锤头型核酶特异性阻断雄激素受体表达对前列腺癌细胞生长的影响

目的：探讨核酶阻断雄激素受体（AR）基因表达治疗前列腺癌的可能性。方法：在脂质体介导下，锤头型抗雄激素受体核酶表达载体转染前列腺癌细胞，采用逆转录－聚合酶链反应（RT－PCR）检测ARmRNA，四甲基偶氮唑盐（MTT）法检测细胞增殖活性，流式细胞仪（FCM）检测细胞周期时相，原位末端转移酶标记法检测细胞凋亡。结果：核酶表达载体转染1－5d后，前列腺癌细胞AR mRNA水平降低32．6％－40．7％（P＜0．05），细胞生长抑制率18．28％－35．34％（P＜0．05），细胞周期阻滞于G2／M期，诱导细胞凋亡，细胞凋亡比率增加20．70％（P＜0．01）。结论：应用核酶特异性切割ARmRNA，能有效阻断AR基因的表达，诱导前列腺癌细胞凋亡，有可能成为前列腺癌基因治疗的有效方法之一。     

锤头状核酶对短片段雄激素受体 mRNA的体外切割作用

目的 研究锤头状核酶对雄激素受体(AR)mRNA片段的体外切割活性，探讨抗雄激素基因治疗的新途径。方法 计算机模拟AR mRNA片段二级结构，选择第1018位GUC为核酶切割位点，设计特异性切割AR的锤头状核酶(RZ)，合成含T7启动子的核酶和底物模板链，体外转录RNA，37℃条件下行核酶体外切割反应。结果 核酶在体外37℃反应1h，可将98％的底物RNA(255nt)切割为168nt和87nt2个片段。结论 特异性切割AR的锤头状核酶在体外对底物RNA有良好的切割活性，为应用核酶阻断雄激素受体基因表达奠定了基础。     

反义寡核苷酸阻断雄激素受体表达对前列腺癌细胞生长活性的影响

目的　探讨反义技术阻断雄激素受体 (AR)基因表达对前列腺癌细胞生长的影响。　方法　设计、合成针对AR基因反义寡核苷酸 (ASODN) ,在脂质体介导下转染前列腺癌细胞。采用RT PCR方法检测AR基因表达 ;MTT比色法检测细胞增殖活性 ;透射电镜、TUNEL检测细胞凋亡 ;流式细胞术分析细胞周期时相。　结果　 0 .5～ 2 .0 μmol LASODN作用 12～ 36h ,癌细胞ARmRNA水平降低 10 .45 %～ 82 .10 %(P <0 .0 5 ) ,细胞增殖活性抑制 11.8%～ 5 6 .9%(P <0 .0 5 ) ,细胞周期阻滞于G2 M期 ,部分细胞呈现典型凋亡形态学改变 ,凋亡比率为 34.2 5 %(P <0 .0 1)。　结论　应用ASODN封闭AR基因表达能抑制前列腺癌细胞体外生长活性 ,有望成为前列腺癌基因治疗的有效策略。     

抗Fas核酶抑制Fas介导的小鼠肝脏细胞凋亡的研究

目的 研究抗Fas锤头状核酶对原代培养的小鼠肝脏细胞Fas表达及其凋亡的影响。方法 采用胶原酶灌流法分离小鼠肝脏细胞，同时构建了针对Fas mRNA的锤头状核酶真核质粒，经纳米载体Effectene将其导入肝细胞，通过RT-PCR、Western blot检测细胞Fas的表达，以Caspase-3活性检测试剂盒测转染前后细胞Caspase-3活性的变化，Annexin V-FITC凋亡检测试剂盒测转染前后细胞凋亡，MTT法测细胞的增殖。结果 小鼠原代肝细胞上表达Fas，抗Fas核酶能显著降低肝细胞上Fas水平，经抗Fas抗体作用后，转染核酶的细胞Caspase-3活性明显降低，该细胞增殖活性较对照组增强，而凋亡率明显降低。结论 抗Fas核酶能显著降低小鼠原代肝细胞上Fas的表达，使其免于Fas途径的凋亡。     

