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。     

体外诱导骨髓间充质干细胞向肝细胞分化的实验研究

目的：探讨HGF和EGF对骨髓间充质干细胞向肝细胞方向的诱导分化作用，探索出合适的诱导条件，为肝细胞移植、生物人工肝、组织工程构建等提供基础。方法：骨髓来自杂种犬的髂骨，采用全血贴壁法分离培养骨髓间充质干细胞，流式细胞仪检测细胞CD标记；用含HGF和EGF的α—MEM培养液进行诱导培养，并于诱导后7、14、21d留取细胞；用免疫组化检测AFP、CK18，免疫荧光检测ALB，PAS反应检测糖原；流式细胞仪检测细胞分化率。结果：分离的骨髓间充质干细胞表面CD13、CD34和CD45皆为阴性，CD29为阳性。诱导至7d出现AFP表达．14d表达增高，21d时表达下降。CK18、ALB和糖原14d时出现表达，并随时间延长表达逐渐增加：21d时ALB表达阳性率可达50％以上。结论：HGF和EGF有诱导骨髓间充质干细胞向肝细胞分化的作用。     

骨髓基质干细胞分化来源肝细胞移植的细胞准备及组织学观察

目的 探讨大鼠骨髓基质干细胞向肝细胞分化后体外标记方法及移植肝细胞的肝内组织学表现。方法 分离大鼠骨髓基质细胞，在体外诱导分化为成熟肝细胞。将5-溴脱氧尿嘧啶核苷（BrdU）掺入后的肝细胞移植入已行部分肝切除大鼠体内，分别应用免疫组织化学和免疫荧光方法观察受体肝脏内移植细胞的形态和功能。结果分化成熟肝细胞在BrdU掺入培养后细胞核染色可见特异性棕褐色标记；肝细胞移植后肝组织切片BrdU染色可定位移植细胞；白蛋白抗体染色显示移植细胞具有功能活性。结论 骨髓基质干细胞分化来源的肝细胞移植后形态功能稳定，是进行肝细胞移植的理想细胞来源。     

肝细胞生长因子诱导骨髓间充质干细胞向肝细胞分化的实验研究

目的探讨成年大鼠骨髓间充质干细胞(MSCs)在体外是否可定向诱导分化为肝细胞及其方法。方法采用梯度离心法,分离纯化SD大鼠的MSCs,流式细胞仪检测和碱性磷酸酶染色鉴定细胞类型。根据培养基中肝细胞生长因子(HGF)浓度不同,将MSCs分为4组进行诱导分化：A组0 ng／ml,B组10ng／ml,C组20ng／ml,D组40ng／ml。倒置显微镜连续观察细胞分化过程的形态学变化。于培养的1,3,7,14,21,28 d,分别以逆转录聚合酶链反应(RT-PCR)和细胞免疫组化检测各组细胞甲胎蛋白(AFP)、细胞角蛋白18(CK18)和白蛋白的基因和蛋白表达。结果分离纯化的SD大鼠MSCs的表面标志为CD29^ ,CD44^ ,CD34^-,CD45^-和CD90^ ,细胞的碱性磷酸酶染色阴性,细胞纯度达98％以上。C组与D组的MSCs,于培养第7天出现AFP基因表达,第14天表达增强,第28天表达减弱;第14天始出现白蛋白、CK18基因表达,而后持续。B组和A组在培养过程中,未出现AFP、CK18和白蛋白的表达。C组与D组MSCs的AFP细胞免疫组化染色培养第7天即出现阳性,第14天白蛋白和CK18免疫组化染色阳性。A组和B组的MSCs的AFP、白蛋白和CK18的免疫组化染色均阴性。结论成年大鼠MSCs在较高浓度的HGF的诱导下,可分化为肝细胞。     

