自体骨髓干细胞移植治疗下肢缺血性疾病的实验与临床分析

目的研究自体骨髓干细胞移植分离及治疗下肢缺血性疾病的效果。方法利用流式分析法检测CD34+细胞数;利用内皮细胞集落培养法观察干细胞的内皮分化能力;根据患者症状、体征的改善率及下肢血管新生率观察移植治疗效果。结果分离纯化的骨髓液中CD34+细胞浓度为(4.499±0.612)×105/mL,单个核细胞(MNC)浓度为(3.051±0.803)×107/mL,内皮形成集落数为(9.02±3.11),患者临床症状、体征都有不同程度的改善,血管新生率达31.25%。结论分离纯化的骨髓干细胞对下肢缺血性疾病的移植治疗是有效的。     

间充质干细胞体外扩增联合自体外周血造血干细胞移植治疗恶性血液病

目的　探讨体外培养扩增临床规模的人骨髓间充质干细胞(MSCs)的可行性及其联合自体造血干细胞(APBSCs)移植治疗恶性血液病的可行性、安全性和对造血重建的影响。方法　骨髓单个核细胞(MNC)来自12例正常人及恶性血液病患者,采用无血清培养基体外培养人骨髓间充质干细胞(MSCs)。MSCs联合APBSCs移植治疗4例恶性血液病。结果　能够从所有2 6 .3±8.8ml骨髓样本中提取MSCs,其中10例成功扩增。4例患者接受扩增后MSCs联合APBSCs输注。MSCs输注无明显不良反应。中性粒细胞≥0 .5×10 9/L和血小板≥2 0×10 9/L中位时间分别为9(8～11)d和8.3(7～10 )d。结论　应用无血清培养体系可培养扩增临床规模的人MSCs。MSCs联合APBSCs共移植安全可行。快速的造血恢复提示在清髓性治疗后输注MSCs可促进造血。     

定向诱导兔骨髓间质干细胞分化为神经元样细胞的研究

目的 :体外定向诱导兔骨髓间质干细胞 (MSC)分化为神经元样细胞。方法 :采用Ficoll Paque液 (1 .0 83g/L)离心分离兔MSC ,体外扩增 ,硫代甘油等试剂的无血清达乐伯克改良必需基本培养基 (DMEM)诱导MSC分化为神经元。免疫组化鉴定神经元烯醇化酶 (NSE)、神经丝蛋白 (NF)、胶质纤维酸性蛋白 (GFAP)的表达。结果 :兔骨髓间质干细胞在体外扩增原代可获得 5× 1 0 5、1 0代可获得 2× 1 0 1 0 个细胞。加入硫代甘油诱导后 ,MSC胞体收缩 ,突起伸出 ;免疫组化显示诱导出的神经元样细胞NSE、NF、表达阳性 ,GFAP阴性。结论 :兔骨髓间质干细胞在体外可以分化为神经元样细胞     

成人血源性间充质干细胞的分离和鉴定

目的建立一种分离、培养扩增成人血源性间充质干细胞(MSCs)的方法进行鉴定,并与骨髓源性MSCs比较生物学特性。方法30名成年志愿者随机平分为二组,一组直接抽静脉血,并细分为全血细胞法组和外周血密度梯度离心法组,同时抽骨髓为骨髓密度梯度离心法组;另一组为血细胞分离机法组。各组进行细胞存活率、集落形成率、形态学、流式表型分析、胶原染色等研究。结果外周血密度梯度离心法分离成人静脉血后培养,贴壁细胞呈梭形,集落形成率为(0.12±0.08)/106单个核细胞,传代扩增到第5代时每份平均细胞数达51.4674×106,表达CD44、CD54、CD105和CD166,不表达CD14、CD34、CD45和CD31,细胞分泌Ⅰ、Ⅲ型胶原。全血细胞法和血细胞分离机法获得的细胞不能连续多次传代。结论外周血密度梯度离心法比全血细胞法和血细胞分离机法简单,贴壁细胞培养扩增容易,获得的血源性MSCs与骨髓源性MSCs的生物学特性相似。     

自体外周血干细胞移植物中单个核细胞计数与CD34+细胞的相关性

目的探讨自体外周血干细胞移植物中的单个核细胞(MNC)计数和CD34+细胞计数之间的关系. 方法行自体外周血干细胞移植(APBSCT)的患者16例,采用化疗联合粒细胞集落刺激因子(G-CSF)或粒单细胞集落刺激因子(GM-CSF)动员,用血细胞分离机进行外周血MNC分离,对每份采集物计数MNC和流式细胞仪计数CD34+细胞,进行直线相关分析. 结果 16例患者的MNC为0.10×108～2.62×108/kg,CD34+细胞为0.50×106～35.25×106/kg,移植物中的MNC计数和CD34+细胞计数呈正相关(r=0.694,P<0.001). 结论 MNC计数和CD34+细胞计数均能可靠预测自体外周血干细胞移植后的造血重建.     

