靶向性双自杀基因治疗载体pcDNA3.1(-)Cp-CD-TK构建的初步研究

目的 构建由癌胚抗原启动子(CEA promoter,Cp)驱动的靶向性双自杀基因治疗载体pcDNA3.1(-)Cp-CD-TK,并观察其在CEA阳性大肠癌细胞中专一性表达和对增殖的影响.方法 采用PCR分别扩增出Cp、CD、TK三种目的 基因并采用双酶切、连接依次将Cp、CD、TK基因插入pcDNA3.1(-)质粒;将靶向性载体pcDNA3.1(-)Cp-CD-TK分别转染CEA阳性的人大肠癌SW480细胞和CEA阴性的Hela细胞,采用RT-PCR检测CD-TK基因的表达.用MTT法检测转染pcDNA3.1(-)Cp-CD-TK后的SW480细胞对化疗前药5-氟胞嘧啶(5-Fc)和丙氧鸟苷(GCV)的敏感性.结果 Cp基因片段、CD基因片段和TK基因均克隆正确.靶向性基因治疗载体pcDNA3.1(-)Cp-CD-TK经凝胶电泳和DNA测序证实构建正确.转染了靶向性载体pcDNA3.1(-)Cp-CD-TK的SW480细胞经过RT-PCR鉴定证实有CD-TK基因的表达,CEA阴性Hela细胞则没有CD-TK基因的表达.细胞培养试验中,转染了靶向性载体pcDNA3.1(-)Cp-CD-TK的SW480细胞对前药5-Fc和GCV敏感.结论 正确构建了靶向性双自杀基因治疗载体pcDNA3.1(-)Cp-CD-TK,靶向性双自杀基因治疗载体pcDNA3.1(-)Cp-CD-TK能够使CD-TK基因在CEA阳性细胞中专一性表达,达到靶向杀伤大肠癌细胞目的 .     

CD/5-FC系统对结肠癌细胞的杀伤作用

目的 :探讨组织特异性胞嘧啶脱氨基酶 (cytosinedeaminase,CD) 5 氟胞嘧啶 (5 fluorocytosine,5 FC)系统对不同分泌癌胚抗原 (carcinoembryonicantigen ,CEA)的大肠癌细胞LoVo和SW4 80的靶向杀伤作用 .方法 :脂质体法将CEA基因顺式转录调控序列(TRS)驱动CD基因的组织特异性逆转录病毒载体G1CEACDNa及非组织特异性逆转录病毒载体pCD2分别转导入大肠癌细胞LoVo和SW4 80 ,以G4 18筛选阳性克隆扩增后给予前药 5 FC进行敏感试验 .结果 :LoVo CEACD及LoVo CD比LoVo对 5 FC的敏感性明显提高 (P <0 .0 1,t=5 .6 88,n =9;P <0 .0 1,t=3.136 ,n =9) ,SW4 80 CEACD及SW4 80 CD比SW4 80对 5 FC的敏感性明显提高 (P <0 .0 1,t=3.4 37,n=9;P <0 .0 1,t=3.5 16 ,n =9) ,LoVo CEACD比LoVo CD对 5 FC的敏感性明显增强 (P <0 .0 5 ,t=2 .183,n =9) ,而SW4 80 CEACD对 5 FC的敏感性小于SW4 80 CD ,SW4 80 CEACD对前药 5 FC的敏感性低于LoVo CEACD(P <0 .0 5 ,t=2 .5 0 4 ,n =9) ,转CD基因之LoVo和SW4 80细胞体外实验均可观察到明显的旁观者效应 .结论 :组织特异性CD 5 FC系统对LoVo和SW4 80细胞均有明显的靶向杀伤效果 ,但对SW4 80细胞的杀伤作用小于LoVo细胞 .     

腺病毒介导的CD基因在有胰腺癌细胞中的靶向性表达

目的：探讨腺病毒介导的大肠杆菌胞嘧啶脱氨酶（Ｅ．ｃｏｌｉ ＣＤ）基因在胰腺癌细胞中靶向性表达的可能性及其作用。方法：将含癌胚抗原（ｃａｒｃｉｎｏｅｍｂｒｙｏｎｉｃ ａｎｔｉｇｅｎ ＣＥＡ）启动子的重组腺病毒感梁人胰腺癌ＳＷ１９９０细胞（ＣＥＡ＾＋）、Ｃａｐａｎ－２细胞（ＣＥＡ＾－）和入宫颈癌Ｈｅｌａ细胞，观察ＣＤ基因在３种细胞中的表达及细胞对５－ＦＣ的敏感性差异。结果：腺病毒介导ＣＤ基因仅在ＳＷ１     

