LHR真核表达载体构建及表达

目的构建黄体生成素受体（LHR）真核表达质粒载体和建立稳定高表达LHR的乳腺癌细胞株。方法KpnI／Hpa工双酶切LHR-cDNA质粒载体获得目的基因,定向克隆构建pcDNA3．1（4-）真核表达载体,酶切图谱和序列分析鉴定;重组质粒载体经脂质体转染MCF-7乳腺癌细胞,G418筛选,RT-PCR、细胞内cAMP测定,筛选鉴定高表达LHRMCF-7细胞。结果重组质粒pcDNA3．1（4-）-LHR酶切图谱分析结果、序列分析证明pcDNA3．1（4-）-LHR载体中LHR序列与GenBank的LHR基因序列完全相符。RT-PCR检测MCF-7细胞LHRmRNA,MCF-7细胞内cAMP浓度测定,证实MCF-7细胞高表达LHR。结论构建并转染pcDNA3．1（4-）-LHR真核表达载体,在MCF-7细胞中稳定表达,为探讨hCG对乳腺癌细胞的作用提供实验基础。     

人喉癌细胞端粒酶催化亚基cDNA片段的扩增与克隆

目的 :测定人喉鳞状上皮癌 hep- 2细胞的端粒酶活性水平并获得 hep- 2细胞中 h TERT c DNA片段的克隆。方法 :采用 TRAP- ELISA法测定酶活性水平 ,RT- PCR技术扩增获得目的片段。利用酶切法、PCR法和序列分析法对所获克隆进行检测。结果 :hep- 2细胞具有较高的端粒酶活性水平 ,获得含h TERT c DNA片段的克隆。结论 :利用 RT- PCR法能够从具有较高端粒酶活性水平的 hep- 2细胞中扩增获得 h TERT c DNA片段     

膝骨关节炎关节液和滑膜中IL-18与MMP-13的表达及临床意义

目的：探讨膝骨关节炎(KOA)患者的关节液和滑膜中白细胞介素(IL)-18与基质金属蛋白酶(MMP)-13的表达及其临床意义。方法选取2011年1月至2013年12月期间我院收治的76例KOA患者作为观察组,76例患者中,轻度30例,中度26例,重度20例。所有受试者软骨损伤分度为Ⅱ度26例,Ⅲ度28例,Ⅳ度22例;另选择32例膝关节半月板损伤患者为对照组。检测受试者膝关节关节液、滑膜中IL-18、MMP-13表达情况。结果观察组患者的膝关节关节液和膝关节滑膜中的IL-18、MMP-13水平均高于对照组,差异均有显著统计学意义(P<0.01);随着KOA程度的加重,关节液和滑膜中的IL-18、MMP-13水平呈上升趋势,差异均有显著统计学意义(P<0.01);随着KOA分度的增加,关节液和滑膜中的IL-18、MMP-13水平呈上升趋势,差异均有显著统计学意义(P<0.01);关节液IL-18、MMP-13水平与滑膜IL-18、MMP-13水平分别呈正相关(r=0.361,0.371,P=0.021,0.016);关节液IL-18水平与关节液MMP-13水平呈正相关(r=0.369,P=0.019);滑膜IL-18水平与滑膜MMP-13水平呈正相关(r=0.378,P=0.015);关节液和滑膜中IL-18、MMP-13水平与KOA程度、KOA分度均呈正相关(P<0.05)。结论 MMP-13、IL-18在KOA关节液和滑膜中均高表达;两者呈现出显著正相关,共同在KOA的发病及病情进展中起作用。     

Detection of renal allograft dysfunction with characteristic protein fingerprint by serum proteomic analysis

This study aimed to diagnose renal allograft dysfunction with specific biomarkers by serum proteomic analysis. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) and bioinformatics (support vector machine and leave-one cross validation) were used to analyze serum proteome. Enrolled patients included 38 biopsy-proved acute rejection (BPAR), 10 acute tubular necrosis (ATN), 24 subclinical rejection (SCR) and 29 stable control recipients verified by protocol biopsy. A characteristic protein profile can be detected in each renal allograft dysfunction group. BPAR patients were differentiated from stable patients with markers of 9710.1, 4971, 6675.5, 8563.8, 6709.2, 9319 and 4476.7 Da with high sensitivity and specificity. ATN can be clearly distinguished from BPAR and stable control. Subclinical rejection differentiated from stable control with markers of 9193.1, 2759.1, 8464.6 Da. The independent blind test yielded with high specificity and sensitivity for each group. Serum proteome analysis by SELDI-TOF MS combined with bioinformatics in renal allograft dysfunction is valuable and promising. Specific markers were detected in each group. Identification of these proteins may prove useful as diagnostic markers for allograft dysfunction and better to elucidate the mechanism of acute rejection.     

