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1.
目的 构建针对端粒酶hTERT的双H1启动子SECs,并探讨其转录生成的siRNA对HepG2细胞端粒酶活性的抑制作用.方法 利用融合PCR技术,构建2个针对人端粒酶hTERT基因外显子不同片断的双H1启动子SECs,分别转染人肝癌细胞HepG2,转录生成siRNAs.用TRAP法和PCR-EIA法检测端粒酶活性,分析双H1启动子SECs对HepG2细胞端粒酶表达的干扰作用.结果 成功构建针对hTERT的双H1启动子SECs,其转录产物siRNA可明显抑制HepG2细胞端粒酶的活性.结论 siRNA SECs对HepG2细胞端粒酶hTERT基因表达有明显的干扰作用,为临床开展对肿瘤端粒酶基因干扰抑制的实验研究奠定了基础.  相似文献   

2.
目的:探讨针对端粒酶hTERT基因的小片段发夹RNA对人肿瘤细胞(HeLa)端粒酶基因的干扰及对肿瘤端粒酶的抑制作用。方法:体外合成制备shRNA,并用磷酸钙转染法转染HeLa细胞,分别用TRAP-银染法和PCR-EIA法检测端粒酶活性。结果:将制备的46个碱基的小片段发夹RNA转染HeLa细胞后端粒酶活性明显下降。结论:shRNA对肿瘤端粒酶hTERT基因表达有明显的干扰作用, 可望为临床开展对其他肿瘤端粒酶基因干扰抑制的实验研究奠定基础。  相似文献   

3.
目的 应用小干扰RNA(siRNA)技术特异性抑制人端粒酶逆转录酶(hTERT)基因在胃癌SGC7901细胞中的表达,观察其对细胞增殖的影响.方法 hTERT基因的RNA干扰表达重组体用脂质体介导方法转染SGC7901细胞.荧光定量聚合酶链反应(PCR)检测hTERT基因表达.TRAP-PCR法检测端粒酶活性、噻唑蓝(MTT)比色法检测细胞增殖.结果 SGC7901细胞转染前后hTERT基因的表达强度分别为:转染pU6组平均表达量为6.5×105拷贝/μg RNA;转染pU6-hTERT-siRNA Ⅰ组平均表达量为4.1 × 104拷贝/μg RNA;转染pU6-hTERT-siRNAⅡ组平均表达量为5.4×104拷贝/μg RNA.转染pU6组与其余两组P值<0.01;转染pU6-hTERT-siRNA Ⅰ组和转染pU6-hTERT-siRNAⅡ组P值>0.05.端粒酶活性下降,细胞生长速度明显减慢.结论 小干扰RNA能有效抑制SGC7901细胞中hTERT基因表达,hTERT基因表达下调影响SGC7901细胞增殖.  相似文献   

4.
目的 观察RNA干扰对大肠癌细胞中人端粒酶逆转录酶基因(hTERT gene)的抑制效应.方法 大肠癌细胞株HT-29分为Control组、N-hTERT组、Lipofectamine组和sihTERT组;将靶向于hTERT基因的小干扰RNA,转染大肠癌细胞;分别检测大肠癌细胞转染前后的端粒酶活性,hTERTmRNA和蛋白水平,细胞生长增殖和凋亡情况.结果 sihTERT转染大肠癌细胞效率为60%;sihTERT组的端粒酶活性值、hTERT mRNA表达、hTERT蛋白表达、细胞凋亡率分别为0.73±0.14、0.47±0.08、0.37±0.07、49.5%,sihTERT组与其他三组比较差异有统计学意义(P<0.01).结论 靶向于hTERT的siRNA能特异性沉默大肠癌细胞hTERT基因,下调hTERT基因mRNA及蛋白表达水平,降低端粒酶活性,抑制癌细胞生长增殖从而促进大肠癌细胞凋亡.  相似文献   

