的表达。结果大鼠骨髓基质干细胞在诱导后细胞形态变化，突起交织；免疫组织化学发现分化细胞NSE、NF-200表达阳性率分别为（68．0±1．7）％，（76．2±2．9）％，少数GFAP阳性。结论胚胎脊髓组织匀浆可诱导骨髓基质干细胞分化为神经元样细胞。     

三磷酸胞苷二钠诱导骨髓间充质干细胞分化为神经元样细胞的研究

目的:探讨三磷酸胞苷二钠体外诱导大鼠骨髓问充质干细胞(MSCs)向神经细胞分化的条件.方法:从大鼠双侧股骨中分离获取MSCs,体外培养扩增纯化后传代于塑料培养皿中,以含有三磷酸胞苷二钠(MSCs)的培养液对传至3～5代的细胞进行体外诱导分化,采用免疫细胞化学法对诱导后的细胞鉴定.结果:CTP浓度为0.2 mg/ml时,能够诱导NF、GFAP 和 NSE 阳性细胞比例为(32.73±0.63)%、(62.77±2.45)%、(30.41±0.65)%,显著高于对照组(P<0.01).结论:MSCs可通过体外培养并纯化,CTP可以诱导MSCs向神经细胞和神经胶质细胞分化,最佳诱导浓度为0.2mg/ml.     

人嗅鞘细胞与骨髓间充质干细胞体外共培养的实验研究

[目的]通过体外共培养观察人嗅鞘细胞(OECs)对骨髓间充质干细胞(MSCs)增殖分化的影响.[方法]取5个月以上引产胎儿嗅球及骨髓,分离培养及鉴定OECs及MSCs,携带绿色荧光蛋白(GFP)基因的腺相关病毒转染标记OECs,双苯亚甲胺荧光染料标记MSCs,将两种细胞按1∶1比例进行共培养,MSCs单独培养组为阴性对照组,免疫细胞化学方法检测MSCs表达巢蛋白(nestin)、神经元特异性核蛋白(NeuN)、神经元特异性烯醇化酶(NSE)、神经丝蛋白(NF-M)、胶质纤维酸性蛋白(GFAP)情况.[结果]MOI值为1×106时OECs表达GFP阳性率为84.89％,对细胞生长活性无明显影响.共培养时两种细胞生长良好,共培养7d未见形态学改变,经复方丹参注射液诱导后部分MSCs呈现神经元样改变,表达nestin、NSE、NeuN、NF-M、GFAP特异性标志,且共培养组的分化率明显高于单独MSCs培养组,有显著性差异.[结论]OECs可通过分泌可溶性物质及细胞之间的相互作用启动MSCs分化程序,提高MSCs向神经细胞分化率.     

大鼠骨髓间充质干细胞体外成脂分化能力的实验研究

目的:研究大鼠骨髓间充质干细胞体外成脂分化能力与传代次数的关系。方法:用全骨髓培养法,分离骨髓间充质干细胞,加含有地塞米松、胰岛素、3-异丁基-1甲基黄嘌呤、吲哚美辛诱导剂的培养液进行诱导,诱导第10天进行细胞形态学观察和油红O染色鉴定,并进行光密度值测定。结果:随着传代次数增加,油红O染色细胞计数值与光密度值均下降。结论:大鼠MSCs成脂能力随传代次数增加而降低。     

骨髓间充质干细胞分化为神经细胞研究进展

骨髓间充质干细胞(BMSC)分化为神经细胞的研究旨在诱导分化出具有分泌神经递质和电生理功能的成熟神经细胞,并提高神经细胞分化率.细胞因子诱导、中药及其提纯物诱导、细胞共培养诱导及提高细胞内环磷腺苷诱导等方案有助于BMSC分化为神经细胞,但体内实验诱导分化率远较体外实验低.不同浓度的细胞因子有不同的诱导分化率,中药及其提...     

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.     

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.     

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.     

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.     

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.     

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.     

Systemic analgesia adapted to the children's condition

A concept of balanced analgesia using nonsteroidal anti-inflammatory drugs (NSAIDs), paracetamol (acetaminophen), opioids, and corticosteroids can also be used in patients with pre-existing illnesses. NSAIDs are the most effective treatment for acute pain of moderate intensity in children; however, these drugs should be avoided in patients at increased risk for serious side effects, e.g. patients with renal impairment, bleeding tendency, or extreme prematurity. NSAIDs can be given with minimal risks to the younger child with mild to moderate asthma, and, in these patients, the use of steroids can be encouraged; in addition to their antiemetic and analgesic action, a beneficial effect on asthma symptoms can be expected. In the non-intubated child with cerebral trauma, exaggerated sedation caused by opioids and increased bleeding tendency caused by NSAIDs must be avoided. In neonates and small infants, the oral administration of sucrose or glucose is helpful to minimize pain reaction during short uncomfortable interventions.