构建由缺氧反应元件修饰的hTERT核心启动子引导FCY1基因表达的复制缺陷型腺病毒

目的：构建由缺氧反应元件串联重复体修饰的人端粒酶逆转录酶（hTERT)核心启动子引导酵母胞嘧啶脱氨酶FCY1基因表达的复制缺陷型重组腺病毒。方法：人工设计缺氧反应元件串联重复序列，克隆后插入hTERT启动子上游并经测序证实。将修饰后的hTERT启动子和酵母胞嘧啶脱氨酶基因FCY1插入穿梭质粒，与辅助质粒共转染HEK293细胞，重组产生复制缺陷型腺病毒。结果：获得了由3倍和6倍缺氧反应元件修饰的hTERT核心启动子引导FCY1基因表达的复制缺陷型重组腺病毒。结论：基于Cre重组酶／loxP的腺病毒载体构建系统操作简便、重组效率高。     

修饰hTERT核心启动子靶向转录FCY1的重组腺病毒的构建及鉴定

目的 :构建并鉴定雌激素反应元件与缺氧反应元件共修饰人端粒酶逆转录酶 (hTERT)核心启动子靶向转录酵母胞嘧啶脱氨酶 (FCY1)的复制缺陷型重组腺病毒 .方法 :构建由雌激素反应元件 (ERE)和缺氧反应元件 (HRE)串联重复序列共修饰的hTERT核心启动子 ,将该启动子序列与酵母胞嘧啶脱氨酶 (FCY1)基因插入穿梭质粒 ,用穿梭质粒与辅助质粒共转染HEK 2 93细胞 ,获得重组腺病毒载体Ad EHhT FCY1,经PCR鉴定证实后 ,采用终点稀释试验测定病毒滴度 .结果 :经 2 4HRE与 8ERE共修饰的hTERT核心启动子序列经正反向测序均正确 ;穿梭质粒和辅助质粒共转染的HEK 2 93细胞 ,在转染后 7～ 10d出现了典型的细胞病变反应 (CPE) ,PCR反应能扩增出 12 2bp的目的片段 ,终点稀释试验测定得到的病毒滴度为 6 2× 10 7pfu·mL-1.结论 :成功构建并获得高滴度的ERE与HRE串联重复序列共修饰的hTERT核心启动子引导FCY1转录的复制缺陷型重组腺病毒 .     

负载hTERT复制缺陷型腺病毒的包装及鉴定

目的:构建负载人端粒酶逆转录酶(hTERT)基因的复制缺陷型腺病毒.方法:将克隆有正义hTERTcDNA的腺病毒穿梭质粒pDC315-hTERT与腺病毒包装质粒pBHGlox-DeltaE1,3Cre共转染人胚肾293细胞(HEK293),包装负载hTERT基因的复制缺陷型腺病毒表达载体(Ad-hTERT).对Ad-hTERT扩增、纯化并浓缩后,进一步行感染性鉴定、电镜鉴定及双引物PCR鉴定.结果:纯化浓缩后的Ad-hTERT滴度可达5×1012pfu/L.电镜下可见在HEK293细胞中形成的病毒颗粒,PCR双引物鉴定Ad-hTERT可扩增出Ad及hTERT特异片段,而对照则不能扩出hTERT片段.结论:成功构建了负载hTERT基因的复制缺陷型腺病毒.     

新型Smad7 重组腺病毒载体的构建

目的构建鼠Smad7重组复制缺陷型腺病毒载体(AdSmad7).方法采用常规分子生物学方法,抽提SD大鼠肝脏总RNA,RT PCR获得鼠Smad7 cDNA片段,插入腺病毒穿梭载体质粒pAdDTrack CMV启动子下游,构建重组穿梭载体pAdTrack CMV Smad7,线性化后与腺病毒骨架载体质粒AdEasy 1在细菌BJ5183内同源重组,鉴定后在HEK293细胞包装成重组腺病毒AdSmad7.RT PCR检测AdSmad7在HEK293中的表达情况.结果该重组缺陷型腺病毒载体经测序、限制性内切酶酶切分析、PCR等鉴定,与预期结果一致;重组腺病毒质粒转染HEK293细胞后3 d可观察GFP明显表达;RT PCR检测证实Smad7表达增强.结论利用新型腺病毒载体AdEasy系统可快速构建同时表达GFP和Smad7的重组复制缺陷型腺病毒AdSmad7,为进一步研究TGFβ信号转导和肝纤维化的基因治疗奠定基础.     

