CD34~+脐血造血干细胞的体外培养及其生物学特性研究

目的:建立稳定的CD34+脐血造血干细胞(CD34+UHSC)的体外分离、培养扩增体系,并研究其生物学特性。方法:密度梯度离心及免疫磁珠法从脐血中分离CD34+UH-SC,加入细胞因子SCF、IL-3和IL-6进行体外培养扩增,观察细胞形态及其生长特性。应用流式细胞仪测定细胞周期及其CD34分子的表达,研究其增殖、生长和分化特性。以RT-PCR法检测干细胞高表达基因ABCG2的转录表达。结果:在体外培养条件下,CD34+UHSC悬浮生长,约1周后易形成克隆球,增殖旺盛,呈现指数生长期;细胞增殖周期随体外培养时间延长而逐渐趋缓;CD34+UHSC表面标志亦随体外培养时间延长而表达下降,同时ABCG2基因的表达亦逐步下调。结论:体外获得纯化的CD34+UHSC,生长稳定、增殖能力强,但随体外培养时间的延长,CD34+分子标志逐步丢失,提示在增殖旺盛的指数生长期7～21d内最适用于各项相关实验研究。     

人类胚胎干细胞向造血干细胞分化的微环境研究

目的 探讨胎肝基质细胞和骨髓基质细胞在诱导胚胎干细胞向血液血管干细胞分化中的作用,分析基因表达差异.方法 通过将分化4 d的EBs分别接种到以下两组中:(1)胎肝基质细胞层+细胞因子;(2)骨髓基质细胞层+细胞因子:诱导结束后,流式细胞术检测各组中血液血管干细胞标志KDR阳性细胞数以及造血干细胞标志CD34阳性细胞数,计数细胞分化过程中形成的造血干细胞样的集落数,比较诱导分化效果;收集胎肝基质细胞和胚胎骨髓基质细胞,采用cDNA芯片分析基因表达差异.结果 流式细胞术检测发现,两组中KDR+细胞的比例为分别为:(1.06±0.20)%、(8.8±1.49)%;CD34+细胞比例为分别为:(1.25±0.16)%、(9.19±2.10)%;血岛样集落数量分别为0.9±0.36、10.6±0.63;上述结果均以骨髓基质细胞联合细胞因子诱导方法效率最高.基因芯片(hBMSCs/hFLSCs)中总共有240条基因在骨髓基质细胞中高表达,397条基因的在肝脏基质细胞中高表达.对细胞因子、细胞黏附分子以及细胞外基质蛋白的基凶加以分析,结果发现在hBMSCs高表达的相关的21条基因,推测可能与造血分化相关.结论 人类胚胎十细胞向造血干细胞分化过程中,涉及到细胞与细胞之间的相互作用,相关的细胞因子、细胞黏附分子、细胞外基质蛋白等可能起重要的作用.     

脐血CD_(34)~+细胞体外扩增新进展

造血干细胞 (hematepoieticstemcell,HSC)是各种血细胞和免疫细胞的始祖 ,具有极重要的生理功能。脐带已经成为近年来研究的热点 ,但单份脐血中造血干细胞理论上不少 ,但实际上数量往往不足以支持成人的造血重建 ,需要体外加以扩增。影响脐血CD+34 细胞体外扩增的因素有很多 ,现就几种关键因素对脐血CD+34 细胞体外扩增作用的研究新进展综述如下。1　细胞因子造血干细胞的自我更新及分化增殖受多种细胞因子调节。近年来 ,有关学者已经相继建立了一些造血细胞因子组合 ,使造血干细胞扩增取得了很大进展。Ng等[1 ] 用FL(FLT3配基 ) +TP…     

脐带血来源基质细胞培养及造血微环境的建立

目的：研究脐带血来源的基质细胞对脐血CD34⁺ 细胞体外扩增的促进作用。 方法：应用MACS磁珠分离系统分离脐血CD34⁺细胞，通过细胞因子对脐血前体基质细胞体外诱导，使其产生基质细胞和形成造血微环境。观察基质细胞的生长状态和细胞表面分子的表达，并建立以该基质细胞为滋养层的脐血造血干细胞体外扩增体系。结果：细胞因子联合培养对CFU-GM增长没有明显影响，但对于CFU-E的形成和细胞增长的影响细胞因子组和细胞因子+基质细胞组所培养的CPU-E与对照组比较差异有显著性(P＜0.05或P＜0.01 ）。脐带血来源的基质细胞分泌细胞因子的时间-效应曲线显示，脐血基质细胞培养3、7、10、14和20 d后，TPO、SCF浓度在培养第7天时达到最高，而GM-CSF在第7天以后则逐渐降至最低。在脐血基质细胞作为滋养层的扩增体系中，加入相应的细胞因子可以使脐血造血细胞明显扩增，其中细胞因子(SCF+IL-3+EPO+TPO+GM-CSF)联合基质细胞培养体系的扩增作用最强。结论： 应用细胞因子可以体外培养脐血前体基质细胞产生脐血基质细胞,并可在该基础上形成造血微环境支持体外造血。     

VEGF,b-FGF诱导人脐血单个核细胞向血管内皮祖细胞分化的研究

目的:研究VEGF, b-FGF诱导下人脐血单个核细胞向血管内皮祖细胞(EPCs)分化.方法:从新鲜脐血中用Ficoll密度梯度离心法获取单个核细胞,在加有VEGF,bFGF的M199培养基中培养,培养7 d,免疫组化(SABC法)和免疫荧光检测细胞表面抗原的表达.结果:人脐血血单个核细胞(MNCs)经体外VEGF, b-FGF的诱导分化,表达内皮祖细胞的表面标志KDR, CD1133和CD34.结论:人脐血单个核细胞在一定的培养条件下可诱导分化为内皮祖细胞.     

