沉默树突状细胞恒定链对其活力和分化成熟的影响

目的 观察siRNA沉默树突状细胞(DCs)的恒定链(Ii)转染后对DCs活力及分化成熟的影响.方法 从小鼠骨髓分离骨髓前体细胞,细胞经100 ng/mL 粒细胞-巨噬细胞集落刺激因子(GM-CSF)和100 ng/mL白细胞介素-4(IL-4)诱导培养6 d后,转染针对DCs Ii链特异的小分子干扰RNA(Ii siRNA),转染后加用(或不加)50 ng/mL 肿瘤坏死因子-α(TNF-α)继续诱导成熟48 h,分别通过Western blotting检测沉默效果,光镜观察DCs的活力和形态,流式细胞仪检测细胞的凋亡和表面分子标志(MHCⅡ、CD86、CD40),ELISA检测细胞IL-12p70的释放.结果 Ii siRNA能够明显抑制DCs Ii的表达,转染Ii siRNA后对DCs的活力、形态、细胞表型以及细胞因子IL-12p70的分泌均没有影响.结论 通过siRNA沉默DCs的Ii链不会影响DCs的活力及分化成熟,该策略能够用于进一步的DCs肿瘤疫苗的研究.     

沉默树突状细胞恒定链对其活力和分化成熟的影响

目的观察siRNA沉默树突状细胞(DCs)的恒定链(Ii)转染后对DCs活力及分化成熟的影响。方法从小鼠骨髓分离骨髓前体细胞,细胞经100ng/mL粒细胞-巨噬细胞集落刺激因子(GM-CSF)和100ng/mL白细胞介素-4(IL-4)诱导培养6d后,转染针对DCsIi链特异的小分子干扰RNA(IisiRNA),转染后加用(或不加)50ng/mL肿瘤坏死因子-α(TNF-α)继续诱导成熟48h,分别通过Western blotting检测沉默效果,光镜观察DCs的活力和形态,流式细胞仪检测细胞的凋亡和表面分子标志(MHCⅡ、CD86、CD40),ELISA检测细胞IL-12p70的释放。结果IisiRNA能够明显抑制DCsIi的表达,转染IisiRNA后对DCs的活力、形态、细胞表型以及细胞因子IL-12p70的分泌均没有影响。结论通过siRNA沉默DCs的Ii链不会影响DCs的活力及分化成熟,该策略能够用于进一步的DCs肿瘤疫苗的研究。     

结肠癌细胞总RNA电穿孔法体外转染成熟树突状细胞

目的：探讨结肠癌细胞总RNA电穿孔法转染成熟树突状细胞（mDCs）的转染效率及对树突状细胞(DCs）特征和功能的影响,为DCs核酸肿瘤疫苗的制备及其在临床应用奠定基础。方法： 用重组人粒细胞-巨噬细胞集落刺激因子（rhGM-CSF）、重组人白细胞介素4（rhIL-4）将人外周血单核细胞(PBMC)诱导成未成熟树突状细胞（imDCs）,肿瘤细胞坏死因子-α（TNF-α）作用24 h刺激其成熟。实验设为imDCs组和mDCs组。Trizol法提取特异表达癌胚抗原(CEA)的结肠癌细胞SW480总RNA,电穿孔法将总RNA转染mDCs,设为RNA转染DCs组。流式细胞术检测各组细胞表面分子标志变化和转染DCs组的CEA蛋白表达情况。ELISA法检测各组细胞IL-12分泌水平。结果：rhGM-CSF、rhIL-4、TNF-α能将PBMC诱导成具有典型毛刺样突起形态学特征的DCs。imDCs组DCs表面CD1a表达呈阳性,CD80弱阳性,CD83阴性。mDCs组DCs表面CD1a、CD83、CD80表达均呈阳性。RNA转染DCs组DCs表面CD1a、CD83、CD80表达均呈阳性。总RNA转染mDCs后4 h检测到CEA蛋白表达。mDCs组、RNA转染DCs组IL-12分泌量与imDCs组比较均显著升高（P＜0.01), 而mDCs组与RNA转染DCs组比较差异无显著性（P>0.05)。结论：结肠癌细胞总RNA可通过电穿孔法转染mDCs并表达CEA蛋白,电穿孔不改变mDCs的表面分子特征及功能。     

