激光共聚焦显微镜观察FITC-Annexin V/PI双染色法检测人肺癌细胞凋亡

目的激光共聚焦显微镜观察FITC-Annexin V／PI双染色法检测人肺癌细胞凋亡的形态。方法对不同剂量含药血清处理的体外培养人肺癌PGLH7细胞株进行Annexin V／PI双染,激光共聚焦显徽镜观察细胞凋亡的形态特征。结果Annexin V／PI双染色法加激光共聚焦显微镜观察,经含药血清作用的人肺癌细胞ee,凋亡早期细胞呈AnnexinV高染而PI低染,细胞膜呈绿色而细胞核不着色;凋亡中、晚期细胞则AnnexinV和PI均为高染,细胞膜呈绿色而细胞核呈红色;部分细胞的核呈碎片状或梅花状,为典型的细胞凋亡形态。结论FITC—AnnexinV／PI双染色法加激光共聚焦显微镜观察凋亡细胞是目前观察凋亡的理想方法,特别适用于凋亡细胞的早期检测。     

小鼠生精细胞体外长期培养及超微结构分析

目的初步探讨新生小鼠睾丸组织中生精细胞在体外培养条件下的增殖分化条件,建立有效的生精细胞体外成熟模型。方法用酶法和Percoll不连续密度梯度法分离纯化新生小鼠睾丸生精细胞,获取富精原细胞层,采用自制小鼠睾丸组织培养液对分离纯化后的细胞进行体外培养。用电子显微镜观察培养的细胞超微结构。结果新生小鼠生精细胞在用成年小鼠睾丸组织提取液配制的培养基中存活达194d。电子显微镜观察可见,培养后的细胞中含有精子细胞顶体样结构。结论采用小鼠睾丸组织制备的培养液培养的小鼠生精细胞可体外长期培养并显示出一定的分化趋势。     

大鼠精原细胞的分离纯化

目的：探讨大鼠精原细胞的分离纯化。方法：采用酶消化法制备10d Wister大鼠睾丸单细胞悬液；Percoll液不连续密度梯度分离精原细胞；用贴壁速度的差异纯化精原细胞。光电镜观察精原细胞的形态结构特征；应用酪氨酸蛋白激酶(c-kit)免疫化学鉴定精原细胞。结杲；精原细胞主要分布在28％～36％(Ⅱ带)间的Percoil梯度；分离纯化的精原细胞形态结构与组织切片上精原细胞一致，其纯度达55．68％；c-kit在精原细胞呈阳性表达，在睾丸体细胞呈弱阳性或阴性表达。结论：利用酶消化法、Percoll液不连续密度梯度及细胞贴壁速度的差异，分离纯化的精原细胞满足体外实验要求；精原细胞表达特异的c-kit受体。     

酸性成纤维细胞生长因子对庆大霉素诱导的海马星形胶质细胞凋亡的影响

目的：通过建立海马星形胶质细胞（ AS）的体外培养模型,从细胞凋亡的模块探讨酸性成纤维细胞生长因子（ aFGF）对庆大霉素（ GM）诱导的海马AS损伤的保护作用。方法体外培养海马AS,采用胶质纤维酸性蛋白免疫荧光进行鉴定。将海马AS随机分为3组,分别予正常培养（正常对照组）、2 g／L GM作用24 h（损伤组）及加入4.25μg／L aFGF 24 h后再加2 g／L GM作用24 h（保护组）。通过应用TUNEL结合DAPI核染色法和Annexin V－FITC／PI双染法反映细胞的凋亡状况。结果损伤组的细胞生长受抑制,细胞凋亡,保护组的AS凋亡率下降,其中损伤组TUNEL染色阳性的细胞数较正常对照组显著增加（ P＜0.01）,保护组TUNEL染色阳性的细胞数较损伤组显著减少（ P＜0.01）;保护组与正常对照组之间差异无统计学意义（ P＞0.05）;而通过Annexin V－FITC／PI双染法可见：正常对照组、损伤组、保护组细胞的总凋亡率分别为8.37％、28.66％、25.55％。其中,保护组细胞的早期凋亡率与损伤组比较差异无统计学意义,而其晚期凋亡率与损伤组相比有所降低。结论 aFGF具有减轻GM所致AS凋亡的作用,可能阻碍细胞早期凋亡的进一步发展。     

新生小鼠精原细胞分离和纯化的实验研究

目的：分离和纯化7～8d新生雄性小鼠精原细胞,为深入研究生精机理及其影响因素提供细胞来源和技术支持。方法：采用组合酶消化法制备7～8d雄性小鼠的生精细胞悬液：Percoll密度梯度离心法分离精原细胞,贴壁培养法进一步纯化精原细胞：滴片进行碱性磷酸酶染色：取同时间段雄性小鼠睾丸组织冰冻切片进行碱性磷酸酶染色。结果：精原细胞主要分布于45％～55％梯度间的Percoll中,经贴壁培养后纯度达75．2％。结论：用上述方法成功地分离和纯化了小鼠精原细胞。     