5.
目的 构建人端粒酶逆转录酶(hTERT)基因启动子调控Fas相关死亡结构域蛋白(FADD)肿瘤细胞特异性表达载体,观察其对人结肠癌细胞凋亡的作用.方法 利用hTERT基因核心启动子和FADD基因片段,构建hTERT基因启动子调控FADD 的肿瘤细胞特异性表达载体.用脂质体转染法,将其分别转染人结肠癌细胞和正常细胞,观察人结肠癌细胞和正常细胞的端粒酶活性、细胞周期及凋亡.结果 hTERT基因核心启动子调控FADD 表达载体,在所检测的肿瘤细胞中具有明显的转录活性;而在正常细胞中则无明显的转录活性.Colon320细胞、LoVo细胞、SW480细胞与WI-38细胞凋亡率在不同转染时间比较,差异有统计学意义(P均<0.01).WI-38转染pLNCX-hTERT-FADD与pLNCX-hTERT-GFP在24、48、72、96、120h凋亡率比较,差异均无统计学意义(P均>0.05).结论 hTRET基因核心启动子具有肿瘤特异性,构建hTERT基因核心启动子调控FADD 表达载体可能是一种新的肿瘤治疗途径.  相似文献   

6.
目的 研究野生型p53基因对人胃癌细胞端粒酶逆转录酶(hTERT)mRNA转录及端粒酶活性的影响。方法 将含人野生型p53基因的pcDNA3—p53转染人胃癌细胞系BGG—823,通过半定量逆转录—聚合酶链反应(RT—PCR)检测转染后细胞hTERT基因mRNA的转录,聚合酶链式反应结合酶联免疫吸附(PCR ELISA)方法检测端粒酶活性。结果 转染野生型p53基因后48、72h BGC-823细胞的hTERT基因mRNA转录量由对照组的( )下降为( )和( );转染前BGC—823细胞表达高水平的端粒酶活性(3.049),而转染48、72h后的端粒酶活性分别为1.678和0.757。结论 野生型p53基因能够显著抑制人胃癌细胞hTERT基因mRNA的转录,通过下调hTERT、基因mRNA的转录来抑制胃癌细胞的端粒酶活性。  相似文献   

7.
目的 探讨转染缺失突变的人端粒酶逆转录酶 (hTERT)基因对膀胱癌细胞株T2 4端粒酶活性和体外增殖的影响 ,为膀胱肿瘤基因治疗提供新的基因靶点。 方法 采用DNA 磷酸钙共沉淀法 ,将绿色荧光蛋白基因标记的含突变型hTERT真核表达载体 pEGFP hTERT导入人膀胱癌细胞株T2 4中。应用荧光显微镜、端粒酶PCR ELISA法、与衰老相关的 β 半乳糖苷酶染色、软琼脂集落形成试验、裸鼠皮下成瘤试验等方法动态观察转染细胞中端粒酶活性及对细胞恶性表型的影响。 结果 在转染 pEGFP hTERT细胞中可见与突变型hTERT基因融合的绿色荧光蛋白稳定表达于细胞核内 ,转染细胞端粒酶活性降低 ,衰老相关 β 半乳糖苷酶表达增加 ,软琼脂中集落形成减少 ,裸鼠成瘤性降低。与转染空载体组及未转染组细胞相比 ,差别有显著性意义 (P <0 .0 5 )。 结论 转染突变型人端粒酶逆转录酶基因hTERT能抑制膀胱癌细胞T2 4的端粒酶活性 ,促进其衰老并逆转膀胱癌细胞的恶性表型 ,对膀胱肿瘤基因治疗具有潜在的临床应用价值  相似文献   

8.
目的了解端粒酶逆转录酶(hTERT)、siRNA对GBC-SD细胞代谢、侵袭和端粒酶活性、hTERT mRNA、hTERT蛋白、β-catenin mRNA、β-catenin蛋白的作用,并探讨其相关机制。方法在质粒pGCsi—H1/GFP-hTERT干扰后,采用端粒酶TRAP、半定量逆转录-聚合酶链反应(RT—PCR)和Western blot方法检测GBC-SD端粒酶活性、hTERT基因和β—catenin基因mRNA、蛋白质表达水平。噻唑蓝(MTT)比色法检测琥珀酸脱氢酶(SDH)活性、运用Transwell小室了解癌细胞侵袭情况。结果pGCsi—H1/GFP—hTERT对GBC-SD端粒酶活性、SDH活性、侵袭、hTERT mRNA、hTERT蛋白均有抑制作用并随浓度升高而增强。pGCsi—HI/NEGative对上述指标无明显抑制作用,与pGCsi—H1/GFP—hTERT抑制作用比较差异有统计学意义(P〈0.01)。结论pGCsi—H1/GFP—hTERT可以抑制端粒酶、SDH活性及其侵袭力,其机制与hTERT mRNA及蛋白表达降低有关。  相似文献   