KDR-CDglyTK融合基因系统对结肠癌细胞增值的影响

目的 探讨腺病毒介导KDR启动子驱动的融合基因体系对人结肠癌细胞SW620增值的影响.方法 重组腺病毒AdEasy-KDR-CDglyTK体外感染表达KDR的SW620细胞株和对照组不表达KDR的LS174T细胞株,并给予不同浓度的前药GCV(ganciciovir)和/或5-FC(5-fluorocytosine).观察该体系对SW620细胞生长增值的影响.结果 携带双自杀基因(CDglyTK)和报告基因(GFP)的重组腺病毒载体,感染复数为100时,95%以上的受感染SW620和LS174T细胞中有GFP表达.已转染腺病毒的SW620和LS174T细胞同未转染的各自细胞在细胞生长方面无显著性差异(P>0.05).在前药应用下,已转染腺病毒的SW620和LS174T细胞表现出对前药不同的敏感性:表达KDR的SW620细胞对前药具有较高的敏感性,与前者相比,不表达KDR的LS174T细胞对前药不敏感(P<0.01).融合基因的疗效优于单一自杀基因(P<0.01).结论 KDR基因启动子可以调控融合基因体系选择性地杀伤人结肠癌SW620细胞,抑制其增值.     

胞嘧啶脱氨酸基因治疗人胰腺癌的实验研究

探讨腺病毒介导的大肠杆菌胞嘧啶脱氨酶基因用于人胰腺癌基因疗法的可行性。方法将含癌胚抗原启动子的重组腺病毒感染人胰腺癌ＳＷ１９９０细胞和Ｃａｐａｎ－２细胞以及人宫颈癌Ｈｅｌａ细胞,用逆转录聚合酶链反应（ＲＴ－ＰＣＲ）和Ｗｅｓｔｅｒｎｂｌｏｔ检测ＣＤ基因在细胞中的表达,以ＭＴＴ法比较细胞对５－氟胞嘧（５－ＦＣ）的敏感性差异。建立胰腺癌裸鼠皮下移植瘤模型,观察ＣＤ基因的原位治疗效应及安全性。结果腺病毒介     

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.     

人胰腺癌干细胞对吉西他滨的敏感性及其分子机制研究

目的 检测人胰腺癌干细胞对吉西他滨的敏感性,分析其差异性基因的表达,初步阐明其耐药分子机制.方法 运用流式技术从人原代胰腺癌和细胞系SW1990中分选CD24+CD44+ESA+细胞,行NOD/SCID鼠移植瘤实验验证其肿瘤干细胞特性;运用吉西他滨进行体内、外干预,检测胰腺癌干细胞凋亡和表达率变化情况.采用人类全基因组表达谱Affymetrix U133 plus2.0芯片对胰腺癌干细胞和非干细胞进行差异基因筛选.结果 人原代胰腺癌和细胞系SW1990中分选到CD24+CD44+ESA+(0.8%)和CD24+CD44+(3.6%)细胞,移植瘤实验证实其为胰腺癌干细胞.肿瘤干细胞凋亡率及表达率检测提示胰腺癌干细胞对吉西他滨显著耐药.与胰腺癌非干细胞比对,基因表达谱芯片筛查到胰腺癌干细胞820条差异基因,其中上调表达281个,下调表达539个.结论 胰腺癌干细胞对吉西他滨耐药,具有特征性基因表达谱,为阐明胰腺癌多药耐药机制及靶向治疗奠定基础.     

XAF1基因对胰腺癌细胞SW1990增殖和凋亡的影响

目的 探讨Ad5/F35腺病毒介导的X染色体连锁凋亡抑制蛋白相关因子1(XAF1)基因对胰腺癌细胞株SW1990增殖和凋亡的影响.方法 构建和包装Ad5/F35- XAF1重组腺病毒并感染胰腺癌细胞SW1990(Ad5/F35-XAF1组),设立对照病毒感染组(Ad5/F35-Null组)和空白组.运用RT-PCR技术和Western blotting分析对感染细胞进行鉴定.流式细胞仪检测细胞凋亡并分析细胞周期,TUNEL法测定细胞凋亡率;MTT法检测细胞增殖.结果 Ad5/F35-XAF1组SW1990细胞XAF1mRNA和蛋白表达水平均明显上调.与空白对照组和Ad5/F35-Null组比较,Ad5/F35-XAF1组细胞凋亡率明显增加,增殖活性明显降低,G2/M期细胞比例显著增加,组间比较差异均有统计学意义(P<0.05).结论 Ad5F35-XAF1重组腺病毒感染胰腺癌细胞SW1990后,能促进细胞凋亡并抑制细胞增殖.     

