脐血单个核细胞分离方法的比较及形态学改变

目的:探索一种简便、高效的脐血单个核细胞分离方法。方法:以Ficoll密度梯度离心法与羟乙基淀粉(HES)分离法进行对比.比较两种分离方法分离脐血所得的单个核细胞(MNC)细胞数,并观察两组单个核细胞不同培养时间下形态学变化。结果:两种分离方法在分离脐血MNC方面存在着显著性差异,其中以HES法效果较好,但HES法所得细胞培养后生长状态欠佳。结论:HES法是一种高效的脐血单个核细胞分离方法,但该法所得细胞的生长状态欠佳,需进一步实验证实。     

两种分离人胎盘组织单个核细胞方法的比较

目的探讨从人胎盘组织制备高丰度单个核细胞(MNC)的方法.方法采用机械法加胶原酶消化法制备人胎盘组织单个细胞悬液,再分别用羟乙基淀粉(6%HES)和Ficoll分离其中的MNC,并对分离效果进行比较.结果胎盘组织单个细胞悬液经6%HES分离后MNC的回收率为(45.3±11.7)%,而经Ficoll分离为(24.1±5.5)%,两者比较差异有统计学意义(P＜0.01),前者分离MNC的回收率明显高于后者.结论 6%HES分离法优于Ficoll法,其可从胎盘组织获得高丰度的MNC.     

一种富集脐血造血干/祖细胞的简易方法

脐血造血干细胞的分离与冻存是建立脐血干细胞库、进行干细胞移植的关键。我们采用不同比重的淋巴细胞分离液（Ficoll液）经密度梯度离心法分离脐血单个核细胞（MNC），计数并用单抗经流式细胞仪进行表型分析。结果显示：每份脐血平均采集量为99.8±7.3ml，单个核细胞计数平均为（4．15±0．67）×106／ml；使用比重为1.064的Ficoll液比使用常规比重1．077的Ficoll液所分离的造血干细胞数量多，前者CD34 细胞百分数约为后者的3倍。     

当归多糖对脐血造血细胞的抗冷冻损伤作用

目的：探讨当归多糖（APS）对脐血造血细胞的抗冷冻损伤作用.方法：采用细胞计数法比较羟乙基淀粉（HES）、明胶、淋巴细胞分离液（Ficoll）分离脐血单个核细胞（MNC）的回收率.应用细胞计数法、台盼蓝拒染法、集落形成实验和流式细胞术,检测冻存1,3,6mo脐血MNC的生物学活性,将脐血MNC与不同浓度APS共培养24h后冻存,1mo后复苏,观察APS对脐血MNC抗冷冻损伤的作用.结果：HES法、明胶法的MNC回收率均大于85%,显著高于Ficoll法（50.00±4.00）%（P〈0.05）;冻存1,3,6mo组MNC,CFU-Mix,CD34＋细胞回收率及台盼蓝拒染率差异均无显著性,且脐血MNC的损伤与冻存时间不相关.APS50,100,200,400mg/L不同浓度APS组MNC,CFU-Mix回收率和台盼蓝拒染率除APS400mg/L组下降外,其余各组明显上升（P〈0.05）;各组组间CD34＋细胞回收率差异无显著性.结论：APS可有效提高脐血造血细胞的抗冷冻损伤能力.     

脐血CD34^＋细胞分离方法比较

目的：比较不同方法分离脐血单个核细胞（MNC）及纯化CD34＋细胞效果。方法：采集脐血48份，在室温下保存12、24、48小时，分别用密度离心法和羟乙基淀粉（HES）沉淀法分离脐血MNC，再用免疫磁珠法纯化CD34^＋细胞，以台盼蓝拒染法检测细胞存活率。结果：脐血在室温下保存48小时分离MNC和CD34^＋细胞数最多．其次为12小时，在24小时分离所得最低（P〈0．05）；HES沉淀法较密度离心法分离所得MNC和CD34^＋细胞数明显增多（P〈0．05）；6组细胞存活率差异无统计学意义（P〉0．05）；免疫磁珠法纯化CD34^＋细胞可达（88±5）％的纯度。结论：HES沉淀法优于密度离心法，且48小时分离为优，免疫磁珠法纯化CD34＋细胞可提高纯度：各种方法对细胞存活率无明显影响。     

两种从骨髓中分离人间充质干细胞方法的比较研究

目的比较Ficoll(1.073g/ml)密度梯度离心法分离人骨髓间充质干细胞(human bone marrow derived mesen-chymal stem cells,hBMMSCs)与常用的Percoll分离法之间的差异.建立一种简便、实用的hBMMSCs分离方法.方法分别应用上述两种方法分离hBMMSCs,比较用两种方法从骨髓中分离的有核细胞得率及死亡率、贴壁细胞克隆数、细胞形态以及细胞表面标志.结果两种方法获得的有核细胞得率无显著差异(P＞0.05);Ficoll(1.073g/ml)密度梯度离心法获得的有核细胞死亡率显著小于Percoll分离法(P＜0.01);Ficoll(1.073g/ml)密度梯度离心法获得的hBMMSCs原代培养5 d的贴壁细胞克隆数/37.5 cm2显著多于Percoll分离法(P＜0.05).Ficoll(1.073g/ml)密度梯度离心法与Percoll分离法获得的hBMMSCs细胞形态均一,为纺锤形或三角形;Fieoll(1.073g/ml)密度梯度离心法与Percoll分离法获得的原代hBMMSCs CDl05阳性表达率[(94.0±2.0)%vs(95.8±1.5)%]及CD34阴性表达率[(96.0±1.2)%vs(97±1.0)%]无显著差异(P＞0.05).结论与Percoll分离法相比较,Ficoll(1.073g/ml)密度梯度离心法分离hBMMSCs具有细胞死亡率较小,细胞得率较多的优点,是一种较好的hBMMSCs分离方法.     