9.
目的:构建针对hTERT基因的人工miRNA表达框架,并验证其对HepG2细胞端粒酶活性的抑制作用。 方法:应用融合PCR技术针对不同位点设计并构建3个靶向hTERT的人工miRNA表达框架,对各表达框架鉴定后,将其单独或两种共转染HepG2细胞,采用TRAP-银染法和TRAP-二聚体蝎形探针荧光定量PCR法检测细胞端粒酶活性。 结果:3个针对hTERT基因的人工miRNA表达框架均成功构建,单独或共转染HepG2细胞后,HepG2细胞的端粒酶活性均被不同程度的抑制,且两种表达框架共转染的抑制作用明显大于单独转染,差异均有统计学意义(均P<0.05)。 结论:靶向hTERT基因的人工miRNA表达框架能有效而特异抑制HepG2细胞端粒酶活性,多位点联合抑制是一种有效的实验方案。  相似文献   

10.
反义人端粒酶逆转录酶基因转染对人胃癌细胞系的影响   总被引:2,自引:0,他引:2  
目的探讨反义人端粒酶(hTERT)基因治疗的可行性。方法构建hTERT基因的反义表达载体,经脂质体介导转染人未分化胃癌细胞系HGC-27,通过Southernblot检测外源反义基因的整合;RT-PCR及DNA测序法检测反义基因的转录;RT-PCR半定量方法检测被封闭目的基因mRNA的转录水平;TRAP及PCRELISA方法检测细胞的端粒酶活性;流式细胞仪检测细胞周期变化。结果外源反义hTERT基因已整合入细胞并获稳定转录,且能显著封闭目的基因转录的mRNA,并显著抑制HGC-27细胞的端粒酶活性,抑制HGC-27细胞的增殖并促进其凋亡。结论端粒酶反义hTERT基因可有效地应用于胃癌的基因治疗。  相似文献   

11.
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 (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.  相似文献   

12.
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.  相似文献   

13.
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.  相似文献   

14.
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.  相似文献   

15.
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.  相似文献   

16.
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.  相似文献   

17.
Background: The efficacy of intraoperative salvage and washing of wound blood and the predictors of allogeneic red cell transfusions in prosthetic hip surgery are insufficiently known.
Methods: In 96 patients, undergoing primary or revision surgery, salvaged and washed red cells and, if necessary, allogeneic blood were used to keep haematocrit not lower than 33%. The bleeding of red cells during hospital stay was calculated from the red cell balance. The preoperative red cell reserve (millilitres of red cells in excess of a haematocrit of 33%) was estimated and the difference between this volume and the total bleeding of red cells was retrospectively used to classify patients with regard to the need for red cells. Stepwise regression analysis was used to define patient-related variables associated with allogeneic blood transfusion.
Results: Preoperative knowledge of the type of operation (primary, revision), the preoperative red cell reserve, and the body mass could predict roughly half of the need for banked blood (r2=0.45). Only one-third of the total bleeding of red cells was retransfused. For complete avoidance of allogeneic blood, autotransfusion was most effective in patients with a moderate need (0–4 u). However, 32% of such patients required allogeneic blood.
Conclusions: Autotransfusion has a limited efficacy to decrease the need for allogeneic blood, and other blood-saving methods should be added for this purpose. It is difficult to predict the need for allogeneic blood preoperatively.  相似文献   

