CArG调控元件辐射诱导活性的研究

目的构建含有人工合成CArG调控元件的荧光素酶报告基因重组质粒,研究该元件在多种肿瘤细胞中不同剂量放射线照射下的放射诱导活性.方法将含有9个串联的CCATATAAGG结构的CArG增强子与CMV IE basal gene promoter构成的嵌合调控元件插入到pGL3-Basic质粒,构建荧光素酶报告质粒.利用脂质体将该报告质粒瞬时转染肿瘤细胞株HeLa、A549、HepG2,给予不同剂量137Cs γ射线照射,36 h后检测荧光素酶活性,观察该调控序列的放射诱导活性.结果该CArG调控元件在低剂量放射线的诱导下能明显增强下游基因的表达,在3 Gy的γ射线照射下诱导活性最强.结论人工合成的CArG调控元件在低剂量的放射线作用下能有效诱导下游基因的表达,在肿瘤的放射-基因治疗中具有较强的应用潜力.     

伽马射线对重组质粒(CArG)_6-hTERTp-SEA-GPI在人肿瘤A549细胞和成纤维细胞WI-38中的SEA表达的影响

目的:观察γ-射线对重组质粒(CArG)6-hTERTp-SEA-GPI在人肿瘤A549细胞和人正常细胞WI-38中的SEA表达的影响。方法:重组质粒(CArG)6-hTERTp-SEA-GPI分别转染A549和WI-38细胞,转染后12h2Gyγ-射线照射肿瘤细胞和正常细胞,转染后24h后采用RT-PCR法检测SEA mRNA的表达,转染后48h采用Western blot法检测SEA蛋白的表达。结果:(CArG)6-hTERTp-SEA-GPI能够在肿瘤细胞A549细胞中表达SEA,正常细胞WI-38细胞中不能表达SEA;2Gyγ-射线可以诱导转染(CArG)6-hTERTp-SEA-GPI质粒的A549细胞SEA mRNA和蛋白的表达,表达量分别增加115.4%和24.2%。结论:重组质粒(CArG)6-hTERTp-SEAGPI能够在肿瘤细胞A549中选择性表达SEA,且2Gyγ-射线可以增强SEA的表达水平。     

小鼠 Egr-1启动子的克隆及其辐射诱导特性的研究

目的 :扩增 Egr- 1启动子并构建 Egr- PGL重组质粒 ,探讨不同剂量 X射线诱导被转染的NIH3T3细胞荧光素酶表达活性。方法 :利用 PCR方法扩增 Egr- 1p与 PGL3- E表达载体连接 ,用发光仪测定荧光素酶与底物反应的发光值 ,分析 X射线诱导荧光素酶表达。结果 :对 PCR产物进行测序 ,序列基本正确 ;电泳检测重组质粒 ,有正确的特异性片段 ;重组质粒转染小鼠 NIH3T3细胞鉴定 Egr- 1p的辐射调节特性 ,发现照射组发光值均高于假照组 ,75m Gy照射组的表达量约为假照组的 5倍 ,2、4、6、8和 10 Gy照射组的表达量分别约为假照组的 5～ 7.5倍。结论 :扩增得到的 Egr- 1启动子在不同剂量 X射线照射下均具有诱导下游基因表达增强作用 ;低剂量照射诱导 Egr- 1启动子下游基因表达增强作用的发现可能在肿瘤基因治疗中更具有实用价值。     

小鼠Egr-1启动子的克隆及其辐射诱导特性的研究

目的：扩增Egr-1启动子并构建Egr-PGL重组质粒,探讨不同剂量X射线诱导被转染的NIH3T3细胞荧光素酶表达活性。方法：并利用PCR方法扩增Egr-1p与PGL3-E表达载体连接,用发光仪测定荧光素酶与底物反应的发光值,分析X射线诱导荧光素酶表达。结果：对PCR产物进行测序,序列基本正确;电泳检测重组质粒,有正确的特异性片段;重组质粒转染小鼠NIH3T3细胞鉴定Egr-1p的辐射调节特性,发现照射组发光值均高于假照组,75mGy照射组的表达量约为假照组的5倍,2、4、6、8和10Gy照射组的表达量分别约为假照组的5～7．5倍。结论：扩增得到的Egr-1启动子在不同剂量X射线照射下均具有诱导下游基因表达增强作用;低剂量照射诱导Egr-1启动子下游基因表达增强作用的发现可能在肿瘤基因治疗中更具有实用价值。     