RNA转染树突状细胞诱导特异性抗前列腺癌免疫反应的实验研究

目的 研究前列腺癌细胞RNA转染树突状细胞(DCs)诱导的细胞毒T淋巴细胞(CTL)对前列腺癌细胞的特异性杀伤作用.方法 联合应用粒细胞-巨噬细胞集落刺激因子(GM-CSF)、白细胞介素-4 (IL-4) 诱导培养小鼠骨髓单个核细胞向DCs分化,脂质体法转染RNA,流式细胞术测定转染后树突状细胞共刺激分子表达的变化,MTT法测定DCs对T淋巴细胞的刺激能力.ELISA法检测IFN-γ的分泌水平.LDH法测定DCs诱导的CTL对前列腺癌细胞的杀伤效果.结果 用GM-CSF和IL-4诱导培养小鼠骨髓来源的单核细胞,体外诱导培养6 d后获得大量DCs;RNA转染后,DCs表面共刺激分子CD40、CD80、CD86和MHC-Ⅱ分子表达提高(P<0.05),刺激T淋巴细胞增殖能力明显提高(P<0.01);前列腺癌RNA转染的DCs诱导CTL对前列腺癌细胞和肝癌细胞的杀伤作用有明显差异(P<0.01).结论 前列腺癌细胞RNA转染DCs后诱导的CTL对前列腺癌有特异性杀伤作用,为进一步抗肿瘤疫苗的实验研究奠定了基础.     

以GFP为标记的肝癌细胞RNA转染树突状细胞的体外效应

目的:探讨GFP作为标记观察肝癌细胞RNA转染DCs效果的可行性及肿瘤细胞RNA转染DCs制备疫苗的可行性. 方法:绿色荧光蛋白质粒载体 pGFP-C3稳定转染肝癌细胞HepG2,Trizol法提取筛选后细胞HepG2-GFP总RNA;分离肝癌患者外周血单核细胞体外诱导DCs细胞,总RNA转染DCs,荧光显微镜下观察转染效果,流式细胞仪检测转染前后DCs表型变化;ELISA法检测转染前后上清中IL-12变化情况;MTT法检测效应细胞对靶细胞的杀伤率. 结果:pGFP-C3稳定转染肝癌细胞HepG2后可得到稳定表达GFP的细胞HepG2-GFP,荧光显微镜下呈绿色荧光;总RNA转染的DCs荧光显微镜下呈绿色荧光,CD80(13.2%上升至86.7%),HLA-DR(38.9%上升至97.9%),CD83(0.9%上升至97.1%),CD86(31.2%上升至92.5%)表达明显升高,上清IL-12分泌量ng/L显著增高(61.3±8.1→287.4±29.3,P＜0.05);诱导的CTL能够对肝癌细胞株HepG2起特异性杀伤作用. 结论:GFP可以作为肿瘤细胞RNA转染树突状细胞的观察标记,肿瘤细胞RNA转染DCs可作为一种有效的肿瘤疫苗.     

siRNA沉默livin基因对恶性黑素瘤LiBr细胞凋亡、周期及增殖的影响

目的：观察沉默livin基因对恶性黑素瘤LiBr细胞凋亡和周期及增值的影响．方法：采用siRNA-3转染人恶性黑素瘤LiBr细胞后分别应用TUNEL法和AnnexinV-FITC／PI双染流式细胞术检测其对细胞凋亡的影响,应用PI单染流式细胞术分析对细胞周期的改变,应用免疫荧光细胞化学技术检测siRNA对凋亡效应蛋白Caspase-3表达的影响及应用MTY法检测siRNA对细胞增殖的作用．结果：siRNA．3转染LiBr细胞72h可诱导细胞凋亡（P〈0．05）和引起细胞G0／G1期阻滞（P〈0．05）;可下调Caspase-3蛋白表达（P〈0．05）及抑制细胞增殖（P〈0．05）．结论：Livin可做为应用RNA干扰技术探索恶性黑素瘤基因治疗的潜在靶基因．     

血管内皮生长因子RNA干扰效应对鼻咽癌生长的影响

目的 研究RNA干扰（RNA interference）效应对人鼻咽癌低分化上皮细胞株CNE血管内皮生长因子（vascular endothelial growth factor,VEGF）基因表达及肿瘤生长的抑制作用.方法 体外转录试剂盒合成VEGF siRNA;荧光素标记试剂盒标记siRNA;脂质体法将siRNA转入CNE细胞株.荧光显微镜下观察siRNA的转染效率;ELISA检测siRNA对VEGF蛋白表达的抑制作用;用VEGF siRNA转染过的CNE细胞注射入裸鼠,监测裸鼠肿瘤的大小,并采用HE染色,光镜下观察VEGF siRNA对肿瘤组织的影响.结果 在VEGF siRNA转染后的CNE细胞株,VEGF的表达得到有效地抑制.对鼻咽癌异种移植肿瘤裸鼠内的治疗观察表明,肿瘤生长在治疗3周后显著减少,病理切片HE染色siRNA组肿瘤的坏死灶增多.结论 体外转录合成的siRNA能特异有效地下调VEGF基因的表达,提示用2＇-脱氧修饰的VEGF特定siRNA能作为一种有效的肿瘤制剂,为抗鼻咽癌基因治疗提供了新的思路.     