癌胚抗原启动子调控融合自杀基因yCDglyTK载体的构建及其应用研究

目的构建靶向性基因治疗载体pcDNA3．1（-）CryCDglyTK，研究癌胚抗原（CEA）启动子是否能控制新型融合自杀基因yCDglyTK在CEA阳性结肠癌细胞中专一性表达和杀伤作用。方法采用PCR、RT—PCR、融合PCR、酶切、连接等技术构建由CEA启动子、CMV增强子驱动的融合自杀基因pcDNA3．1（-）cvvCDglyTK表达载体和分别由CEA启动子和巨细胞病毒（CMV）增强子驱动的融合自杀基因pcDNA3．1（-）CEAyCDglyTK、pcDNA3．1（-）CMVyCDglyTK表达载体，以磷酸钙纳米为载体分别转染CEA阳性的人结肠癌细胞株LOVO细胞和CEA阴性的Hela细胞，采用RT—PCR、免疫荧光法检测感染细胞中yCDglyTK基因的表达，并用MIT法检测感染后细胞对5-氟胞嘧啶（5-FC）的敏感性。结果LOVO细胞在感染以上三种质粒表达载体后均有yCDglyTKmRNA表达，且对5-FC的敏感性明显增强，Hela细胞在纳米-pcDNA3．1（-）CMVyCDglyTK复合物感染后有yCDglyTKmRNA表达，对5-FC的敏感性增强，而在纳米-pcDNA3．1（-）CEAyCDglyTK及纳米-pcDNA3．1（-）CVyCDglyTK复合物感染后则没有yCDglyTKmRNA表达，5-FC对其亦无杀伤作用。结论该实验构建的由CEA启动子、CMV增强子驱动的融合自杀基因yCDglyTK靶向性基因治疗载体。能使融合自杀基因在CEA阳性细胞中专一性表达，从而达到靶向治疗肿瘤的目的。     

KDR启动子驱动的双自杀基因对人结肠癌细胞的特异性杀伤作用

目的探讨腺病毒介导的KDR启动子驱动的双自杀基因系统CDglyTK对人结肠癌细胞SW480的特异性杀伤作用。方法用腺病毒AdKDR-CDglyTK感染表达KDR的SW480细胞和不表达KDR的LS174T细胞,观察其感染效率并以RT-PCR方法检测重组腺病毒及转基因细胞CDglyTK的表达,然后给予不同浓度的前药5-FC及GCV处理,MTT法检测该系统对不同细胞株的杀伤效应和旁观者效应;用流式细胞术观察细胞内DNA含量和细胞周期的变化。结果携带双自杀基因和报告基因(GFP)的重组腺病毒载体,感染复数为100时,95%以上的受感染SW480和LS174T细胞中有GFP表达。RT-PCR检测发现SW480细胞有目的基因的表达,而LS174T细胞无表达。在前药应用下,已转染腺病毒的SW480和LS174T细胞表现出对前药不同的敏感性:表达KDR的SW480细胞对前药具有较高的敏感性,而不表达KDR的LS174T细胞对前药不敏感(P<0.01)。将感染腺病毒的细胞与未感染细胞以不同比例混合培养,观察到该系统有明显的旁观者效应。用流式细胞仪测定给药组出现典型的凋亡峰,细胞周期显示治疗组细胞G0-G1期比率增多,S期细胞...     

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.     

Development on Adjustable Calibration Marker for Shock Wave Focus

Shock wave lithotripsy （SWL） is a treatment of choice for upper urinary stones. However, this procedure is inappropriate for obese patients because the focus is often unable to reach the target owing to the limited focal distance in shock wave source. Although treating such patients in a blast path may increase the application length of shock wave source, it＇s difficult to find this path on the lithotripter monitor. For this reason, we invented an adjustable calibration marker in order to set an effective focus in the shock wave hath.     

Study on the TCM Pathogenesis of Angiopathic Complications of Type II Diabetes

Excess production of reactive oxygen species(ROS)of mitochondrion mediated by hyperglycemia is the common pathogenesis of angiopathic complications of diabetes.TCM holds that the damp from the dysfunction of spleen.kidney and liver is the causative factor of complications of diabetes.This is similar to the mechanism of Ros resulting in angiopathic complications of diabetes.When the angiopathic complications of type II diabetes mellitus(T2DM)are difierentiated as caused by turbid damp in TCM can be explained as ROS.Since the obstruction of pathogenic damp in channels and collaterals is said to be the main pathogenesis,the treating principle should be dissolving the damp to remove the obstruction.     

Leptin, Ghrelin and TNF-α before and after Hypo-caloric Traditional Chinese Diet and Auricular Acupuncture

INTRODUCTION Obesity is a complex emergent problem, which can be possibly solved not only by the diet but also by the life style and promotion of a constant physical exercise. 1, 2 No doubt careful attentions must be given to the nutritional condition of obese people, the dietary habits, the somatic build (i.e. distribution of fat mass) and the organic functions linked to formation of the fat mass. All the parameters should be constantly monitored before, during and after a diet treatment. 3, 4, 5     

Relationship between Dysglycemia and Carotid Atherosclerosis in Tibetan Population

People with dysglycemia are at high risk for atherosclerotic diseases. This study aims at investigating the atherosclerotic vascular damage in dysglycemia and its metabolic origin in Tibetan population.     

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.