骨髓源性肝干细胞的筛选、扩增和分化

目的 探讨采用含淤胆血清的培养体系直接从体外全骨髓细胞培养中筛选、扩增和分化骨髓源性肝干细胞的可行性.方法 制备含不同浓度淤胆血清的条件筛选培养液,常规培养大鼠全骨髓细胞,贴壁后换用条件筛选培养液,根据筛选的结果确定最佳的淤胆血清浓度.筛选到的骨髓源性肝干细胞分别采用扩增培养液和分化培养液进行扩增和诱导分化.传代细胞应用流式细胞仪检测干细胞标记.采用免疫组织化学、RT-PCR和电镜等方法对骨髓源性肝干细胞进行形态学以及表型特征的鉴定.以糖原染色和尿素分析的方法对诱导分化的细胞进行代谢功能的测定.结果 筛选培养结果,含50 ml/L的淤胆血清培养液的筛选效果最佳:骨髓源性肝干细胞能够生存,而其他非肝干细胞因不能适应而凋亡.纯化的肝干细胞能在扩增培养体系中传代6代并且维持稳定的细胞表面特征.更换为分化培养体系后,可形成肝细胞样集落形成单位.肝细胞样集落形成单位的细胞表达胎肝细胞的标志(AFP,白蛋白和细胞角蛋白8/18),胆管细胞的标志(细胞角蛋白19),肝细胞的功能蛋白(甲状腺素转运蛋白和细胞色素P450-2 b1),以及肝细胞核因子(HNF-1α和HNF-3 β).同时具有糖原储存和尿素合成等肝细胞特有功能.结论 含淤胆血清的筛选培养液能从全骨髓细胞培养中有效地筛选骨髓源性肝干细胞,并且纯化的肝干细胞能传代6代.肝干细胞分化后能形成具有肝细胞样的表型和功能.骨髓源性肝干细胞为解决临床肝细胞治疗的肝细胞来源问题提供了一个新的方法.     

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。     

体外诱导小鼠骨髓干细胞转化为肝细胞的实验研究

目的模拟体内肝脏发生发育的环境和条件，建立以细胞因子为主的体外诱导培养体系，探讨骨髓干细胞体外转化肝细胞的可行性。方法获取小鼠骨髓干细胞，建立以细胞因子为细胞诱导的培养体系。在细胞培养过程中，观察细胞形态和数量，逆转录-聚合酶链反应（RT—PCR）检测肝细胞特异性基因的表达。Western blot和流式细胞仪检测ALB和CK18在蛋白水平的表达情况。糖元染色法行细胞糖原染色、尿素合成试验检测细胞的合成和代谢功能。结果在诱导培养12d，可以观察到多极性的肝细胞样细胞。且细胞逐渐增多、集落不断增大。诱导细胞在培养7d开始表达AFP mRNA并维持到第21天。此后表达逐渐减弱；培养7天开始表达ALB mRNA和CK18 mRNA。随着培养时间的延长表达不断增强；培养14d开始表达TTR mRNA，随着培养时间的延长表达不断增强。通过Western blot检测，诱导21d的细胞表达ALB和CK18蛋白，流式细胞术分析ALB阳性细胞的比例为60．45％，CK18阳性细胞的比例为67％。诱导培养21d，细胞胞浆内可见红染的糖原颗粒；诱导培养6d，细胞开始合成尿素，尿素合成功能随诱导时间的延长而增强，于第15天达到高峰。结论我们建立的以细胞因子FGF、HGF、OSM、EGF为主的细胞诱导培养体系能促使骨髓干细胞定向转化为肝细胞。     

肝细胞生长因子联合成纤维细胞生长因子-4诱导人骨髓间充质干细胞分化为肝样细胞的实验研究

目的探讨肝细胞生长因子（hepatocyte growth factor，HGF）联合成纤维细胞生长因子4（fibroblast growth factor-4，FGF4）诱导人骨髓间充质干细胞（human bone marrow mesenchymal stem cells，HMSCs）向肝细胞方向分化的能力。方法分别用HGF、FGF4、HGF＋FGF4诱导HMSCs分化为肝样细胞。在不同分化阶段用免疫细胞化学染色法检测甲胎蛋白（alpha fetoprotein，AFP）和细胞角蛋白18（cytokeratin 18，CK18）；免疫荧光细胞化学染色法检测AFP、白蛋白（albumin，ALB）的表达；RT—PCR检测AFP和ALB mRNA表达的情况。结果HMSCs经诱导向肝样细胞转化。免疫细胞化学染色各诱导组均可检测出AFP和CK18表达，图像分析表明HGF＋FGF4联合诱导分化的AFP、CK18阳性率最高，HGF次之，FGF4则较弱；免疫荧光细胞化学染色发现各诱导组AFP、ALB均为阳性表达；各诱导组RT—PCR均可检测出AFP和ALBmRNA的表达，而阴性对照组则没有表达。结论HGF、FGF4、HGF＋FGF4均能诱导HMSCs分化为肝样细胞，分化的肝样细胞能分泌肝细胞特异性产物AFP、ALB、CK18等。以HGF＋FGF4诱导HMSCs分化为肝样细胞的阳性率最高。     