放射线对肝癌细胞中端粒酶启动子活性的影响

目的:研究放射线对不同的肝癌细胞株中人端粒酶逆转录酶(hTERT)启动子活性的影响。方法:报告质粒phTERTp-Luc经脂质体METAFECTENE介导瞬时转染4种不同的肝癌细胞株后,给予2 Gyγ射线照射,观察照射组与非照射组中荧光素酶的表达。结果:瞬时转染4种不同的肝癌细胞株后,检测到照射可经hTERT启动子而诱导荧光素酶的表达增强,即hTERT启动子在2 Gy照射组中比在非照射组中具有更高的活性。结论:2Gyγ射线可以诱导肝癌细胞中hTERT启动子活性增强,有望用于增强肝癌细胞中hTERT启动子介导的靶向基因治疗,并为进一步深入研究奠定了基础。     

Egr-1启动子辐射诱导的基因表达特性

目的:研究电离辐射作用下早期生长反应因子1(Egr-1)启动子调控增强绿色荧光蛋白(EGFP)报告基因在人结肠癌细胞SW480中的表达。方法:利用基因重组手段构建质粒Egr-1-EGFP;阳离子脂质体介导转染人结肠癌细胞SW480,给予不同剂量γ射线照射,流式细胞仪检测照射后不同时间点EGFP的表达。结果:电离辐射能够诱导Egr-1启动子调控下游基因的表达,且6 Gy照射后最为明显,在照射后24 h达到高峰。结论:Egr-1启动子具有电离辐射诱导特性。     

Egr-1启动子辐射诱导的基因表达特性

目的：研究电离辐射作用下早期生长反应因子1（Egr-1）启动子调控增强绿色荧光蛋白（EGFP）报告基因在人结肠癌细胞SW480中的表达。方法：利用基因重组手段构建质粒Egr-1-EGFP;阳离子脂质体介导转染人结肠癌细胞SW480,给予不同剂量γ射线照射,流式细胞仪检测照射后不同时间点EGFP的表达。结果：电离辐射能够诱导Egr-1启动子调控下游基因的表达,且6 Gy照射后最为明显,在照射后24 h达到高峰。结论：Egr-1启动子具有电离辐射诱导特性。     

放射诱导启动子的合成与鉴定及质粒pcDNA3.1(+)/E-CDglyTK的构建

[目的]合成放射诱导启动子序列并构建其调控双自杀基因的真核表达质粒pcDNA3.1( )/ECDglyTK.[方法]利用人工寡核苷酸片段合成放射诱导启动子,以绿色荧光蛋白(GFP)作为报告基因转染Tca8113细胞,经流式细胞仪鉴定其辐射诱导特性.将pCEA-CDglyTK中双自杀基因CDglyTK亚克隆到pcDNA3.1( ),然后把合成启动子插入到CDglyTK上游构建质粒pcDNA3.1( )/E-CDglyTK并酶切鉴定,阳离子脂质体介导其转染舌鳞癌Tca8113细胞,RT-PCR观察CDglyTK表达及诱导放疗的增效作用.[结果]合成启动子序列分析与设计序列完全一致;低剂量放射线照射可诱导这种人工启动子增强GFP在Tca8113细胞中的表达;重组质粒的酶切图谱与预期一致;3 Gy放疗显著增强CDglyTK在Tca8113细胞中的表达.[结论]成功合成放射诱导启动子并构建由其调控双自杀基因的真核表达质粒pcDNA3.1( )/E-CDglyTK,为进一步研究肿瘤放射-基因治疗奠定了基础.     

缺氧/辐射双敏感性启动子的构建及其转录调控特性

目的: 构建缺氧/辐射双敏感性启动子,阐明其转录调控特性. 方法: 通过基因重组将缺氧反应元件(HREs)序列插入早期生长反应基因-1(Egr-1)启动子的上游,构成缺氧/辐射双敏感性HRE-Egr启动子及其报告载体pHEL,转染肺癌A549细胞. 在2, 4, 6, 8, 10 Gy照射(γ-射线)后及合并缺氧(氧浓度1, 5, 10, 25, 50 mL/L)时,检测转染细胞内报告基因荧光素酶(luciferase, Luc)表达活性,以及在6 Gy照射合并氧浓度10 mL/L时Luc表达的动态变化. 结果: 成功构建了HRE-Egr启动子及其报告载体,转染A549细胞,发现常氧下HRE-Egr启动子照射后表达活性迅速升高,呈辐射剂量依赖性,与Egr-1启动子相一致. 缺氧时,Egr-1启动子辐射诱导活性明显下降(P<0.05),而HRE-Egr启动子活性则显著升高(P<0.01),同时后者活性持续时间(32 h)明显长于前者(16 h). 结论: HRE-Egr启动子具有辐射/缺氧双重敏感性,在缺氧条件下其辐射诱导活性和持续时间明显增强和延长.     

