脐血CD34+细胞体外扩增和分化调节的研究

目的探讨脐血体外扩增的最佳造血生长因子组合及时间.方法脐血CD34+细胞培养于含不同造血生长因子组合的无血清培养液.对其有核细胞数(NC)、表面抗原以及集落形成能力进行监测.结果同对照组相比,扩增组的NC和CD34+细胞均有不同程度的扩增,扩增高峰分别为第10d和第6d;其中含SCF、IL-3、IL-6、TPO、Flt3-L、G-CSF及Epo的E组扩增的细胞数最多.第6d,CD34+细胞、NC及CFC总数分别扩增了9.90、101.80和31.18倍,第10d分别累积扩增了3.56、357.42和73.11倍.而D组(含SCF、IL-3、IL-6、TPO及Flt3-L)扩增的细胞中CD34+及CD34+CD38-含量最高.提示E组细胞分化较D组明显.结论体外扩增可望解决单份脐血造血干/祖细胞数量不足的问题,但扩增中应注意其过度分化,扩增时间以6～10d为宜.     

造血细胞生长因子对脐血CD34+细胞短期体外扩增的作用

目的 探讨造血细胞生长因子(HGF)的不同组合对脐血CD34 细胞短期体外扩增的作用．方法 用免疫磁珠法分高纯化脐血CD34 细胞，培养于无血清培养液中。加入不同的细胞因子，于第4，7，10，14天检测CD34 细胞百分率和有核细胞(NC)总数。结果 和对照组相比，各组的NC扩增倍数随着培养时间的延长虽不同程度的增加，干细胞在FL、TPO，GM-CSF、SCF、IL-6、G-CSF、EPO和IL-3等因子作用下，NC扩增倍数在培养第14天达高峰，为328．96倍；而CD34 细胞扩增倍数在培养第7天达最高峰，但在FL、TPO、GM-CSF、SCF，IL-6、G-CSF等因子作用下，CD34 细胞扩增倍数最大，7d后达25．23倍，而后随着培养时间的延长而逐渐下降。结论 脐血CD34 细胞在HGF作用下，扩增时间以7-10d为最佳。     

TPO、PDGF、IL-11在脐血巨核细胞生长和分化中的作用

目的 比较血小板生成素（TPO）、血小板源性生长因子（PDGF）、白细胞介素-11（IL—11）在促进巨核系增殖、分化及成熟过程中的不同效果，初步探讨巨核细胞分化过程中相关信号通路蛋白的表达。方法用免疫磁珠法分离纯化脐血CD34^＋细胞，在液体培养体系中经TPO、PDGF-BB、干细胞因子（SCF）、IL-1β、IL-3、IL-6及IL—11等细胞因子组合的诱导，检测有核细胞（NC）总数和CD41^＋、CD61^＋表达，蛋白印迹实验（Westernblot）分析巨核细胞分化过程中相关信号通路蛋白的表达。结果 TPO对巨核系的扩增效果明显强于PDGF和IL-11，而PDGF、IL—11的扩增效果差别不大；含IL—11的因子组，NC及CD41^＋、CD61^＋细胞的总数都有明显增加，但CD41^＋、CD61^＋细胞比例不高；脐血CD34^＋细胞在TPO、SCF、IL-1β、IL-3、IL-6及IL—11等因子作用下，随着培养时间的延长，促分裂原活化蛋白激酶42／激酶44（MAPKp42／p44）蛋白表达并无明显改变，蛋白激酶B蛋白亦未见明显变化，但磷酸化的p42和p44的表达却明显增加。结论 在体外诱导脐血CD34^＋细胞向巨核系定向分化过程中，TPO起着最重要的作用；TPO联合其他造血生长因子刺激脐血CD34^＋细胞向巨核细胞分化和增殖过程中，刺激了MAPKp42／p44分子的磷酸化，从而激活和维持巨核细胞的增殖。     

多种细胞因子组合对脐血CD34+细胞的体外扩增

目的 探讨不同细胞因子组合在无血清、无基质条件下对脐血干／祖细胞的调控作用。方法 将SCF、Flk2／Flt3配体(FL)、TPO、IL-6及IL-6受体的可溶性形式(sIL-6R)进行不同组合扩增脐血CD34^ 细胞。结果 ①SCF FL ／-TPO组合能有效快速扩增脐血CD34^ 、CD34^ Thy-1^ 、CD34^ CD33^ 及长期培养启始细胞(LTC—IC)；②该组合同时加入IL-6及sIL-6R后，脐血干／祖细胞显著增殖；单独加入IL-6(不含sIL-6R)时，早期CD34^ 细胞和LTC—IC含量无明显增加，而且CFU-Mix、BFU—E的扩增不如CFU—GM明显；③通过细胞凋亡早期膜变化标记FITC—Annexin—V检测细胞凋亡率，加入Flt3L和／或IL-6 sIL-6R后Annexin-V阳性细胞由15．2％～19．1％降至2．8％～3．5％。结论 在SCF TPO Flt3L IL-6 sIL-6R组合的无基质、无血清悬浮体系中，脐血既能产生大量定向祖细胞，又能保持一定数量的早期造血细胞，这一体外培养体系具有重要的临床意义。     