不同外周血单个核细胞分离方法对流式细胞术分析结果的影响

目的 探讨3种外周血单个核细胞(PBMC)分离方法对后续流式细胞术分析结果的影响,并比较PBMC冻存前后流式细胞术分析结果。方法 2019年5月,选择四川大学华西医院人类疾病和免疫治疗研究室6位体检健康的工作人员为受试对象,采集每位受试者外周血15 mL,平均分为3份,分别采用Ficoll离心法、羟乙基淀粉(HES)沉降法和HES+Ficoll法分离PBMC。显微镜下观察3种方法分离所得PBMC的形态,并采用细胞计数仪进行细胞计数。分离所得PBMC于冻存前和冻存6个月后行流式细胞术分析,计算PBMC中淋巴细胞所占比例、淋巴细胞中活细胞(FVS780荧光强度小于102为活细胞)所占比例(细胞存活率)和活细胞中CD4⁺T细胞数与CD8⁺T细胞数的比值(CD4⁺T/CD8⁺T)。结果 3种分离方法得到的PBMC形态均一致。与其他2种分离方法相比,HES沉降法分离得到的PBMC除淋巴细胞外还有部分红细胞。HES沉降法所得PBMC数量显著多于Ficoll离心法与HES+Ficoll法(P<0.05);F...     

混合脐血干细胞移植的研究进展

自198 8年Gluckman等[1 ] 用脐血代替骨髓对1例小儿Fanconi贫血患者进行抑制并重建其造血功能以来,脐带血移植(umbilicalcordbloodtransplant,UCBT)已应用于数十种疾病的治疗[2 - 4] 。UCBT的成功率取决于输入脐血单个核细胞(MNC)的数量,对于人组织相容性白细胞抗原(HLA)相匹配的供者脐血MNC至少需2×10 7 kg以上,低于这一剂量,植入率降低,不足1×10 7 kg则难以植入[5,6 ] 。脐血移植后造血重建较骨髓移植慢,这亦与脐血所含CD+34 细胞数量少有关[4 ,5] 。单份脐血造血干细胞数少,在体外对脐血造血干细胞扩增后,只有造血祖细胞和成…     

脐血造血干细胞不同温度长期冻存效果的实验研究

目的通过观察在不同温度条件下长期冻存的效果,以探讨脐血造血干细胞适宜的保存时间。方法以终浓度为5%二甲基亚砜(DMSO)、3%羟乙基淀粉(HES)和4%人血白蛋白(HSA)作为细胞冷冻保护剂,分别采用-80℃冰箱直接冻存和程控降温-196℃液氮冻存,冻存前及冻存3、6及12个月后,应用全自动血细胞分析仪测定脐血单个核细胞(MNC)计数,台盼蓝拒染法显微镜下计数细胞存活率及流式细胞仪测定CD34 细胞计数。结果在-80℃冰箱冻存3个月后,脐血MNC计数、台盼蓝拒染率及CD34 细胞计数冻存前后比较差异均无显著性。冻存6个月后,脐血台盼蓝拒染率较冻存前差异有显著性(P<0.05)。冻存12个月后,脐血各项指标较冻存前差异均有显著性,其中台盼蓝拒染率差异存在非常显著性(P<0.01)。在-196℃液氮冻存3、6及12个月后,脐血MNC计数、台盼蓝拒染率及CD34 细胞计数冻存前后比较均无显著差异。结论以终浓度为5%二甲基亚砜(DMSO)、3%羟乙基淀粉(HES)和4%人血白蛋白(HSA)作为细胞冷冻保护剂,脐血造血干细胞在-80℃冰箱中可以有效保存3个月,6个月后存在部分细胞损害,此时的脐血不适合作干细胞移植。脐血经程控降温-196℃液氮冻存12个月后,造血干细胞仍保存完好,适宜长期保存。     

造血干细胞采集、分离与冻存的研究

目的 :研究外周血造血干细胞的采集、分离、冻存及干细胞活性的检测。方法 :用封闭式动脉采血法采集 10例脐血 ,经Ficoll溶液分离获得干细胞 ,分成 3组 ,分别用 10 %二甲亚矾 (DMSO)、自配的 5 % DMSO+6 %羟乙基淀粉 (HES) +4 %人体白蛋白及 CP- 13种不同的冷冻保存液冻存于 196℃的液氮和 80℃的低温冰箱中 ,对冷冻后的样本进行单个核细胞 (MNC)计数。锥虫蓝拒染试验及流式细胞仪 CD+ 34 细胞计数。结果 :采集的血量平均数为 (72 .2± 17.3) ml,3种不同冷冻保存液对造血干细胞的保存效果在 1、3、6个月差异无显著性 (P>0 .0 5 )。随着冷冻时间的延长 ,特别是 6个月后 ,流式细胞仪对 CD+ 34 细胞检测的相对数逐渐增高。在 6个月内 ,80℃低温冰箱中的冻存与 196℃液氮内的冻存相比 ,对造血干细胞的影响差异无显著性 (P>0 .0 5 )。结论 :自配的冷冻保存液效果良好 ,短期造血干细胞的冻存可以在 80℃低温冰箱内进行 ,冷冻保存造血干细胞 1、3、6个月对细胞的活性影响不大。     

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.