激光扫描共聚焦显微镜观察转基因马铃薯中hIL-12的表达

目的观察外源hIL-12基因在转基因马铃薯的不同组织部位的表达情况。方法采集转基因马铃薯的茎和块茎组织进行研究，以野生马铃薯为阴性对照，用间接荧光免疫标记法、激光共聚焦显微镜观察hIL-12在转基因马铃薯不同组织的表达情况。结果经激光共聚焦显微镜检测，转基因马铃薯茎与块茎中均有hIL-12的表达，而野生马铃薯中无hIL-12的表达。结论本课题组建立的人白介素-12转基因马铃薯表达体系中茎和块茎均能表达hIL-12。     

从转基因马铃薯中纯化的人白细胞介素-12的生物学活性分析

目的研究从转人白细胞介素-12（hIL-12）基因马铃薯中纯化的hIL-12的生物学活性。方法ELISA法测定纯化的hIL-12诱使外周血单个核细胞产生IFN-γ的量。结果纯化的hIL-12在体外能刺激PBMC产生IFN-γ，而野生对照组无作用。结论本实验室从转hIL-12基因马铃薯中纯化的hIL-12具有生物学活性。     

TLR9在非小细胞肺癌患者外周血单个核细胞抗肿瘤免疫中的调控作用

目的 研究TLR9激动剂--含非甲基化胞嘧啶-鸟嘌呤二核苷酸序列的寡脱氧核苷酸(CpG ODN)对非小细胞肺癌(NSCLC)患者外周血单个核细胞(PBMC)抗肿瘤免疫的影响.方法 分离36例NSCLC患者PBMC和肺癌细胞,RT-PCR检测TLR9 mRNA表达.PBMC分别经空白培养、不含非甲基化胞嘧啶-鸟嘌呤二核苷酸序列的寡脱氧核苷酸(non-CpG ODN)和CpG ODN共培养72 h.3H-TdR掺入法测定PBMC增殖变化.流式细胞仪检测T细胞表面CD69分子变化、T细胞亚群比例以及CD8+T胞内IFN-γ和IL-4的表达.ELISA法测定PBMC上清液IFN-α的浓度,并评价抑制性ODN和氯喹对IFN-α产生的影响.分别以自体肿瘤细胞和K562细胞为靶细胞(T),以PBMC为效应细胞(E)流式细胞仪检测不同E/T时PBMC的细胞毒活性.结果 NSCLC患者PBMC表达TLR9 mRNA,其表达强度(0.76±0.09)与健康者(0.77±0.09)差异无统计学意义(t=0.00,P=1.00).CpG ODN诱导肺癌患者PBMC增殖(P<0.01),上调CD3+T细胞表面CD69分子表达(P<0.01),增加CD4+T/CD8+T比值(P<0.01),增强CD8+T产生IFN-γ的能力(P<0.01).CpG ODN促进PBMC分泌IFN-α(P<0.01),抑制性ODN和氯喹抑制CpG ODN诱导产生的IFN-α.CpG ODN增强PBMC对K562和自体肿瘤细胞的细胞毒活性(均P<0.01).结论 TLR9参与调控NSCLC患者的抗肿瘤免疫,TLR9的激活效应主要包括促进PBMC活化、增殖并诱导产生IFN-α,增加PBMC中CD4+T的比例并促进CD8+T分泌IFN-γ,增强PBMC对肿瘤细胞的细胞毒活性.     

人白介素-12转基因马铃薯表达体系中hIL-12的表达

目的 研究本实验室初步建立的人白介素12（hIL-12）转基因马铃薯表达体系中hIL-12的表达。方法 ELISA法检测转基因马铃薯中hIL-12的表达情况。结果 2株转基因马铃薯茎叶中有hIL-12的表达，与对照组相比有显著性差异（P〈0.01）；转基因马铃薯5～7子代中hIL-12的表达量无显著性差异。结论 本实验室建立的人白介素-12转基因马铃薯表达体系能表达hIL-12，并且遗传性状相对稳定。     

SEA-McAb融合蛋白对SMMC-7221细胞的抑制作用及机制

目的 研究葡萄球菌肠毒素A(SEA)与单克隆抗体(McAb)融蛋白的抗肝癌细胞活性及其机制.方法 制备健康人外周血单个核细胞(PBMC)作为效应细胞.MTT法测定SEA-McAb融合蛋白对人肝癌细胞SMMC-7221的体外抑制率;采用荧光抗体流式细胞术测定肿瘤细胞HLA-I和HLA-DR抗原分子的表达;酶联免疫吸附(ELISA)法检测上清液中细胞因子IL-2、IL-12、TNF-α和IFN-γ的水平.结果 肿瘤细胞在上清波作用后,HLA-I和HLA-DR的表达均有明显上升(P<0.01).分泌IL-2、IL-12、TNF-α和IFN-γ均较对照组显著增加(P<0.01).SMMC-7221明显被抑制(P<0.01).结论 SEA-McAb通过激发PBMC的杀伤作用,对肝癌细胞株表现出较强的靶向抑制作用.     