以手部皮神经伴行血管为蒂的岛状皮瓣的临床应用

目的应用手部皮神经伴行血管蒂岛状皮瓣修复手指指背软组织缺损。方法采用手部桡神经浅支、尺神经手背支伴行血管蒂的岛状皮瓣，即拇指桡、尺侧皮神经伴行血管岛状皮瓣修复拇指；第一掌背皮神经伴行血管岛状皮瓣修复示指；第三、四掌背皮神经伴行血管岛状皮瓣修复中、环指；小指尺背侧皮神经伴行血管岛状皮瓣修复小指。共５种１５块，修复指部皮肤缺损１５例。皮瓣切取的最大面积达５ｃｍ×３ｃｍ。结果１５例皮瓣全部成活，效果满意。结论该类皮瓣血供可靠，创伤小，操作简单。特别适用于一期修复伴有伸肌腱缺损的指背创面     

Therapeutic Effectiveness of Sustained Low‐Efficiency Hemodialysis Plus Hemoperfusion and Continuous Hemofiltration Plus Hemoperfusion for Acute Severe Organophosphate Poisoning

There is no report on the effects of sustained low‐efficiency dialysis (SLED) plus hemoperfusion (HP) (SLED + HP) in patients with acute severe organophosphate (OP) poisoning (ASOPP). This study was designed to compare the therapeutic effectiveness between SLED + HP and continuous hemofiltration (CHF) plus HP (CHF + HP) in patients with ASOPP. In order to assess the two treatment methods, 56 patients with ASOPP were divided into CHF + HP group and SLED + HP group. The biochemical indicators, in‐hospital duration, hemodynamic parameters, Acute Physiology, and Chronic Health Evaluation (APACHE II) score, and survival and mortality rates were compared. In both groups after treatment, the levels of serum creatine kinase isozyme MB, creatine kinase, creatinine, glutamic‐oxalacetic transaminease, and glutamate‐pyruvate transaminase, and the APACHE II scores on the first, second, and seventh day decreased (P < 0.05), whereas the levels of serum acetylcholinesterase increased. The two groups showed no statistical differences in in‐hospital duration, biochemical indicators, APACHE II score, hemodynamic parameters, survival rate, or the mortality rate (P > 0.05). In conclusion, SLED has similar hemodynamic stability to CHF and the two treatment methods have similar effects on ASOPP patients. More importantly, SLED plus HP is relatively economical and convenient for patients with ASOPP in clinical practice.     

术中胃减压对神经内镜经鼻颅底手术后恶心呕吐的影响

目的探讨术中胃减压对行神经内镜经鼻颅底手术患者手术后恶心呕吐(PONV)的影响。方法回顾性分析2019年1—8月在复旦大学附属华山医院神经外科接受神经内镜经鼻颅底手术的357例患者的临床资料。根据术中是否进行胃减压,分为胃减压组(146例)和对照组(211例)。比较两组的临床特征和术后24 h内PONV发生率的差异。对于包括胃减压在内的各因素对行神经内镜经鼻颅底手术患者PONV发生的影响,采用单因素和多因素logistic回归分析法探讨。结果所有患者术后24 h内的PONV发生率为11.8%(42/357)。胃减压组的女性比例[58.2%(85/146)对比46.4%(98/211),P=0.029]和Apfel评分为2~3分的比例[64.4%(94/146)对比50.2%(106/211),P=0.008]均高于对照组,而胃减压组PONV的发生率低于对照组[7.5%(11/146)对比14.7%(31/211),P=0.039]。单因素logistic回归分析结果提示,术中采用脂肪填塞修补脑脊液漏、术中采用黏膜瓣修补脑脊液漏、术中胃减压和术中失血量是神经内镜经鼻颅底手术患者PONV发生的影响因素(均P<0.05)。多因素logistic回归分析结果提示,术中胃减压(OR=0.397,95%CI:0.185~0.852,P=0.018)和术中失血量(OR=1.133,95%CI:1.026~1.250,P=0.013)为神经内镜经鼻颅底手术患者PONV发生的独立影响因素。结论实施术中胃减压可降低神经内镜经鼻颅底手术患者PONV的发生率。     