小鼠骨髓干细胞体外定向诱导为肝细胞样细胞的实验研究

目的来源于骨髓干细胞的有功能的干细胞,可能成为生物人工肝和肝细胞移植的细胞来源。我们建立恰当的体外诱导培养体系,促使骨髓干细胞转化为肝细胞。方法获取骨髓干细胞,建立以FGF、HGF、OSM、EGF为主的细胞诱导培养体系。在细胞分化过程中,观察细胞形态和数量的变化,RT-PCR检测基因表达的变化,免疫荧光染色技术在蛋白水平检测ALB和CK18表达情况。PAS染色检测其糖代谢功能。结果在诱导过程中,多极性的肝细胞样细胞在12 d内可以观察到,且其随着细胞分化过程逐渐增多、集落增大。诱导细胞在7 d内表达AFPmRNA并维持到第21天,但后期表达减弱;在7天内表达ALBmRNA和CK18mRNA,随着分化时间的延长,其表达不断增强;在14 d内表达TTRmRNA,随着分化时间的延长,其表达不断增强。免疫荧光染色进行定位:在诱导组,诱导21 d的细胞表达ALB和CK18,其中ALB表达于胞浆和胞膜,而CK18表达于胞浆。糖原染色发现诱导组中,细胞胞浆内出现大小不等的红染的糖原颗粒。结论我们建立的以细胞因子FGF、HGF、OSM、EGF为主的细胞诱导培养体系是有效的,通过诱导骨髓干细胞,我们获得了有部分肝细胞形态结构和功能的肝细胞样细胞。     

Reduction of Plasma Fibrinogen, Immunoglobulin G, and Immunoglobulin M Concentrations by Immunoadsorption Therapy with Tryptophan and Phenylalanine Adsorbents

Abstract Immunoadsorption (1A) therapy with tryptophan (TR-350) or phenylalanine (PH-350) adsorbents has been used to reduce the concentration of serum antibodies in human lymphocyte antigen (HLA)-immunized patients. Other forms of plasma purification have been reported to reduce the level of fibrinogen, which affects the blood properties. In this study we investigated the effects of IA therapy using both adsorbents on plasma fibrinogen and immunoglobulins G and M in 13 patients (8 patients were treated with TR-350, and 5 patients were treated with PH-350). During each session 1 plasma volume (2.8 ± 0.4 L of plasma) was processed through the immunocolumn and then returned to the patient together with the blood cells. Compared with the pretreatment values, the plasma fibrinogen, IgG, and IgM concentrations were significantly reduced after IA therapy (p < 0.01 for TR-350; p < 0.04 for PH-350). There was a positive correlation between the degree of reduction of plasma proteins and the number of IA treatments given. A nonpara-metric test (Wilcoxon's signed-rank test or the Mann-Whitney test) was used for statistical analysis. We conclude from our study that IA therapy effectively lowers the plasma levels of fibrinogen, IgG, and IgM and thus can be considered a valuable alternative to other blood purification methods.     

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.     

The multiply injured child

Blunt trauma is the principal cause of childhood death in many developed countries. This review outlines the differences between adults and children with respect to resuscitation and treatment of orthopaedic injuries in a child with polytrauma. Recent advances in techniques of fracture stabilization are reported.     

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.     

The analgesic effect of racemic ketamine in patients with chronic ischemic pain due to lower extremity arteriosclerosis obliterans

Background : Ketamine in sub-dissociative doses has been shown to have analgesic and phantom-Limb pain, where conventional treatment has often failed. Chronic ischemic pain due to lower extremity arteriosclerosis obliterans often responds poorly to analgesics, and the pain-generating mechanisms are not well understood.
Methods : Eight patients with rest pain in the lower extremity due to arteriosclerosis obliterans were given sub-dissociative doses of 0.15, 0.30, or 0.45 mg/kg racemic ketamine and morphine 10 mg as a 5-min infusion on four separate days in a cross-over, double-blind, randomised protocol. Plasma levels of (S)- and (R)-ketamine and their nor-metabolites were analysed with an enantioselective high-performance liquid chromatography (HPLC) method. Pain levels were evaluated with a visual analogue scale (VAS).
Results : Individual pain levels were highly variable during and after all the infusions but the pooled pain levels showed a dose-dependent analgesic effect of ketamine with a transient but complete pain relief in all patients at the highest dose (0.45 mg/ kg). Side-effects, mainly disturbed cognition and perception, were pronounced and dose-dependent. Morphine 10 mg had an analgesic peak at 20 min and 5/8 patients had complete pain relief. The remaining 3 patients also had high baseline pain scores, indicating a higher analgesic potency for the 0.30 and 0.45 mg/ kg ketamine doses than for morphine 10 mg.
Conclusion : We have demonstrated a potent dose-dependent analgesic effect of racemic ketamine in clinical ischemic pain. Due to a narrow therapeutic window, this analgesic effect is probably best utilised in combination with other analgesics.     