新型Smad7 重组腺病毒载体的构建

目的构建鼠Smad7重组复制缺陷型腺病毒载体(AdSmad7)。方法采用常规分子生物学方法，抽提SD大鼠肝脏总RNA，RT-PCR获得鼠Smad7 cDNA片段，插入腺病毒穿梭载体质粒pAdDTrack CMV启动子下游，构建重组穿梭载体pAdTrackCMV-Smad7，线性化后与腺病毒骨架载体质粒AdEasy-l在细菌BJ5l83内同源重组，鉴定后在HEK293细胞包装成重组腺病毒AdSmad7。RT-PCR检测AdSmad7在HEK293中的表达情况，结果该重组缺陷型腺病毒载体经测序、限制性内切酶酶切分析、PCR等鉴定，与预期结果一致；重组腺病毒质粒转染HEK293细胞后3d可观察GFP明显表达；RT-PCR检测证实Smad7表达增强。结论利用新型腺病毒载体AdEasy系统可快速构建同时表达GFP和Smad7的重组复制缺陷型腺病毒AdSmad7，为进一步研究TGFl3信号转导和肝纤维化的基因治疗奠定基础。     

hTERT启动子调控腺病毒介导的CD基因治疗卵巢癌的实验研究

目的:构建人端粒酶逆转录酶(hTERT)启动子驱动的含胞嘧啶脱氨酶(CD)基因的复制缺陷型腺病毒载体,使CD基因只能在端粒酶阳性的细胞表达.方法:采用细胞内同源重组法构建出hTERT启动子调控的携带CD基因的复制缺陷型腺病毒Ad-hTERTp-CD,经PCR鉴定正确后进行扩增、纯化和滴度测定.分别将腺病毒感染人卵巢癌细胞株SKOV3及人胚肺成纤维细胞,MTT法检测受染细胞的存活率.结果:构建的重组腺病毒中带有hTERT启动子及CD基因.用不同滴度的重组腺病毒以及不同浓度的5-FC作用于SKOV3细胞后,MTT法检测到细胞的存活率随着病毒滴度和5-FC浓度的增加而不断降低;而同样的处理对人胚肺成纤维细胞的也有部分杀伤作用,但远较SKOV3细胞弱,差异有统计学意义(P＜0.01).结论:本实验构建的由hTERT启动子调控的携带CD基因的复制缺陷型腺病毒Ad-hTERTp-CD对SKOV3细胞具有靶向杀伤的能力.     

Betatrophin重组腺病毒过表达载体的构建

［摘要］目的: 应用基因同源重组技术,构建及鉴定携带小鼠betatrophin基因Gm6484(NM_001080940)的复制缺陷型重组腺病毒载体,为进一步研究betatrophin的功能奠定基础。方法: 根据基因库中登录的小鼠betatrophin基因Gm6484(NM_001080940)序列,经化学合成得到含BamHⅠ/ AgeⅠ酶切位点的小鼠betatrophin基因全长cDNA质粒,将其酶切后插入到带有增强型绿色荧光蛋白（enhanced green fluorescent protein,EGFP）基因的GV314载体CMV MCS 3FLAG SV40 EGFP中,得到重组病毒穿梭质粒pGV314 betatrophin;经BamHⅠ/ AgeⅠ双酶切后,与线性化的pDC315病毒基因组质粒体外同源重组,构建含有目的基因的腺病毒载体Ad betatrophin,酶切线性化重组腺病毒质粒后转染HEK293T细胞包装成重组病毒颗粒,经在HEK293T细胞反复扩增数代后,利用聚合酶链式反应（PCR）、蛋白质印迹法及测序鉴定重组的腺病毒。结果： 阳性克隆质粒Ad betatrophin在HEK293T细胞中成功包装出重组病毒,经PCR、蛋白质印迹及测序检测表明重组腺病毒包装成功。结论： 成功构建了携带小鼠betatrophin基因的复制缺陷型重组腺病毒过表达载体。     