Lack of therapeutic effects of gabexate mesilate on the hepatic encephalopathy in rats with acute and chronic hepatic failure

Background and Aim: Inflammation plays a pivotal role in liver injury. Gabexate mesilate (GM, a protease inhibitor) inhibits inflammation by blocking various serine proteases. This study examined the effects of GM on hepatic encephalopathy in rats with acute and chronic liver failure. Methods: Acute and chronic liver failure (cirrhosis) were induced by intraperitoneal TAA administration (350 mg/kg/day for 3 days) and common bile duct ligation, respectively, in male Sprague‐Dawley rats. Rats were randomized to receive either GM (50 mg/10 mL/kg) or saline intraperitoneally for 5 days. Severity of encephalopathy was assessed by the Opto‐Varimex animal activity meter and hemodynamic parameters, mean arterial pressure and portal pressure, were measured (only in chronic liver failure rats). Plasma levels of liver biochemistry, ammonia, nitrate/nitrite, interleukins (IL) and tumor necrosis factor (TNF)‐α were determined. Results: In rats with acute liver failure, GM treatment significantly decreased the plasma levels of alanine aminotransferase (P = 0.02), but no significant difference of motor activity, plasma levels of ammonia, IL‐1β, IL‐6, IL‐10 and TNF‐α or survival was found. In chronic liver failure rats, GM significantly lowered the plasma TNF‐α levels (P = 0.04). However, there was no significant difference of motor activity, other biochemical tests or survival found. GM‐treated chronic liver failure rats had higher portal pressure (P = 0.04) but similar mean arterial pressure in comparison with saline‐treated rats. Conclusions: Chronic GM treatment does not have a major effect on hepatic encephalopathy in rats with TAA‐induced acute liver failure and rats with chronic liver failure induced by common bile duct ligation.     

Simvastatin effects on portal‐systemic collaterals of portal hypertensive rats

Background and Aim: Portal‐systemic collateral vascular resistance and vasoconstrictor responsiveness are crucial in portal hypertension and variceal bleeding control. Statins enhance vasodilators production, but their influence on collaterals is unknown. This study aimed to survey the effect of simvastatin on collaterals. Methods: Partially portal vein‐ligated rats received oral simvastatin (20 mg/kg/day) or distilled water from ?2 to +7 day of ligation. After hemodynamic measurements on the eighth postoperative day, baseline perfusion pressure (i.e. an index of collateral vascular resistance) and arginine vasopressin (AVP, 0.1 nM–0.1 µM) responsiveness were evaluated with an in situ perfusion model for collateral vascular beds. RT‐PCR of endothelial NO synthase (eNOS), inducible NOS (iNOS), cyclooxygenase‐1 (COX‐1), COX‐2, thromboxane A₂ synthase (TXA₂‐S) and prostacyclin synthase genes was performed in parallel groups for splenorenal shunt (SRS), the most prominent intra‐abdominal collateral vessel. To determine the acute effects of simvastatin, collateral AVP response was assessed with vehicle or simvastatin. SRS RT‐PCR of eNOS, iNOS, COX‐1, COX‐2 and TXA₂‐S, and measurements of perfusate nitrite/nitrate, 6‐keto‐PGF1_α and TXB₂ levels were performed in parallel groups without AVP. Results: Acute simvastatin administration enhanced SRS eNOS expression and elevated perfusate nitrite/nitrate and 6‐keto‐PGF1_α concentrations. Chronic simvastatin treatment reduced baseline collateral vascular resistance and portal pressure and enhanced SRS eNOS, COX‐2 and TXA₂‐S mRNA expression. Neither acute nor chronic simvastatin administration influenced collateral AVP responsiveness. Conclusion: Simvastatin reduces portal‐systemic collateral vascular resistance and portal pressure in portal hypertensive rats. This may be related to the enhanced portal‐systemic collateral vascular NO and prostacyclin activities.