Intravenous endotoxin does not increase tissue extravasation of albumin in rats

Background : It is unclear whether activation of the inducible nitric oxide synthase (iNOS) increases or decreases the extravasation of plasma.
Methods : Chloralose anaesthetised male Wistar rats received E. coli lipopolysacharide (LPS), 3 mg kg^-1 i.v., or the corresponding volume of saline, 3 or 5 h before the end of the experiment. Mean arterial pressure (MAP) and heart rate (HR) were recorded. Tissue clearance of radio-labelled albumin, during the last 2 h of each experiment, was determined by a double-isotope method. In separate animals, the serum concentration of nitrite and nitrate was determined, 5 h after LPS or the solvent.
Main Results : LPS initially decreased MAP and lastingly increased HR. In the 3-h LPS animals (n=8), tissue plasma clearance was lower in the heart and calf muscle and increased only in diaphragm, compared to corresponding control animals (n=8). In the 5-h LPS rats, clearance was lowered (n=8) in the entire gastrointestinal tract and in testes, compared to controls (n=8). The serum nitrite/nitrate concentration was higher in animals given LPS (n=6) than in controls (n=6).
Conclusion : After LPS, tissue clearance of albumin was not increased in any major tissue, in spite of increased serum levels of NO end products. Apparently, after activation of iNOS, the augmented release of NO is not necessarily associated with increased albumin extravasation.     

Adrenaline markedly improves thoracic epidural analgesia produced by a low-dose infusion of bupivacaine, fentanyl and adrenaline after major surgery: A randomised, double-blind, cross-over study with and without adrenaline

Background: Basic pharmacological research indicates that there are synergistic antinociceptive effects at the spinal cord level between adrenaline, fentanyl and bupivacaine. Our clinical experience with such a mixture in a thoracic epidural infusion after major surgery confirms this. The objectives of the present study were to evaluate the effects on postoperative pain intensity, pain relief and side effects when removing adrenaline from this triple epidural mixture. Methods: A prospective, randomised, double-blind, cross-over study was carried out in 24 patients after major thoracic or abdominal surgery. Patients with only mild pain when coughing during a titrated thoracic epidural infusion of about 10 ml · h^?1 of bupivacaine 1 mg · ml^?1, fentanyl 2 μg · ml^?1, and adrenaline 2 μg · ml^?1 were included. On the 1^st and 2^nd postoperative days each patient was given a double-blind epidural infusion, at the same rate, with or without adrenaline. The effect was observed for 4 h or until pain when coughing became unacceptable in spite of a rescue analgesic procedure. Rescue analgesia consisted of up to two epidural bolus injections per hour and i.v. morphine if necessary. All patients received rectal paracetamol 1 g, every 8 h. Fentanyl serum concentrations were measured with a radioimmunoassay technique at the start and end of each study period. Main outcome measures were extent of sensory blockade and pain intensity at rest and when coughing, evaluated by a visual analogue scale, a verbal categorical rating scale, the Prince Henry Hospital pain score, and an overall quality of pain relief score. Results: The number of hypaesthetic dermatomal segments decreased (P <0.001) and pain intensity at rest and when coughing increased (P <0.001) when adrenaline was omitted from the triple epidural mixture. This change started within the first hour after removing adrenaline. After 3 h pain intensity when coughing had increased to unacceptable levels in spite of rescue analgesia (epidural bolus injections and i.v. morphine). Within 15–20 min after restarting the triple epidural mixture with adrenaline, pain intensity was again reduced to mild pain when coughing. Serum concentration of fentanyl doubled from 0.22 to 0.45 ng · ml^?1 (P <0.01), and there was more sedation during the period without adrenaline. Conclusions: Adrenaline increases sensory block and improves the pain-relieving effect of a mixture of bupivacaine and fentanyl infused epidurally at a thoracic level after major thoracic or abdominal surgery. Serum fentanyl concentrations doubled and sedation increased when adrenaline was removed from the epidural infusion, indicating more rapid vascular absorption and systemic effects of fentanyl.