树突状细胞肺癌疫苗制备的实验研究

①目的 探讨具有较强免疫活性树突状细胞（DC）的制备及其鉴定。②方法 采用3M KCL提取法和弱酸洗脱法获得肺癌细胞GLC-82的可溶性抗原多肽，并定量抗原多肽浓度；用GLC-82细胞抗原多肽；中击致敏，从人外周血PBMC中用GM—CSF、IL-4和TNF—α体外诱导扩增并经鉴定的DC；利用负载GLC-82细胞抗原多肽的DC，刺激同种异体外周血T淋巴细胞活化增殖，诱导产生具有识别肺癌细胞抗原的特异性CTL；③结果 人体外周血PBMC体外经7天诱导出的DC，具有典型的树突状细胞特性，经GLC-82抗原致敏的DC诱导同种异体T淋巴细胞活化增殖产生CTL，高表达CD3^＋、CD4^＋、CD8^＋，且随培养时间延长，CD3^＋和CD8^＋ T 细胞百分率增加，CD4^＋／CD8^＋比例下降，动态观察CTL增殖倍数迅速增加；④结论 利用3Mkcl单盐提取法制备肿瘤可溶性抗原，并以终浓度50ng,／mL冲击致敏，从外周血PBMC中分离、扩增、活化的DC，使其负载肿瘤抗原，体外诱导能产生特异性CTL。     

鼻咽癌可溶性抗原负载DC诱导CTL杀伤鼻咽癌细胞的体外研究

目的：用负载鼻咽癌细胞(CNE)可溶性抗原的树突状细胞(dendritic cells，DC)诱导自身淋巴细胞，使之活化为鼻咽癌(nasopharyngeal carcinoma，NPC)特异性细胞毒性T细胞(cvtotoxic T lymphocyte，CTL)，观察其对鼻咽癌细胞的体外特异性杀伤作用。方法：用GM-CSF、IL-4和TNF-α体外诱导健康成人外周血单核细胞，使其分化为高纯度DC。用鼻咽癌细胞(CNE)可溶性抗原负载成熟DC，流式细胞仪检测负载前后DC的表型特征，MLR检测负载前后DC对同种异体T淋巴细胞增殖的能力。在白细胞介素-2(IL-2)的作用下，用负载鼻咽癌细胞可溶性抗原的DC诱导自身淋巴细胞成NPC特异性CTL，通过杀伤活性实验观察它对鼻咽癌细胞(CNE)的特异性杀伤效应。结果：人外周血单核细胞体外在GM-CSF、IL-4、TNF-α诱导下获得具典型表型特征的成熟DC。负载CNE可溶性抗原对成熟DC表面共刺激分子及特异性表面标志无影响，仍有较强的刺激同种异体淋巴细胞增殖能力，所诱导的CTL对鼻咽癌细胞(CNE)有明显的特异性杀伤作用。结论：负载鼻咽癌细胞(CNE)可溶性抗原的DC所诱导的CTL在体外对鼻咽癌细胞有特异性杀伤作用，对NPC的临床治疗有潜在的应用价值。     

肿瘤抗原冲击DC诱导淋巴细胞对小鼠胃肠癌的杀伤作用

目的：用胃肠癌冻融抗原冲击树突状细胞（DC）诱导荷瘤小鼠脾细胞，观察DC及细胞毒T细胞（CTL）的生物学功能及对胃肠癌靶细胞体外杀伤作用。方法：小鼠骨髓细胞经GM-CSF、IL-4诱导和胃肠癌细胞系 KATO3、CT26细胞冻融抗原（Ag）的冲击，诱生致敏的DC与活化脾淋巴细胞共培养，获得的CTL对相应靶细胞及对照黑色素瘤细胞进行体外杀伤。结果：制备的DC与CTL具有良好的生物学活性，胃肠癌 Ag冲击的DC诱导的CTL对相应靶细胞的杀伤率分别平均75.98%和86.75%，且效靶比显示量效关系；对无关靶细胞A375为10.57%和11.53%。结论：胃肠癌Ag冲击致敏的DC能诱导CTL产生特异性杀伤作用。     

