大鼠肺血管内皮细胞培养及血管紧张素转换酶表达特点的初步研究

目的:分离大肺血管内皮细胞,建立研究ACE表达调控的体外模型。方法:分离大鼠肺血管内皮细胞采用活体胶原酶灌注法;用胰酶不完全消化和无血清培养相结合的方法得到纯化的内皮细胞;用定量PCR技术检测血管紧张素转换酶(ACE)mRNA水平的变化,速率法测定ACE酶活性。结果:传代细胞第四代后细胞增殖速度趋于稳定,内皮细胞纯度达到95%以上,ACE的活性及mRNA的表达也处于稳定期。表皮生长因子(EGF)可以使ACEmRNA表达受到抑制(P=0.025)。冻存后存代复苏率(0.816±0.085)和贴壁率(0.758±0.079)均较高,生长良好。结论:本法分离到的大鼠肺血管内皮细胞,纯度高,ACE活性及其mRNA表达水平在第四代后稳定,以作为进一步研究ACE调控的良好的体外模型     

人脐静脉内皮细胞分离及冷冻保存的技术

目的 研究分离、培养、扩增人脐静脉内皮细胞及进行冷冻保存技术，探讨建立脐带血管内皮细胞库的可能性。方法 新鲜脐带42条，脐静脉内灌注消化酶消化，获得内皮细胞：以含20％的胎牛血清M199液进行培养。采用Ⅷ因子免疫荧光染色及扫描电镜检查，鉴定所获内皮细胞及其纯度：内皮细胞加入含10％DMSO冷冻保存液，置于液氮进行保存，复苏后进行锥虫蓝试验，四氮唑盐(MTT)试验以及细胞凋亡的流式细胞仪检测。结果 血管内灌注消化液法可获取高纯度的内皮细胞，与未冻存细胞相比，复苏的细胞活力保持在95％以上。复苏后的内皮细胞的生长曲线与未冻存细胞的生长曲线无差异。冻存内皮细胞的凋亡率较未冻存细胞高，但无统计学意义。结论 脐静脉灌注酶消化法可获取足够数量及纯度的内皮细胞。冻存的内皮细胞复苏后仍保持较高的活力及体外增殖能力，可作为制备组织工程学血管的种子细胞来源。     

人脐静脉血管内皮细胞培养、冻存、复苏与鉴定

目的:探讨人脐静脉血管内皮细胞(HUVEC)体外分离、培养、冻存、复苏与鉴定的可靠方法.方法:采用改良的胶原酶灌注消化法对HUVEC体外分离、培养,按慢冻速融原则对第3代细胞进行液氮冻存、复苏.采用免疫组化Ⅷ因子相关抗原染色和活体细胞荧光DiI-Ac-LDL鉴定.结果:分离的原代HUVEC总数平均为1×106个细胞,最多达3×106个细胞;两种鉴定结果HUVEC均为95%以上阳性;冻存后分3批复苏细胞活性均超过90%.结论:成功建立了人脐静脉血管内皮细胞体外培养、冻存、复苏与鉴定方法,获得了足够数目和纯度的血管内皮细胞,保持了细胞的同源同代性.     

人脐静脉内皮细胞的分离培养和冻存

目的研究体外分离、培养、冻存人脐静脉内皮细胞的有效方法以获得大量人脐静脉内皮细胞，为构建含血管的组织工程皮肤提供种子细胞。方法新鲜脐带内灌注胶原酶消化，获得内皮细胞，用含20％胎牛血清的M199液进行培养。倒置显微镜观察细胞形态，结合Ⅷ因子免疫组化鉴定细胞来源。内皮细胞加入10％甘油的冻存液，分别置于渡氮保存1周和4周，复苏后血球计数板计数，绘制生长曲线。结果脐静脉内灌注胶原酶法可获取高纯度的内皮细胞，与未冻存细胞相比，复苏的细胞活力保持在80％以上，复苏后生长曲线与未冻存细胞相似。结论脐静脉灌注酶消化法可获取大量高纯度的内皮细胞。冻存的内皮细胞复苏后仍保持较高的体外增殖能力，可作为制备含血管的组织工程皮肤的种子细胞来源。     