hTERT核心启动子驱动的条件复制型腺病毒载体的构建及鉴定

目的构建并鉴定一种新型人端粒酶逆转录酶(hTERT)启动子驱动的条件复制型腺病毒载体。方法采用PCR的方法克隆腺病毒E1区基因(E1A,E1B55K)、E1B的启动子(E1Bp)以及人端粒酶逆转录酶亚基启动子(hTERTp),以及2个目的基因:人GM-CSF和GFP(GreenFluorescenceProtein,GFP)。采用一系列分子生物学方法构建pShuttle-GFPSV55K穿梭质粒,应用此穿梭质粒与AdEasy-1腺病毒DNA在BJ5183细菌内重组得到重组腺病毒质粒,将其转染293细胞获得Ad-GFPSV55K重组腺病毒。PCR法鉴定重组腺病毒,噬斑形成实验测病毒滴度。结果经过酶切鉴定成功地构建了分别携带GM-CSF基因和GFP报告基因的腺病毒载体,病毒滴度为7×1010pfu/ml。结论成功获得由hTERT启动子驱动的条件复制型腺病毒载体,为肿瘤的基因治疗打下良好的基础。     

携带内皮型一氧化氮合酶基因重组腺病毒载体的构建

目的：构建带有内皮型一氧化氮合酶（eNOS)基因的重组腺病毒载体。方法：将eNOS cDNA克隆于腺病毒穿梭质粒pCA14，得到重组质粒pCA14-CMV-eNOS。采用磷酸钙－DNA沉淀法将质粒pCA14-CMV-eNOS与腺病毒拯救质粒pBHG10共转染293细胞，通过同源重组，生成带有eNOS基因的复制缺陷型重组腺病毒载体（AdCMVeNOS)。eNOScDNA重组进入腺病毒E1区并受CMV启动子控制。结果：经形态学、病毒DNA酶切、PCR和RT－PCR等方法的鉴定，证实了载体构建的正确性。结论：带有eNOS基因的重组腺病毒载体的成功构建，为心血管等疾病的基因治疗打下了基础。     

人FRNK重组腺病毒的构建

目的:应用AdEasy复制缺陷型腺病毒载体系统构建人FRNK基因重组腺病毒,并在HEK293细胞中扩增制备重组病毒。方法:把人FRNK基因克隆入腺病毒穿梭载体pAdTrack-CMV中,构建腺病毒质粒pAdTrack-CMV-hFRNK,经PmeⅠ内切酶酶切成线性化后,采用电穿孔转化将其转化到含有腺病毒骨架载体pAdEasy-1的感受态大肠杆菌BJ5183中,通过卡那霉素筛选获得阳性腺病毒重组质粒。再将获得的腺病毒重组质粒转染到HEK293细胞进行包装,获得人FRNK重组腺病毒pAdhFRNK。荧光显微镜下观察绿色荧光蛋白的表达。结果:经酶切和绿色荧光蛋白表达证明了hFRNK基因的重组腺病毒载体pAdhFRNK构建成功,并制备出高滴度的重组病毒。结论:成功构建携带人FRNK基因的重组腺病毒pAdhFRNK,为研究FRNK的基因治疗奠定了基础。     

Value of D-Dimers in patients with acute aortic dissection

Objective: To evaluatel the value of D-dimers in patients with acute aortic dissection (AAD). Methods: This study consisted of 16 patients with AAD and 27 non-AAD patients. Serum D-dimets were measured by Sta-Liatest D-DI immunoturbidimetric assay. Results: D-dimer level was higher (P ＜ 0.001) in patients with AAD(7.91 ± 5.52 μg/ml) than that in non- AAD group(1.57±1.24 μg/ml). D-dimer was positive (＞0.4 μg/ml) in all patients with AAD and in 10 control group patients (37%). Among patients with acute AAD, D-dimers tended to be higher in Stanford A than in Stanford B (8.67 ± 4.31 μg/ml vs. 3.24±1.27 μg/ml, P ＜0.01). D-dimer values tended to be higher in more extended disease(3.84 ± 1.65 μg/ml, 8.57 ± 3.58 μg/ml and 11.87 ± 5.69 μg/ml in thoracic aorta, thoracic and abdominal aorta, thoracic and abdominal aorta and iliacal arteries, respectively, P ＜ 0.05 for both 8.57 ± 3.58 and 11.87 ± 5.69 vs. 3.84 ± 1.65 ). Including the control group into the analysis, we found a sensitivity of 100%, a negative predictive value of 100%, and a specificity of 66% and a positive predictive value of 64% for D-dimer in diagnosis of AAD in our patients with suspected AAD. Conclusion: D-dimer was elevated in patients with AAD. A negative D-dimer test result could be useful in excluding AAD.     