抗原致敏树突状细胞诱导CTL对MCF-7细胞的体外杀伤效应

目的:采用MCF-7乳腺癌细胞裂解物致敏树突状细胞(DC),观察其体外诱导人T淋巴细胞产生特异性抗乳腺癌免疫的效能。方法:联合应用细胞因子从外周血单个核细胞(PBMC)中诱导出DC,体外负载MCF-7细胞冻融抗原,活化自身T淋巴细胞,采用ELISA法测定γ-干扰素(IFN-γ)、白介素-12(IL-12)水平,乳酸脱氢酶释放法检测细胞毒性T淋巴细胞(CTL)对MCF-7细胞的杀伤效应。结果:负载抗原DC与T淋巴细胞共培养产生IFN-γI、L-12的水平,显著高于未负载抗原DC组(P<0.05)、单纯抗原组(P<0.01)和对照组(P<0.01);负载抗原DC诱导CTL对MCF-7的杀伤效应,显著高于其他3组(P<0.01)。结论:MCF-7细胞裂解物致敏DC诱导的人CTL,对MCF-7细胞具有明显的特异性杀伤效应。     

肺癌可溶性抗原与树突状细胞构建肿瘤疫苗的杀伤机制体外研究

目的:通过肺癌的可溶性抗原致敏的树突状细胞(DC)激活细胞毒性T细胞 (CTL)的杀伤作用各个环节的研究,初步探讨其作用机制及影响条件. 方法:从健康人外周血中提取外周血单核细胞(PBMC),加入GM-CSF和IL-4后,在第4日加入不同的促成熟因子,观察DC的表型变化及测定上清中的细胞因子情况. 通过DC表面HLA-DR的表达来比较不同抗原致敏DC的优劣. 应用不同浓度的肿瘤可溶性抗原(TSA),DC负载TSA的不同时间,不同活化时间的CTL及不同TAK与GLC-82细胞的效靶比来测定其中各种引起TAK最高杀伤活力的情况. 结果:DC较好的促成熟因子是MCM,第7日时达到高峰;TSA是较好的致敏DC抗原;最佳效靶比为1∶50. 结论:肺癌可溶性抗原是良好的DC致敏物,可在单核细胞条件培养基(MCM)协助下致敏并使DC成熟,表达更多的HLA-DR,高效激活T细胞.     

MAGE-3抗原肽致敏DC疫苗抗膀胱癌细胞的体外实验

目的探讨黑色素瘤MAGE-3抗原肽致敏的树突状细胞(DC)疫苗体外抗膀胱癌细胞增殖作用及其分子机制。方法采用Ficoll法从HLA-A2型外周血中分离单核细胞,细胞因子GM-CSF和白细胞介素4(IL-4) 诱导扩增DC细胞后经MAGE-3抗原肽致敏,致敏DC细胞和同型T淋巴细胞共培养法诱导细胞毒性T淋巴细胞(CTL)。MTT法检测抗原肽致敏DC对T淋巴细胞增殖的影响,CTL对靶细胞BIU-87的杀伤作用。采用流式细胞术检测肿瘤细胞凋亡和凋亡相关蛋白变化。结果MAGE-3抗原肽致敏的DC对T淋巴细胞增殖率具有明显提高作用,高于无关抗原致敏DC及未致敏DC。经MAGE-3九肽冲击的DC可有效诱导对MAGE-3阳性细胞BIU-87杀伤的CTL活性,而无关抗原肽及未经抗原肽致敏的DC则不具备其杀伤能力。结论MAGE-3九肽致敏DC能显著促进T淋巴细胞增殖并诱导有效杀伤靶细胞的CTL作用。     

抗原致敏树突状细胞诱导CTL杀伤K562细胞体外实验研究

目的 研究体外负载K562细胞冻融抗原的树突状细胞（DC）诱导细胞毒性T淋巴细胞（CTL）的特异性杀伤作用。方法 联合应用细胞因子从外周血单个核细胞（PBMC）诱导培养出DC,在体外负载K562细胞冻融抗原。采用乳酸脱氢酶释放法,对比K562冻融抗原致敏DC组、未致敏DC组、淋巴细胞组、前列腺癌PC3冻融抗原致敏DC组分别激活的CTL对K562细胞的杀伤作用。结果 培养出具有典型特征的DC;在效靶比为10：1及20：1时。K562冻融抗原致敏DC组诱导的CTL对K562细胞的杀伤率分别为55．9％土22．0％、90．2％＋24．8％,均高于其他3组（p,0．05）。结论 K562细胞冻融抗原致敏DC可诱导激活CTL,具有明显杀伤K562细胞作用。并具有特异性。     