脐血CD34+细胞巨核系定向诱导分化的研究

目的研究TPO与SCF、IL-11协同在体外促进入脐血CD34+细胞向巨核系增殖分化的效应.方法利用流式细胞仪分离纯化脐血CD34+细胞,在含血清液体培养体系中、细胞因子诱导下培养14 d.采用流式细胞术测定不同时间点培养体系中CD41+细胞的比例;同时采用甲基纤维素半固体集落培养测定CFU-MK的数量.结果经14 d培养,TPO+SCF+IL-11组可使CD41+细胞和CFU-MK数量分别扩增40.83±12.41倍、7.86±2.63倍,明显高于TPO组的28.69±8.27倍、4.48±1.25倍.结论在体外SCF和IL-11可以协同增强TPO诱导脐血CD34+细胞向巨核系增殖分化的作用.     

人脐血CD34+细胞体外诱导分化为树突状细胞及其扩增

目的 采用脐血CD34+细胞在rhGM-CSF、rhTNF-α诱导下体外分化扩增为成熟树突状细胞.方法 应用CD34+干细胞分选试剂盒以及免疫磁珠法从脐血中分离CD34+细胞,用rhGM-CSF、rhTNF-α联合诱导分化发育14 d,成为成熟DC,观察细胞形态及检测CD83分子表达.结果 50 ml脐带血可分离出CD34+细胞约1.1×106,在rhGM-CSF、rhTNF-α诱导下CD34+干细胞培养2周,约扩增10～20倍.CD34+干细胞培养第3 d开始出现集落,第8～9天集落最大,CD34+干细胞分化发育为有树突状突起的成熟DC,表面分子CD83阳性率为81.7%.结论 体外联合运用rhGM-CSF、rhTNF-α,能够成功诱导脐血CD34+细胞分化为大量成熟的DCs.     

脐带间充质干细胞对脐血CD_(34)~+祖细胞免疫反应的影响

目的 探讨脐带来源的间充质干细胞(UC-MSCs)是否能有效抑制脐血CD_(34)~+祖细胞的免疫反应,影响反应过程中免疫调节细胞因子的分泌.方法 从脐带中分离、培养MSCs,观察其生长形态,流式细胞术检测表面标志;将不同数量的UC-MSCs加入脐血CD_(34)~+祖细胞、外周血单个核细胞(PBMC)共培养体系中作为实验组,不加UC-MSCs的CD_(34)~+祖细胞、PBMC共培养体系为阴性对照;另设用Tramwell将UC-MSCs和脐血CD_(34)~+祖细胞、外周血单个核细胞共培养体系分隔培养,分别收集各组上清液,ELISA检测IFN-γ和IL-10水平.结果 UC-MSCs高表达CD_(105)、CD_(90)、CD_(29)、CD_(49);不表达CD_(40)、CD_(80)、CD_(86)、HLA-DR;加UC-MSCs组与阴性对照组相比,IFN-γ的分泌量减少(19.30±0.78) vs(27.00±1.11),(P＜0.05);IL-10的分泌量增多(50.28±1.29)、vs(31.68±3.08),(P＜0.05).而且具有数量依赖性;Transwell分隔培养组与阴性对照组相比,IFN-γ的分泌量减少(18.33±0.475)vs(27.00±1.11),(P＜0.05);IL-10的分泌量增多(48.47±1.18)vs(31.68±3.085,(P＜0.05);与接触培养组相比,IFN-γ的分泌量增多(18.33±0.47)vs(7.14±0.13),(P＜0.05),IL-10的分泌量减少(85.19±2.36)vs(48.47±1.18),(P＜0.05).结论 UC-MSCs能抑制脐血CD_(34)~+祖细胞的免疫反应中IFN-γ的分泌,同时促进IL-10分泌,并且这种作用部分依赖于细胞间的相互接触.UC-MSCs和CD_(34)~+祖细胞共移植可作为干细胞移植治疗缺血性心肌病的有效方式.     

诱导人脐血神经干细胞向神经细胞分化的研究

目的:体外诱导人脐血单个核细胞(mononuclear cells,MNCs)中的神经干细胞(neural stem cells,NSCs)向神经细胞分化的研究。方法:体外培养中用表皮生长因子(epithelium growth factor,EGF)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)诱导人脐血NSCs向神经细胞的分化。结果:在EGF和bFGF作用下,实验组Nestin阳性细胞数量显著增多;在EGF和bFGF作用下,Nestin的mRNA表达PCR条带变量两组间有显著性差异。结论:细胞因子EGF和bFGF在体外能促进脐血MNCs中NSCs的分化,联合应用较单独应用具有更强的促进脐血MNCs向神经样细胞分化作用。     

人类脐血集落形成细胞的早期分裂

造血干细胞(hematopoietic stem cell,HSC)体外培养时在不同的细胞因子刺激下形成人们可以辨认的各系细胞集落,被称为各系的集落形成细胞(colong-forming cell,CFC).本研究对人类脐血单个CD34+CD38-细胞进行体外培养,分别与单一黏附因素纤维结合素(Fn)及干细胞支持性基质AFT024体外模拟造血微环境相互作用,研究其集落形成能力与早期分裂模式之间的关联,以及体外模拟的造血微环境对其集落形成能力的影响.旨在从定向造血干细胞早期分裂模式入手,研究调节CFC生长增殖的各种因素,以期为定向造血干细胞体外纯化,扩增以及基因工程提供一条具有实用价值的途径.     

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.     

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.