Research of combined liver-kidney transplantation model in rats

Objective： To set up a simple and reliable rat model of combined liver-kidney transplantation. Methods： SD rats served as both donors and recipients. 4℃ sodium lactate Ringer＇s was infused from portal veins to donated livers,and from abdominal aorta to donated kidneys, respectively. Anastomosis of the portal vein and the inferior vena cava （IVC） inferior to the right kidney between the graft and the recipient was performed by a double cuff method, then the superior hepatic vena cava with suture. A patch of donated renal artery was anastomosed to the recipient abdominal aorta. The urethra and bile duct were reconstructed with a simple inside bracket. Results： Among 65 cases of combined liver-kidney transplantation, the success rate in the late 40 cases was 77.5%. The function of the grafted liver and kidney remained normal. Conclusion： This rat model of combined liver-kidney transplantation can be established in common laboratory conditions with high success rate and meet the needs of renal transplantation experiment.     

APPLICATION OF LORNOXICAM TO PATIENT-CONTROLLED ANALGESIA IN PATIENTS UNDERGOING ABDOMINAL SURGERIES

FOR anesthesiologis s ,treatingpostoperativepainhas alwaysbeen a problem.Althoughopioidshave been provedtobe effective,theirsideeffectscouldnotbeignored.With thedevelopmentofscienceand pharmacology,many drugs with aspectsof satisfactoryanalgesicefficacyand couldbe welltoleratedby patientshave been developed.And lornoxicamisone of them, which isa non-steroidalanti-inflammatorydrug (NSAID ), with analgesic, anti-infl-ammatory,andantipyreticproperties.Itseliminationhalf-time(3 to 5 hours) isle…     

BLOOD PRESSURE CHANGE WITH AGE IN SALT-SENSITIVE TEENAGERS

Objective To observe blood pressure change with age in salt-sensitive teenagers whose salt sensitivity were determined by repeated testing.Methods Salt sensitivity was determined through intravenous infusion of normal saline combined with volume-depletion by oral diuretic furosemide in 55 teenagers. After five years, salt sensitivity was re-examined and subject blood pressure was followed up. Blood pressure changes in salt-sensitive teenagers were compared to that of non-salt sensitive teenagers over five years.Results After 5 years, the repetition rate of salt sensitivity determined by intravenous saline loading is 92.7%. In teenagers with salt sensitivity on the baseline, both the systolic blood pressure increments and increment rates were much higher than non-salt sensitive teenagers (12.7±12.1 mmHg vs. 2.8±5.2 mmHg, P＜ 0.01; 12.2%± 12.0% vs. 2.5% ±4.4%, P＜ 0.001,respectively). There was a similar trend for diastolic blood pressure (8.4 ± 6.4 mmHg vs. 3.7 ± 6.4 mmHg, P = 0.052; 13.2% ±10.6 % vs. 6.8%± 10.1%, P = 0.053, respectively).Conclusions Salt sensitivity determined by intravenous saline loading showed good reproducibility. Blood pressure increments with age were much higher in salt-sensitive teenagers than non-salt sensitive teenagers, especially in terms of systolic blood pressure.     

Acupuncture Combined with Spinal Tui Na for Treatment of Primary Dysmenorrhea in 30 Cases

Objective: To observe the therapeutic effects in acupunture treatment of primary dysmenorrhea combined with spinal Tui Na, and study its mechanism. Methods: Thirty cases of the treatment group were treated by acupuncture combined with spinal Tui Na, and thirty cases in the control group were treated by routine acupuncture. Results: The total effective rate was 93.3% in the treatment group, and 73.3% in the control group, with a significant difference between the two groups (P<0.05). Conclusions: Acupuncture combined with spinal Tui Na has good prospects for treatment of primary dysmenorrhea.     