树突状细胞介导的肝癌免疫治疗实验研究

目的探讨以树突状细胞（DC）为介导的原发性肝癌免疫治疗新方法。方法采用肝癌细胞制备肿瘤细胞性抗原冲击致敏体外培养的DC,体外混合淋巴细胞反应检测抗原致敏DC;刺激同基因T淋巴细胞增殖的能力,观察负载肿瘤抗原的DC;体内外诱导产生肿瘤特异性细胞毒T淋巴细胞（CTL）及其抗肿瘤能力。结果经肝癌细胞抗原致敏的DC;能诱导较强的自体T细胞增殖,且诱导特异性CTL,该CTL对自体肝癌细胞具有很强的杀伤活性,杀伤率明显高于DC和未经肝癌细胞抗原致敏的DC;激活的CTL及T淋巴细胞的杀伤率,而对非同种肿瘤细胞无明显的杀伤作用,经BEL7402肿瘤细胞抗原致敏的DC;经皮下免疫小鼠可诱导有效的免疫保护作用,可抵抗野生型BEL7402细胞的再攻击,肿瘤生长受到明显抑制,瘤体出现延迟,生长减慢。结论DC具有强大的刺激T淋巴细胞增殖和诱导产生特异性CTL的能力,在体内外均能激发特异性抗肿瘤免疫应答。     

鼻咽癌细胞株体外诱导同种异体T细胞TCR VβCDR3谱型和细胞毒性分析

目的 用鼻咽癌细胞株CNE2体外负载同种异体树突状细胞(dendritic cell,DC)诱导T淋巴细胞,使之活化为细胞毒性T淋巴细胞(cytotoxic Tlymphocyte,CTL),观察其体外特异性杀伤和克隆性增殖.方法 用GM-CSF,IL-4和TNF-α体外诱导健康志愿者外周血单核细胞,用CNE2负载,使之分化为成熟DC,流式细胞仪检测负载前后DC的表型特征.在IL-2的作用下,用负载CNE2抗原的DC诱导自身T细胞生成特异性CTL.乳酸脱氢酶释放法检测其特异性杀伤活性;采用免疫谱型分析技术分析T细胞的克隆性.结果 健康志愿者外周血单核细胞体外在GM-CSF,IL-4和TNF-α诱导下获得具有典型表型特征的成熟DC,其诱导的CTL对CNE2有明显的特异性杀伤作用;PBMC的TCR Vβ CDR3谱型呈高斯分布,而诱导后的T细胞部分家族出现优势表达.结论 负载鼻咽癌抗原的同种异体DC所诱导的CTL在体外对鼻咽癌细胞有特异性杀伤作用,优势表达的TCR Vβ CDR3家族对鼻咽癌治疗有潜在的临床应用价值.     

肾癌相关抗原G250致敏的树突状细胞瘤苗体外诱导特异性抗肾癌免疫

目的 探讨肾癌相关抗原G250致敏的树突状细胞(DC)瘤苗体外诱导自体T淋巴细胞特异性抗肾癌免疫效应.方法 自健康人外周血中提取单核细胞,用重组人粒细胞-巨噬细胞集落刺激因子和白细胞介素-4刺激活化、经肾癌相关抗原G250致敏(同时设未致敏的DC为对照),用流式细胞仪检测DC细胞和T细胞表面分子的表达,MTT检测肾癌相关抗原G250致敏的DC刺激自体T淋巴细胞增殖效应,以及诱导的细胞毒性T淋巴细胞(CTL)对不同靶细胞(肾癌细胞株786-0、肺癌细胞株A549)的杀伤活性.结果 两组细胞均高表达CD40、CD80、CD83、CD86、HLA-DR,较低表达CD1a,两组无明显差异(P＞0.05).经G250蛋白致敏的DC激发自体T细胞增殖的能力明显强于未致敏组的DC,由其激活的CTL对786-0的杀伤率明显高于对照组(P＜0.05),而两组对A549细胞均无明显杀伤活性.结论 肾癌相关抗原G250致敏的DC瘤苗体外可有效的诱导特异性抗肾癌效应.     

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.