锤头状核酶靶向抑制HGF／SF的受体c—Met表达可降低前列腺癌细胞的体外侵袭和转移

背景 肝细胞生长因子／扩散因子（HGF／SF）通过激活其酪氨酸激酶受体c—Met,可引发如侵袭和转移等一系列生物学活性。研究已发现,在前列腺癌的发生和进展过程中c—Met过表达。本研究旨在通过转染锤头状核酶抑制人c—Met的表达,来观察其对前列腺癌细胞体外侵袭和转移效应的影响。方法用被修饰的pZeoUl-EcoSpe构建针对c—Met基因的锤头状核酶表达载体Met560,转染前列腺癌细胞DU-145,观察用转染靶向核酶的方法抑制c—Met表达后HGF／SF对前列腺癌细胞的作用。     

核酶切割技术对胆囊癌细胞端粒酶活性抑制作用的研究

目的探讨锤头状核酶切割技术对胆囊癌细胞端粒酶活性的影响及对胆囊癌细胞生长增殖的作用。方法根据拟要切割的胆囊癌端粒酶RNA亚基模板两翼区的序列，体外合成锤头状核酶的基因序列，并构建入pTriEx-4真核表达载体。酶切及测序鉴定正确后，采用脂质转染法导入胆囊癌细胞，观察其对胆囊癌细胞端粒酶活性的影响及对细胞生长、增殖的作用，并设空载体转染组和单纯脂质体转染组作为对照。结果与对照组比较，实验组胆囊癌细胞的端粒酶活性明显减低；核酶基因作用后，G0／G1期细胞比例明显增高，S期细胞比例明显降低；细胞的分裂、增殖受到明显抑制。核酶作用后10d凋亡率为17、10％，13d后凋亡率为31．01％。结论核酶切割技术对胆囊癌细胞端粒酶活性有明显的抑制作用，并能抑制胆囊癌细胞的生长，促进细胞凋亡。     

反基因及反义寡核苷酸体外抗前列腺癌作用的研究

目的探讨三螺旋形成寡核苷酸(TFO)和反义寡核苷酸(ASO)对前列腺癌细胞雄激素受体(AR)表达和细胞增殖的影响。方法设计并合成针对AR基因的TFO和ASO,经脂质体介导转染前列腺癌细胞株LNCaP。MTT法检测细胞增殖活性,RTPCR检测AR基因表达,免疫组化法检测AR蛋白表达,放射免疫分析检测PSA表达。结果转染后24、48、72h,TFO组LNCaP细胞ARmRNA平均表达水平分别为0.25、0.33、0.46,显著低于ASO组的0.44、0.54、0.60,P<0.05;TFO对LNCaP细胞的生长抑制率(51.8%、65.8%、36.2%)显著高于ASO(28.8%、42.6%、17.5%),P<0.05。TFO对AR蛋白表达的抑制作用强于ASO。转染后24hPSA表达水平[(0.5±0.1)ng/ml]显著低于ASO组[(0.8±0.2)ng/ml],P<0.05。结论在LNCaP细胞TFO对AR表达和癌细胞增殖的抑制作用明显强于ASO,反基因策略对于前列腺癌的治疗具有重要研究价值。     

PEG-PLGA纳米粒子介导反基因寡核苷酸体外抗前列腺癌作用研究

目的 验证包载反雄激素受体基因三螺旋形成寡核苷酸(TFO)的聚乙二醇-聚乳酸聚乙醇酸共聚物(PEG-PLGA)纳米粒子(TFO-NPs)对前列腺癌细胞的生长抑制作用.方法 采用改良自乳化溶剂挥发法制备TFO-NPs并进行体外物化表征.将TFO-NPs、脂质体包载的TFO(TFO-Lip)和裸TFO分别转染体外培养的LNCaP细胞,倒置荧光显微镜观察转染效率,MTT法检测细胞增殖活性,RT-PCR和Western blot方法检测AR基因表达.结果 TFO-NPs平均粒径128 nm,载药量为1.02%,包封率为72.28%,在体外具有缓释作用.LNCaP细胞对TFO-NPs和TFO-Lip的摄取率显著高于裸TFO.TFO-NPs和TFO-Lip对LNCaP细胞的抑制率分别为(54.5%±6.2%)和(50.6%±6.1%)显著高于裸TFO,(7.8%±0.8%)(均P＜0.05).TFO-NPs和TFO-Lip处理组LNCaP细胞的雄激素受体表达水平显著低于裸TFO处理组.TFO-NPs和TFO-Lip处理组的转染效率、细胞抑制率和雄激素受体表达水平差异均无显著性.结论 成功制备了TFO-NPs,为反基因治疗前列腺癌的研究提供了有效基因载体.     