CpG ODN增强人外周血单个核细胞对淋巴瘤细胞系Jurket杀伤活性的实验研究

目的 探讨合成含CpG基序的寡核苷酸(CpG ODN)体外刺激人外周血单个核细胞(PBMC)的活性,以及活化后的PBMC对肿瘤细胞的杀伤作用.方法 采用MTT法,ELISA法检测CpG ODN对人外周血单个核细胞(PBMC)的增殖活性以及活化后的PBMC对Jurkat细胞的杀伤活性.结果 CpG ODN组,植物血球凝集素(PHA)组及CpG ODN+PHA组较空白对照组能明显促进PBMC的增殖和干扰素-γ(IFN-γ)、白细胞介素-12(IL-12)的分泌,杀伤活性明显增高(P<0.05);CpG ODN组比PHA组明显促进PBMC的增殖和IFN - γ、IL-12的分泌,且杀伤活性明显增高(P<0.05) ;CpG ODN+PHA组在促进PBMC的增殖和IFN - γ、IL-12的分泌和对Jurkat细胞杀伤活性上明显高于其他组(P<0.05).结论 CpG ODN 具有免疫增强作用,能提高PBMC对肿瘤细胞Jurkat的杀伤作用.     

子宫内膜异位症患者血浆和PBMC培养上清液中IFN-γ、IL-4的表达及其临床意义

目的 检测子宫内膜异位症患者血浆和PBMC培养上清液中IFN-γ、IL-4的表达水平,并分析其临床意义.方法 对25例子宫内膜异位症患者和25例健康生育年龄妇女的外周血单个核细胞(PBMC)进行分离和培养,并采用ELISA方法检测血浆和PBMC培养上清液中IFN-γ、IL-4的表达水平.结果 (1)在PBMC诱生培养上清液中,内膜异位症组IL-4的表达水平为(33.6±11.7)ng/L,明显比对照组[(23.4±4.8)ng/L]升高(P＜0.01),而IFN-γ的表达水平(1 038.8±356.7)ng/L较对照组[(1463.1±348.3)ng/L]明显下降,子宫内膜异位症组的IFN-γ/IL-4比值(38.5±18.2)较对照组(73.2±13.6)也明显下降(P＜0.01).(2)在血浆中,除子宫内膜异位症组IL-4水平[(30.1±10.8)ng/L]高于对照组[(21.4±5.7)ng/L](P＜0.05)外,IFN-γ的表达水平及IFN-γ/IL-4比值与对照组比较,差异均无显著性(P＞0.05).结论 子宫内膜异位症患者体内存在Th1/Th2失衡,使子宫内膜能够逃避免疫监视及杀伤而在异位种植,引起内异症的发生发展.     

肿瘤细胞表达Sema3A对树突状细胞功能的影响

目的:研究肿瘤细胞分泌Sema3A对小鼠树突状细胞(dendritic cells,DCs) 免疫学功能的影响.方法:以Sema3A特异性小干扰RNA片段(Si-Sema)和相应突变片段(Si-mut)分别转染肺腺癌细胞A549,以real-time PCR和Western-blot法检测干扰效果.取未转染和Si-Sema、Si-mut转染细胞的浓缩上清分别作用于DCs,并以流式细胞术分析各组细胞表面MHCⅡ分子及共刺激分子CD40 、CD80的表达;酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)检测DCs表达白细胞介素 (interleukin,IL)-12水平.同时,测定DCs刺激OVA-特异性CD4+T (DO11.10T)细胞表达γ干扰素(interferon-γ,IFN-γ)和IL-2水平.结果:与Si-mut转染组相比,Si-Sema转染的A549细胞表达和分泌Sema3A水平明显下降(93.4±7.3% vs 27.8±5.3%,P<0.01);Sema3A缺失引起DCs表达MHCⅡ分子和共刺激分子水平上升,分泌IL-12 p70量明显提高 (449.3±29.3 pg/ml vs 675.7±60.2 pg/ml,P<0.05),刺激抗原特异性T 细胞表达IFN-γ(639.6 ± 41.9 pg/ml vs 905.6 ± 68.0 pg/ml,P<0.05)和IL-2 (1011.7 pg/ml± 81.4 pg/ml vs 1459.0 pg/ml± 96.8 pg/ml,P<0.05)显著增加.结论:肺腺癌细胞A549通过分泌Sema3A,抑制DCs的成熟和免疫学功能,这可能是目前尚未完全了解的肿瘤免疫逃逸机制之一.     