rhHSP70对食管癌细胞RNA转染的脐血树突状细胞诱导CTL抗肿瘤功能的影响

目的研究重组人热休克蛋白70(rhHSP70)联合食管癌细胞RNA转染脐血树突状细胞(DCs)对细胞毒性T淋巴细胞(CTL)特异性抗肿瘤作用的影响。方法分离脐血单个核细胞,经rhSCF、rhGM-CSF和IL-4诱导和食管癌RNA转染形成成熟DCs,加入rhHSP70,检测DCs表面标志及DCs诱导的T细胞增殖、CTL杀伤活性。结果食管癌RNA转染后DCs高表达抗原提呈分子、协同刺激分子和黏附分子,对T细胞的促增殖作用和CTL杀伤活性显著增强(P<0.01),rhHSP70能显著促进DCs功能。结论 rhHSP70具有增强食管癌细胞RNA转染的DCs对T细胞的促增殖和CTL杀伤活性的作用。     

siRNA干扰下调Moesin表达对U251细胞生长和侵袭能力的影响

目的：研究RNA干扰（RNAi）对人脑胶质细胞瘤（U251）中Moesin蛋白基因表达的抑制作用及其对U251细胞生长、侵袭的影响.方法：以人脑胶质细胞瘤U251细胞系为研究对象,针对Moesin编码基因设计小干扰RNA（siRNA）片段,转染入细胞,通过逆转录聚合酶链反应（RT-PCR）筛选沉默效率最高的siRNA片段,并用WesternBlot技术检测定量转染后U251细胞Moesin蛋白的表达水平.转染后以MTT法检测细胞增殖能力;流式细胞术检测细胞的凋亡水平;Transwell法检测侵袭能力的变化,并以扫描电镜观察转染后的细胞形态变化.结果：RT-PCR,WesternBlot筛选出的MOESIN-139片段沉默效果最好,siRNA转染后U251细胞生长速度减慢,在转染48h时,siRNA组U251细胞生长速度明显低于空白对照组和阴性对照组（P〈0.01）,平均凋亡率siRNA组为（17.30±2.01）%,远高于空白对照组（1.95±0.33）%和阴性对照组（2.02±0.28）%（P〈0.01）.siRNA转染后的U251细胞穿过聚碳酯膜的细胞数量明显减少,由空白对照组26.47±4.07,阴性对照组24.27±3.63下降至siRNA组11.53±2.61（P〈0.01）.细胞形态发生改变,细胞表面伪足数由空白对照组14.20±2.58,阴性对照组15.80±1.30下降至siRNA组6.60±1.82（P〈0.01）.结论：siRNA可下调Moesin的表达,并能有效抑制U251细胞的生长,减低其侵袭性.Moesin有可能成为治疗人脑胶质瘤的靶点分子.     

siRNA对人红白血病细胞株（K562／ADM）阿霉素耐药性的影响

目的：研究小分子干扰RNA片段（small interfering RNA，siRNA）对人红白血病细胞株（K562／ADM）细胞mdr1基因表达及功能的影响，探索一新的耐药的逆转途径。方法：siRNA根椐GeneBank已知序列设计，在脂质体介导下转染K562／ADM细胞：用流式细胞仪检测K562／ADM细胞Pgp的表达及细胞周期的改变；用TUNEL法检测其凋亡；用MTTT检测其对阿霉素ADM的敏感性。结果：siRNA转染后K562／ADM细胞Pgp的表达明显下降，凋亡率明显提高，对阿霉素ADM的敏感性明显提高。结论：siRNA有效逆转了mdr1介导的耐药性，不失为一种新型、有效的肿瘤耐药逆转途径。     

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.     

The clinicopathological and immuohistochemical analysis of solid-pseudopapillary tumor of the pancreas： report of 9 cases

Objective：To investigate the clinical features, pathological characteristics and immunophenotype of solid-pseudopapillary tumor of the pancreas（SPTP）. Methods：Nine surgically treated cases of SPTP were retrospectively reviewed. Hematoxylin and Eosin（HE） staining and immunohistochemical staining were used to analyze all cases, and the general clinical data was collected. Results：Six patients were asymptomatic except for a palpable mass. Two patients complained of vague-epigastric pain. One patient appeared jaundice. The tumor was encapsulated and solid tissues alternately with cystic tissues. Histologically, the histological structure of solid portion was pseudopapillary with a fibrovascular core. Tumor cells were uniform and medium-sized which were arranged in sheets ets or nests or pseudopapillary patterns. Immunohistochemical studies demonstrated that SPTP proved positive in vimentin（9/9 cases）, AAT（9/9 cases）, NSE（9/9 cases）, ACT（7/9 cases）, CK20（2/9 cases）, CgA（1/9 cases）, S-100（3/gcases）, PR（4/gcases）, Syn（3/9 cases） and CD56（5/9cases）, negative in CEA and ER. Conclusion：SPTP is a tumor predominantly occurring in young women frequently without special symptoms. This tumor has various characteristical histological patterns with different immunophenotype.     

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.