锤头状核酶调控CD95介导小鼠皮肤成纤维细胞凋亡

目的　观察抗CD95的锤头状核酶对新生小鼠皮肤成纤维细胞 CD95 表达及其凋亡的影响。方法　采用胶原酶消化法分离新生小鼠皮肤成纤维细胞,并构建了针对 CD95 mRNA的锤头状核酶,经钠米载体 Effectene将其转染至成纤维细胞。通过 RT PCR、流式细胞仪检测成纤维细胞上CD95表达;细胞经抗CD95 抗体(JO2)作用后,通过 Caspase 3 活性检测试剂盒测转染前后细胞Caspase 3活性的变化;MTT法测细胞的增殖;Annexin Ⅴ凋亡检测试剂盒测细胞凋亡。结果　新生小鼠皮肤成纤维细胞上表达CD95,抗CD95核酶能显著降低细胞表面CD95水平;细胞与抗 CD95 抗体孵育后,与空白对照和空载体转染组相比,核酶转染组细胞 Caspase 3 活性和凋亡率明显降低,转染核酶的细胞增殖活性显著增强。结论　抗 CD95 核酶能显著降低新生小鼠皮肤成纤维细胞上CD95表达,使其免于CD95途径的凋亡,为提高皮肤移植物存活提供了实验依据。     

核酶基因抑制肝癌细胞低氧诱导因子-1α表达的实验研究

目的 探讨靶向低氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)核酶基因对肝癌细胞HIF-1α表达的调控.方法 采用脂质体介导的方法,将靶向HIF-1α核酶基因真核表达载体转染肝癌细胞Hep3B2,并予低氧条件诱导.于转染后48 h采用Western Blotting检测Hep3B2细胞中HIF-1α蛋白的表达水平;荧光报告基因方法检测HIF-1转录活性.结果 Hep3B2细胞低氧诱导后,HIF-1α蛋白表达水平、HIF-1转录活性增高(1.0±0.02),转染核酶基因400 μmol/L后48 h,Hep3B2细胞低氧诱导的HIF-1α蛋白表达水平明显下降,HIF-1转录活性下调(0.12±0.025,P＜0.05).结论 核酶基因可特异性抑制低氧诱导的肝癌细胞HIF-1α的表达,降低其转录活性.     

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.     

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.     

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.     

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.     

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.     

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.     

Intrathecal administration of sameridine to patients subjected to arthroscopic knee joint surgery

Background: Sameridine, a new substance with both local anesthetic and opioid effects, was administered intrathecally for the first time to humans, i. e. in patients subjected to arthroscopic knee joint surgery.
Method: A dose-escalating (10, 15, 20 and 25 mg), open study was performed in 33 patients. Only two patients were included in the 25 mg group.
Results: Sameridine provided good quality of surgical anesthesia in all patients except those receiving 10 mg. The maximum level of sensory block, Th5–Th7, was reached within 30 min with a median duration of 3.6–3.9 h. The motor block was more profound with increasing dose, but never lasted longer than the sensory block. The influence on heart rate and blood pressure was minor and atropine and ephedrine were needed in four patients. No clinically significant ECG-changes were detected and no arrhythmias were recorded. Oxygen saturation and respiratory rate did not decrease in a clinically significant way and were not affected by concomitant morphine given i. v. postoperatively. There were few side-effects, the most frequent being mild pruritus (10/33).
Conclusion: Sameridine provided clinically adequate anesthesia for the patients receiving the doses of 15, 20 and 25 mg. Further studies are needed to evaluate the substance and it is of great interest to clinically investigate the opioid component with respect to postoperative analgesia.     

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.