K562细胞survivin基因表达对淋巴细胞增殖和功能的影响

目的探讨K562细胞中survivin基因的表达对淋巴细胞增殖和功能的影响。方法将融合重组质粒pEGFP-C1-survivin和靶向survivin基因的RNAi的重组质粒pTZU6^＋1-survivin分别转染K562细胞,得到K562/survivin^＋和K562/survivin^-2种细胞,用G418筛选K562/survivin^＋细胞,用半定量逆转录-聚合酶链反应（RT-PCR）方法及免疫细胞化学法检测K562/survivin^＋和K562/survivin^-2种细胞中survivinmRNA及蛋白水平的表达,并与K562细胞作对照。将这3种细胞与健康人外周血单个核细胞（PBMC）混合培养（MLTC）,检测混合培养体系中淋巴细胞增殖能力、NK细胞活性及培养上清中IFN-γ水平。结果K562/survivin^＋组的淋巴细胞增殖指数明显低于其余两组。survivin基因的表达水平与NK细胞活性呈负相关。混合培养上清中分泌的IFN-γ,K562/survivin＋组是K562组的1/4.5,是K562/survivin^-组的1/8。结论高表达的survivin可以抑制淋巴细胞的增殖、NK细胞活性及IFN-γ的分泌。     

肺结核患者血清骨桥蛋白和白介素18的检测及意义

目的探讨肺结核患者血清骨桥蛋白(OPN)、白介素18(IL-18)、干扰素-γ(IFN-γ)和超敏C-反应蛋白(hs-CRP)水平的变化及其临床意义。方法选择初治的肺结核患者42例作为肺结核组,健康体检者36例作为对照组,应用酶联免疫吸附法(ELISA)测定血清OPN、IL-18、IFN-γ水平,免疫比浊法测定血清hs-CRP水平。比较肺结核组与对照组、伴有高热者和非高热者、痰菌阳性患者与阴性患者、病变程度严重患者与病情较轻患者OPN、IL-18、IFN-γ和hs-CRP水平。结果肺结核患者组血清OPN、IL-18、IFN-γ和hs-CRP水平明显高于对照组〔分别为(683.21±321.76)ng/ml vs.(168.46±117.31)ng/ml、(428.36±178.54)pg/ml vs.(157.32±83.45)pg/ml、(4.26±2.39)pg/ml vs.(0.17±0.36)pg/ml和(39.67±21.48)μg/ml vs.(3.23±1.34)μg/ml〕,差异均有统计学意义(P<0.05)。伴有高热(体温>39.0℃)患者血清OPN、IL-18、IFN-γ和hs-CRP水平明显高于非高热者〔分别为(743.66±364.23)ng/ml vs.(389.47±164.50)ng/ml、(456.32±261.41)pg/mlvs.(316.35±126.04)pg/ml、(4.67±3.21)pg/ml vs.(2.59±1.98)pg/ml和(51.24±21.62)μg/ml vs.(38.87±14.37)μg/ml〕,差异均有统计学意义(P<0.05);痰菌阳性的患者血清OPN、IL-18和IFN-γ水平明显高于痰菌阴性者〔分别为(612.48±340.33)ng/ml vs.(426.24±154.31)ng/ml、(436.76±211.78)pg/ml vs.(302.34±114.67)pg/ml和(3.81±2.01)pg/ml vs.(2.32±1.61)pg/ml〕,差异均有统计学意义(P<0.05)。病变程度严重的粟粒性肺结核、空洞性肺结核患者血清OPN〔(983.48±431.24)ng/ml、(778.65±214.69)ng/ml〕、IL-18〔(576.26±217.63)pg/ml、(414.57±194.35)pg/ml〕、IFN-γ〔(6.18±2.93)pg/ml、(5.07±2.34)pg/ml〕水平明显高于浸润性肺结核和结核性胸膜炎患者,差异均有统计学意义(P<0.05)。结论肺结核患者血清OPN、IL-18、IFN-γ和hs-CRP水平明显升高,OPN和IL-18可作为判断肺结核病变活动性和病情严重性的敏感指标。     

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.