丹参注射液诱导间质干细胞分化为神经元样细胞

[目的]丹参注射液体外定向诱导成人骨髓间质干细胞(MSC)分化为神经元样细胞.[方法]采用Ficoll-Paque液离心分离成人MSC,体外扩增,流式细胞仪检测MSC表面抗原表达,分别采用含丹参注射液或硫代甘油等试剂的无血清达乐伯克改良必需基本培养基(DMEM)诱导MSC分化为神经元,免疫组化鉴定神经元特异性烯醇化酶(NSE)、神经丝蛋白(NF-M)、胶质纤维酸性蛋白(GFAP)、巢蛋白(nestin)的表达.[结果]成人骨髓间质干细胞在体外扩增原代可获得0.5×106,15代可获得(2～3)×1012个细胞,细胞流式细胞仪检测结果显示CD29、CD44、CD90、CD105、CD166表达阳性,CD11a、CD14、CD34、CD38、CD45、CD80、CD86为阴性.加入丹参或硫代甘油诱导后,MSC胞体收缩,突起伸出;免疫组化显示诱导出的神经元样细胞NSE、NF-M、nestin表达阳性,GFAP阴性.[结论]丹参可以在体外诱导成人骨髓间质干细胞分化为神经元样细胞.     

血清及细胞因子对人骨髓间质干细胞增殖的影响

目的 :探讨影响体外骨髓间质干细胞增殖的因素。方法 :取胸部外科手术无血液系统疾病病人肋骨 ,采用密度梯度离心法分离单个核细胞 ,以每皿 5× 10 5个的细胞浓度接种 ,9d后 ,瑞士 吉姆萨染色 ,计数集落并进行统计分析。结果 :在 5 %～ 30 %的血清浓度范围内 ,骨髓间质干细胞集落数随血清浓度的升高而增多。bFGF ,IL 1α ,IL 3,IL 6等细胞因子可促进骨髓间质干细胞的增殖。结论 :血清浓度与细胞因子bFGF ,IL 1α ,IL 3,IL 6可促进MSC的增殖     

脐血造血干/祖细胞集落体外生物特性的研究

目的探讨脐血造血干/祖细胞CD34+含量、集落生长及产率等生物特性并与骨髓进行相关比较。方法选择脐血标本25份,正常骨髓标本18份,健康成人标本20份,对脐血体积测量和有核细胞计数,用流式细胞仪对脐血、骨髓、外周血中CD34+细胞含量进行检测;采用体外细胞培养法测定脐血和骨髓的粒单系祖细胞集落(CFU-GM)和粒-巨噬细胞与红系细胞混合集落(CFU-GEM)的产率、生长、构成,并进行了相关比较。结果①脐血量平均为96.73 mL(64~150 mL)含有核细胞数为7.84×108[(4.94~11.3)×108];②脐血中CD34+细胞含量0.85%虽低于骨髓的1.68%,但明显高于外周血的0.15%,P均<0.05,差异有显著性;③CFU-GM的产率脐血为(92.9±12.2)/2×105高于骨髓的(73.4±14.1)/2×105,脐血CFU-GEM的产率(11.64±1.86)/2×105高于骨髓的(8.63±1.17)/2×105,差异均有显著性(P<0.01,P<0.05)。结论①脐血中含有丰富的造血干/祖细胞,是造血干细胞移植的较好的来源。②脐血的造血干/祖细胞对外源性集落刺激因子反应较骨髓更敏感,更易在外源条件下增殖分化,体外扩增获得大量脐血造血干/祖细胞以达到移植阈值是完全可行的。     

大鼠骨髓间充质干细胞对同种异体T淋巴细胞的作用及其机制

目的:探讨骨髓间充质干细胞(MSCs)在混合淋巴细胞反应(MLR)中对同种异体T淋巴细胞免疫应答反应的影响,并探讨其作用机制。方法:建立MSCs和同种异体T淋巴细胞共培养体系,反应体系总量250μl。以SD大鼠的脾T淋巴细胞为刺激细胞,以W istar大鼠的脾T淋巴细胞为反应细胞,分为6组。组Ⅰ:对照组,1×105刺激细胞和1×105反应细胞共同培养;组Ⅱ:1×105反应细胞与1×104SD大鼠的MSCs共同培养;组Ⅲ:1×105刺激细胞和1×105反应细胞并加入1×104SD大鼠的MSCs共同培养;组Ⅳ:细胞种类及数量同组Ⅲ,另加1-甲基色氨酸(1-MT)(终浓度1 mol/L);组Ⅴ:细胞种类及数量同组Ⅲ,另加植物刺激素(终浓度2μg/m l);组Ⅵ:每孔加入反应细胞和刺激细胞各1×105及MSC 1×103。混合培养120 h,结束培养前13 h,每孔加入3H-TdR 20μl,以液闪测定仪测定各组的每分钟脉冲数。反相高效液相色谱法检测MSCs和MLR共培养体系中色氨酸含量。结果:MSCs可以抑制混合淋巴细胞培养体系中T淋巴细胞增殖,并呈现出剂量依赖关系;同时MSCs和MLR共培养体系中色氨酸含量明显降低。1-MT可以阻断这一作用。结论:MSCs在体外可抑制同种异体T淋巴细胞的免疫应答,吲哚胺2,3双加氧酶参与了这种免疫抑制作用。     

大鼠骨髓间质干细胞体外分化为成骨细胞的实验研究

目的：建立大鼠骨髓间质干细胞(mesenchymal stem cells，MSCs)体外诱导分化为成骨细胞的模型。方法：分离大鼠骨髓间质干细胞进行体外培养，观察其生物学特性。并选用一定的诱导剂诱导成骨细胞，通过形态学变化、碱性磷酸酶染色及钙沉积对成骨细胞进行鉴定。结果：大鼠MSCs细胞形态呈长梭形，成骨细胞诱导后MSC细胞形态由长梭形向多边形转变，ALP染色阳性，Von Kossa染色阳性，呈现典型的成骨细胞形态和生物学特征。结论：大鼠骨髓MSCs体外能被诱导分化为成骨细胞，为骨组织工程研究的优良种子细胞。     

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.