不同细胞因子对脐血CD34+细胞扩增的影响

目的探讨不同细胞因子促进脐血CD34 细胞扩增的效应,为下一步的临床应用奠定基础。方法采用EasySep免疫磁珠阳性选择系统分离脐血中CD34 细胞,在无血清、无基质细胞培养体系中添加不同细胞因子培养2周,分组:A组,SCF FL TPOB组,SCF FL TPO IL-3C组,SCF FL TPO G-CSF。结果CD34 细胞数目于培养后7d达到峰值,3组间差异无显著性;B组扩增至14d时细胞总数达(384.40±17.48)倍,显著高于另外2组。扩增7d后的脐血细胞经体外培养可形成造血集落。结论体外扩增脐血CD34 细胞理想的细胞因子组合为SCF TPO FL,以7d为宜。     

条件培养液对脐血CD34^＋细胞扩增效应的实验研究

目的：探讨并分析不同条件培养液对脐血CD34^ 细胞的体外扩增效应及影响因素。方法：利用4种条件培养液（胎儿肌肉、胎盘组织、外周血、脐血单个核细胞）对脐血CD34^ 细胞进行2周的体外扩增，并和三细胞因子组合：干细胞因子（stem cell factor,SCF)，血小板生成素（thrombopoietin,TPO),flt-3配基（flt-3 ligand,FL)，对脐血CD34^ 细胞扩增结果进行了比较；利用ELISA方法检测4种条件培养液中细胞因子[白细胞介素－1（interleukin-1,IL-1)、白细胞介素-6(interleukin-6,IL-6)、FL、TPO]的浓度。结果：脐血单个核细胞条件培养液及细胞因子组合对脐血CD34^ 细胞均有明显的扩增效应，脐血组优于细胞因子组合；4种条件培养液中，脐血组细胞因子FL的含量明显高于其余条件培养液组。结论：脐血条件培养液可作为体外培养扩增脐血CD34^ 细胞有效而廉价的扩增剂；细胞因子FL可能是造成不同条件培养液对脐血CD34^ 细胞体外扩增结果差异的主要因素。     

脐血造血干/祖细胞体外扩增的实验研究

目的探讨因子组合FL+TPO+SCF+IL-6对脐血CD34 +细胞的体外扩增效应.方法采用Mini-MACS分离纯化出CD34+细胞, 在无基质接触的液体悬浮培养体系培养,通过间接免疫荧光染色、流式细胞仪技术、集落形成试验等动态测定细胞扩增情况.结果培养4周后,FL+TPO+SCF+IL-6组细胞总数、集落形成数均比扩增前显著增加(P＜0.05),并且能维持一定比例的CD34+、Th y-1+细胞;SCF+IL-3+IL-6+GM-CSF+EPO组在第2周时集落形成数已降低,第4周时集落形成数、CD34+细胞基本检测不到.结论与经典因子组合SCF+IL-3+IL-6+G M-CSF+EPO相比,因子组合FL+TPO +SCF+IL-6能在较长时间内有效扩增脐血造血干/祖细胞, 是一个优化组合方案.     

脐血造血干／祖细胞体外扩增的实验研究

目的 探讨因子组合FL＋TPO＋SCF＋IL－6对脐血CD34^ 细胞的体外扩增效应。方法 采用Mini-MACS分离纯化出CD34^ 细胞,在无基质接触的液体悬浮培养体系培养,通过间接免疫荧光染色、流式细胞仪技术、集落形成试验等动态测定细胞扩增情况。结果 培养4周后,FL＋TPO＋SCF＋IL－6组细胞总数、集落形成数均比扩增前显著增加（P＜0．05）,并且能维持一定比例的CD34^ 、Thy-1^ 细胞;SCF＋IL－3＋IL－6＋GM－CSF＋EPO组在第2周时集落形成数已降低,第4周时集落形成数、CD34^ 细胞基本检测不到。结论 与经典因子组合SCF＋IL－3＋IL－6＋GM－CSF＋EPO相比,因子组合FL＋TPO＋SCF＋IL－6能在较长时间内有效扩增脐血造血干／祖细胞,是一个优化组合方案。     

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.     

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。     

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.