γ射线诱导下PUMA基因在人前列腺癌细胞中的作用

目的研究在γ射线诱导下PUMA基因(p53 up-regulated modulator of apoptosis)在人前列腺癌细胞(p53野生型LNCaP细胞、p53变异型PC-3细胞)中的作用效果。方法采用MTT方法检测在3、5、8Gy照射剂量的γ射线诱导下人前列腺癌细胞的抑制率;采用RT-PCR的方法检测在3、5、8Gy照射剂量的γ射线诱导下PUMA基因的表达情况。结果在γ射线诱导下LNCaP细胞抑制率较PC-3细胞抑制率高(P<0.01);随γ射线照射剂量的增加,LNCaP细胞中PUMA-mRNA的表达越明显(P<0.05)。结论γ射线诱导下PUMA基因可促使人前列腺癌LNCaP细胞凋亡。     

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.     

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.     

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.     

Expression of OPGmRNA and ODFmRNA in the patients of hip fracture due to osteoporosis

Objective：To investigate the gene expression of osteoprotegerin（OPG） and osteoclast differentiation factor（ODF） in the bone tissue of patients with hip fracture due to osteoporosis. Methods：OPGmRNA and ODFmRNA in the bone tissue in 50 cases of osteoporosis sufferers（over 50 years old） with hip fracture（Observer Group） and 30 cases of hip facture sufferers with no osteoporosis（Control group） were analyzed with the Semi-Quantitative RT-PCR method. Results：The mRNA expressed of ODF, OPG were both high in the patients with hip fracture. In the control group, the expression of OPG mRNA was observed, while the expression of ODF mRNA was very slight. Conclusion：Aged patients contained all signals including OPG, ODF that are essential for inducing osteoclastogenesis and promoting bone resorption.     

Dynamic Changes in Serum Estradiol and Progesterone levels in Patients of Premenstrual Syndrome with Adverse Flow of Liver-qi

Objective:To probe into the influence of changes of ovarian hormones on the pathogenesis of the specific sub-type premenstrual syndrome(PMS)and reveal partial microcosmic mechanisms of adverse flow of liver-qi.Methods:Estradiol(E2)and progesterone(P)levels in serum were determined at different phases of menstrual cycle by radioimmunoassay.Results:In the group of PMS with adverse flow of liver-qi.the secretive peak value Of E2 and P at the follicular phase significantly decreased,and the secretive peak value at the luteal phase did not come into being.Conclusions:Low E2 and P secretive peak at the follicular phase and absence of secretive peak at the luteal phase is one of the microcosmic mechanisms of PMS with adverse flow of liver-qi.One of the pathophysiologic mechanisms of specific sub-type PMS is probably the continuous low level of E2and P.     

Real-time Three-dimensional Echocardiography in Assessment of Left Ventricular and Right Ventricular Volumes

Real-time three-dimensional echocardiography (RT3DE)is a new ultrasound technique that enables dynamic threedimensional visualization and quantification of the heart in real time. Investigation of feasibility and methodology of RT3DE in determining left ventricular (LV) and right ventricular (RV) volumes, RT3DE was performed in 35 normal adults using Philips SONOS 7500 system with a 2-4 MHz matrix array transducer. The 60°×60° "pyramid" volume database was obtained and analyzed on a TomTec echo workstation. Both LV and RV volumes were calculated with four 3DE methods (i.e. apical 2, 4, 8, and 16-plane) through manually tracing ventricular endocardial borders in end diastole and end systole. Stroke volumes were then calculated. LV volume was also measured by 2DE Simpson's rule using GE VIVID 7 ultrasound machine.     

SCREENING FOR A 21-CHROMOSOME ABNORMALITY IN PREIMPLANTED EMBRYOS OF ELDERLY WOMEN

Increasing maternal age is the only etiological factor unequivocally linked to Down's syndrome in humans. The occurrence rate of newborns with Down's syndrome is about 1/220 in women over 35 years old. However, the occurrence rate in embryos fertilized in vitro, of the elder woman is unclear. Using FISH we screened the number of chromosome 21 in preimplanted embryos of 5 elderly women (average age, 38.4 years) to study the feasibility and necessity of screening trisomy 21 in embryos in patients over 35 years old at the in vitro fertilization (IVF) center.     

Scientific principles and rigorous processes should be followed in developing clinical guidelines for therapeutic interventions of integrative medicine

A clinical guideline for the therapeutic interventions of integrative medicine may be defined as a written document which states a series of recommendations on therapeutic interventions of integrative medicine for a special disease or condition. The guideline may provide assistance to medical professionals in making clinical decisions aimed at improving the clinical outcome of patients and reducing the costs of medical care（~＇4~. Recommendations issued by a guideline should be based on the best available evidence in both Western and Chinese medicine. For fulfilling this purpose, the development of clinical guidelines for therapeutic interventions in the field of integrative medicine should follow scientific principles and undergo a rigorous processes.