Luo Lingjie's Experience in Treating Chronic Nephropathy

In treating chronic nephropathy,Luo Lingjie,a chief physician,pays attention to regulating the balance between yin and yang,treating infection if present,and removing pathogenic factors.He prescribes gentle drugs and uses carefully strongly warming-tonifying ones,emphasizes the importance of persuading the patient to persist in treatment with medication and nurse one's health for recuperation,and is good at combined use of TCM and western medicine therapy and brings the merits of various therapies into full play,with obvious theraoeutic effects.     

Dr.Zhang Ren's Experience in the Acupuncture Treatment of Different Diseases with the Same Therapeutic Principle

Dr.Zhang Ren,the chief physician,is the chairman of Shanghai Acupuncture and Moxibustion Association.Having been engaged in medicine for about 40 years,he is experienced in treating various intractable diseases.In his long years of clinical practice,he advocates taking the TCM differentiation as the basis to seek for the acupuncture method for treatment of modern intractable diseases.The author of this essay had the fortune to follow Dr.Zhang in study.The following is a summary of Dr.Zhang's experience in the acupuncture treatment for different intractable diseases with the same therapeutic principle.     

安心颗粒对急诊经皮冠状动脉介入术后生活质量影响的研究

目的：评价使用安心颗粒对急诊经皮冠状动脉介入术（PPCI）术后生活质量的影响.方法：将160例接受PPCI的急性ST段抬高型心肌梗死患者随机分为安心颗粒组（术前顿服安心颗粒8.8g,术后安心颗粒4.4 g/次,每日2次）和对照组（仅接受基础药物治疗）.所有患者均服用阿司匹林、氯吡格雷和阿托伐他汀.分别在入院时、出院前1d、出院后180 d时,应用心肌梗死多维度量表（MIDAS）、中文版SF-36评价量表对患者生活质量评分.并观察术后30 d以内的出血并发症、血小板减少症发生情况.结果：入院时和出院前1d,两组患者的心肌梗死MIDAS、SF-36量表评分比较无差异（P＞0.05）;出院后180 d时,与对照组比较,安心颗粒组MIDAS、SF-36评分明显减低（P＜0.05）;组内与入院时比较,两组出院前1d、出院后180 d时,MIDAS、SF-36评分均降低（P＜0.05）.两组患者在随访期间均无大量出血、少量出血、重度和极重度血小板减少症发生,安心颗粒组有4例、对照组有7例发生不明显出血（P＞0.05）.两组发生轻度血小板减少症的患者数比较无差异（P＞0.05）.结论：PPCI使用安心颗粒,能改善急性ST段抬高型心肌梗死患者的生活质量,且不增加出血风险.     

The comparative studies of the influences of Urapidil and Nicardipine on sino-atrial node function, atrio-ventricular node function and hemodynamics

Objective:To investigate the influences of urapidil and nicardipine on rabbit sinus function,atrio-ventricular node function and hemodynamics.Methods:Thirty-two Angora's rabbits were selected and randomly divided into four groups.U1 group:urapidil 0.25 mg/kg;U2 group:urapidil 0.5 mg/kg;N1 group:nicardipine 10 μg/kg;N2 group:nicardipine 20 μg/kg.All these medicine were administrated within 30 seconds.Measurements were taken before and after the administration of urapidil or nicardipine for the following data:mean blood pressure(MAP),heart rate(HR),sino-atrial conduction time(SACT),maximal sinoatrial recovery time(SNRTmax)corrected sinus node recovery time(CSNRT),index of sinus node recovery time(SNRTI),Wenckebach A-V conduction frequency (WB),and P-R interval.Results:Significant MAP and HR changes were identified in all of the four groups before and after administration of both urapidil and nicardipine.No significant changes could be found in the rest of the parameters.Intergroup analysis showed that SACT and CSNRT of N1 and N2 groups were shorter than those of the U2 group(P＜0.01);the MAP decreased(P＜0.01)and the HR increased drastically(P＜0.01).Conclusions:Neither urapidil(0.25 mg/kg,0.5 mg/kg)nor nicardipine(10μg/kg,20μg/kg)has any significant influence on rabbit sinus function or rabbit atrio-ventricular node function.Nicardipine could be a better choice than urapidil for parafunctional sinus node patients.