18.
目的    观察缺氧对肾小管上皮细胞分泌外泌体的影响,探讨外泌体在缺氧致肾脏损伤中的作用及机制。 方法    (1)常氧(21% O2)及缺氧(1% O2)分别处理大鼠肾小管上皮细胞(NRK-52E)48 h,收集细胞上清液并使用高速梯度离心法分离外泌体。采用透射电镜、纳米示踪分析、Western印迹、蛋白浓度定量鉴定并比较两组外泌体的基本特性。(2)在共培养实验中,以不同浓度(1、10、50、100、300 mg/L)的常氧外泌体、缺氧外泌体分别干预脂多糖(LPS)诱导的大鼠原代腹腔巨噬细胞,使用实时荧光定量PCR与酶联免疫吸附试验(ELISA)法分别检测巨噬细胞白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)、诱导型氮氧化物合酶(iNOS)水平;使用Western印迹法检测巨噬细胞磷酸化(p)STAT/STAT及细胞因子信号传导抑制蛋白1(SOCS1)的蛋白表达;最后,使用实时荧光定量PCR法检测常氧外泌体与缺氧外泌体中炎性反应相关微RNA(microRNA,miR)的表达差异。 结果    (1)离心得到的囊泡具有外泌体典型的结构,粒径小于150 nm,表达外泌体标志蛋白CD63,说明分离得到外泌体。缺氧对肾小管上皮细胞分泌的外泌体形态、粒径分布比例无明显影响,但提高了外泌体的分泌量。(2)缺氧外泌体相比于常氧外泌体促进了LPS诱导的M1型巨噬细胞IL-6、TNF-α、iNOS 的表达和分泌(均P<0.01),同时提高STAT的磷酸化水平并减少SOCS1的蛋白表达(均P<0.01);对炎性反应相关microRNA检测发现缺氧外泌体中miR-155、miR-27a表达量较常氧外泌体明显升高(P<0.05)。 结论    缺氧可改变外泌体的生物学功能,表现为协同促进LPS诱导的M1型巨噬细胞的表型转化,这可能是慢性肾脏病微炎性反应状态持续的原因之一。  相似文献   

19.
Abstract While flexible-leaflet, central-flow prosthetic heart valves promise relief from anticoagulation therapy, they continue to be restricted by inadequate durability. In consequence, a novel trileaflet valve, made entirely from polyurethane, has been developed. A batch of 6 consecutively manufactured polyurethane valves was subjected to hydrodynamic function and accelerated fatigue testing. Computerized data acquisition and control systems have been introduced to improve valve testing methodologies. In terms of hydrodynamic function, the polyurethane valve demonstrates transvalvular pressure gradients similar to those for a bioprosthetic valve (Carpentier-Edwards) and levels of retrograde flow significantly less than those for either the bioprosthetic valve or a bileaflet mechanical valve (St Jude Medical). The equivalent of 10 years of cycling without failure has been exceeded by all 6 polyurethane valves in accelerated fatigue tests with 2 valves remaining intact after 674 million cycles (equivalent to approximately 17 years) in continuing tests. Highspeed photography revealed considerable differences in leaflet motion between valves cycled at accelerated and physiological rates.  相似文献   

20.
Background: Ventilation during interventional rigid bronchoscopy (IRB) under general anaesthesia (jet ventilation, positive pressure ventilation and spontaneous assisted ventilation) may offer some difficulties. This study compares the effectiveness during IRB of intermittent negative pressure ventilation (INPV) and spontaneous assisted ventilation (SAV). Methods: Thirty-eight patients submitted to IRB were randomised into two groups: SAV or INPV. All patients received a total intravenous anaesthesia; INPV patients were paralysed. Pre-and intra-operative arterial blood gases and O2 flow through a rigid bronchoscope were assessed. The endoscopist applying a subjective score evaluated the operating conditions. Results: Patients of the INPV group, as compared to the SAV group, required a lower dosage of fentanyl (2.6 ± 1.8 (μg · kg?1· h?1 vs. 6.6 ± 4.8 μg · kg?1· h?1), a lower O2 supply (3.3 ± 2.8 1/min vs. 11.6 ± 3.4 1/min), a shorter recovery time (5.4 ± 2.9 min vs. 9.8 ± 7.1 min) and no manually assisted ventilation (0 ± 0 vs. 1 ± 1.1 nd?/procedure). Intraoperative PaCO2 was higher in the SAV (8.1 ± 1.3 kPa) than in the INPV group (5.0 ± 1.6 kPa) and intraoperative pH differed in the two groups (7.26 ± 0.05, SAV vs. 7.47 ± 0.08, INPV). Operating conditions, as assessed by a subjective score, were considered better with INPV than with SAV (4.9 vs. 4.3). Conclusions: As compared to SAV, INPV in paralysed patients during IRB reduces administration of opioids, shortens recovery time, prevents respiratory acidosis, excludes the need for manually assisted ventilation, reduces 02 need and affords optimal surgical conditions. INPV appears a safe, non-invasive and effective ventilatory management during IRB.  相似文献   

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