原花青素诱导前列腺癌PC-3细胞凋亡的机制研究

目的：探讨原花青素在体外诱导人前列腺癌PC-3细胞凋亡的机制。方法：体外培养人前列腺癌PC-3细胞株,用100,200,300μg／ml原花青素作用24h。荧光素标记的连接素V（Annexin V—fluoresein isothiocyanate,Annexin V—FITC）和碘化吡啶（propidium iodide,PI）双染检测原花青素对PC-3细胞凋亡的影响,采用流式细胞术检测以不同浓度原花青素作用后PC-3细胞线粒体膜电位（△ψm）的变化。结果：100μg／ml原花青素作用细胞24h,5．64％的细胞出现早期凋亡,84．7％的P03细胞呈现线粒体膜电位降低;300vg／ml原花青素组P＆3细胞凋亡率和坏死率分别为44．86％、50．81％。结论：源花青素可在体外诱导PC-3细胞凋亡,其机制与降低线粒体膜电位有关。     

两种鬼臼脂素衍生物的2DNMR研究

观察睾丸炎期间生精细胞凋亡的情况。方法：Ｇｉｅｍｓａ染色、ＴＵＮＥＬ及流式细胞术测定。结果：经Ｇｉｅｍｓａ染色可见一些生精细胞及巨大核细胞的细胞核染色致密,形状不规则。流式细胞仪测定发现正常小鼠睾丸内亚单倍体很少,模型小鼠睾丸内亚单倍体增多。ＴＵＮＥＬ阳性细胞在模型小鼠睾丸内增多,主要为精原细胞。精母细胞及管腔中的巨大核细胞。结论：正常小鼠睾丸中可见少量凋亡的生精细胞。睾丸炎期间凋亡的生精细胞增多。此时出现的一些巨大核细胞也是凋亡中的细胞。     

小鼠生精细胞的体外双室无血清培养

目的 探讨无血清条件下应用插入式细胞培养皿( Transwell 小室)对小鼠睾丸间质细胞 - 支持细 胞 - 生精细胞的双室培养技术。方法 取 60 日龄 C57BL / 6 雄性小鼠睾丸间质细胞和 15 日龄雄性小鼠睾丸支持细 胞与生精细胞混合细胞团双室共培养,不添加血清。每日在倒置显微镜下观察生精细胞的形态和生长情况,苏木 精染色观察生精细胞形态,染色体倍性分析检测细胞分化情况。结果 在培养 1 周后,可见圆形精子细胞出现, 2 周 后可见长形精子细胞, 3 周后可见较短鞭毛,生精细胞可存活 8 周;培养 1 周时,流式细胞术可检测出单倍体细胞, 单倍体细胞百分比随培养时间延长而增加。结论 应用双室无血清培养体系体外培养小鼠生精细胞可获得精子且 生精细胞存活时间较长。     

小鼠实验性睾丸炎期间生精细胞凋亡的观察

目的:观察睾丸炎期间生精细胞凋亡的情况.方法:Giemsa染色、TUNEL及流式细胞术测定.结果:经Giemsa染色可见一些生精细胞及巨大核细胞的细胞核染色致密,形状不规则.流式细胞仪测定发现正常小鼠睾丸内亚单倍体很少,模型小鼠睾丸内亚单倍体增多.TUNEL阳性细胞在模型小鼠睾丸内增多,主要为精原细胞、精母细胞及管腔中的巨大核细胞.结论:正常小鼠睾丸中可见少量凋亡的生精细胞.睾丸炎期间凋亡的生精细胞增多.此时出现的一些巨大核细胞也是凋亡中的细胞.     

大肠杆菌感染对人巨噬细胞系U937凋亡的影响

目的：探讨大肠杆菌（E.coli）感染与人巨噬细胞系U937细胞凋亡的关系.方法：以Annexin V FITC/PI双染流式细胞仪检测及Hoechst 33258荧光染色,Giemsa染色等细胞形态学观察为指标,研究E.coli感染对U937细胞凋亡的诱导作用.结果：Giemsa染色：E.coli感染10,20 min（U937：E.coli=1：20）后偶见细胞凋亡,感染30,60及90min时可见许多细胞有典型的凋亡形态学改变,并可见凋亡小体;Annexin V FITC/PI双染可见U937细胞凋亡百分率随E.coli感染时间的延长而增高.感染30,60及90min时,细胞凋亡率与对照组相比明显增高,有显著性差异（P＜0.001）.Hoechst 33258荧光染色结果表明当细胞与细菌浓度比较低时（1：10）即可引起部分U937细胞发生凋亡,U937细胞凋亡百分率随E.coli感染剂量的增加而增加,且Hoechst 33258荧光染色及流式细胞仪二种方法所得结果一致.结论：E.coli以时间和剂量依赖方式诱导U937细胞凋亡.     

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.