人脐静脉血管内皮细胞培养、冻存、复苏与鉴定

目的：探讨人脐静脉血管内皮细胞（humanumbicicalveinendothelialcell,HUVEC）体外分离、培养、冻存、复苏与鉴定的可靠方法。方法：采用改良的胶原酶灌注消化法对HUVEC进行分离、培养与鉴定,并遵循”慢冻快复”的原则进行冻存与复苏,同时使用Ⅷ因子相关抗原染色法进行鉴定。结果：分离的HUVEC生长良好,细胞总数可达I-3×10^2。Ⅷ因子相关抗原染色鉴定结果为95％以上阳性,冻存后复苏的细胞活性超过90％。结论：HUVEC可以从脐带获得并通过原代培养成为细胞系,获得了人脐静脉血管内皮细胞体外培养、冻存、复苏与鉴定的可靠方法。     

猪血管内皮细胞的培养与表型鉴定

目的: 原代培养猪血管内皮细胞并纯化和成功传代.方法:自乳猪活体状态下获取腹主动脉,以胶原酶消化、培养PAEC,观察细胞形态、贴壁率,以DiI-Ac-LDL吸收试验进行细胞类型的鉴定.结果:原代PAEC贴壁率约90%,培养至第2代即可获得纯化的PAEC,第5代至第12代细胞增殖较快,形态正常,呈圆形、短梭形和三角形,DiI-Ac-LDL吸收试验阳性.结论:通过活体状态下获取血管、控制杂细胞污染的途径以及合理选择酶消化法可以有效快捷地获得纯化的PAEC并稳定传代.     

大鼠乳鼠腹主动脉血管内皮细胞的培养与鉴定

目的：研究大鼠乳鼠腹主动脉血管内皮细胞的培养与鉴定方法。方法：取出生5~7d左右的大鼠乳鼠的腹主动脉血管,用组织块法在含20%胎牛血清及内皮细胞生长添加剂和肝素的DMEM培养基中培养,倒置相差显微镜观察内皮细胞形态和生长特征,免疫荧光方法进行血管内皮细胞Tie2相关抗原的鉴定。结果：来自于大鼠乳鼠腹主动脉的血管内皮细胞在改良的培养基中进行体外培养,能够保持增殖和传代的能力,且Tie2抗原鉴定呈阳性。结论：成功分离大鼠乳鼠腹主动脉组织并且获得体外原代培养大鼠乳鼠腹主动脉内皮细胞。用组织培养方法可以获得较满意的血管内皮细胞。     

树鼩主动脉内皮细胞株的建立及其生物学特性的研究

将树鼩胸主动脉分层剥离,用组织块贴壁法,体外培养出主动脉内皮细胞,历经一年,传至23代,命名为TSaec-8910。倒置显微镜下细胞单层生长,呈铺路石样镶嵌排列,第Ⅷ因子相关抗原阳性,透射电镜观察,细胞质内未找到Weibel-Palade小体。细胞生长曲线及分裂指数示9～12d汇合成单层,按1:2或1:3传代,传代间隔为lO～14d,细胞冻存复苏后接种存活率为31.5％,细胞染色体检查为二倍体细胞,2n=62,雄性。内皮细胞生长因子、上皮细胞生长因子、条件培养基和附着底物对TSaec-8910细胞有明显影响,细胞在玻璃瓶壁上贴壁时间为24～18h,而在涂有鼠尾胶瓶壁则为4h左右,内皮细胞生长因子、上皮细胞生长因子能促进TSaec-8910细胞贴壁和增殖,20％TSaec-8910细胞条件培养基亦能良好地维持细胞形态。     

大鼠胸主动脉内皮细胞培养方法的改进

目的：建立一种简单易行的SD大鼠胸主动脉的内皮细胞培养方法。方法：分离大鼠胸主动脉,清除血管外结缔组织,将其剪成厚约1.5mm的血管环,以15-20个血管环密度接种于25 cm^2培养瓶中,以含20%胎牛血清的DMEM液培养,60h后去掉血管环。待细胞生长达单层融合时,消化传代。对P2代细胞应用抗Ⅷ因子抗体进行鉴定。结果：60h后,细胞呈集落样散在分布于瓶底;7-9d细胞汇合成单层,呈铺路石状。经Ⅷ因子相关抗原免疫组化鉴定,所培养细胞为内皮细胞。结论：血管环贴壁法培养SD大鼠胸主动脉内皮细胞简易可行。     

组织贴块法培养大鼠主动脉平滑肌细胞及鉴定

目的探讨以原代培养的方法获得活力稳定、高度纯化的大鼠血管平滑肌细胞的体外模型,并为相关研究提供所需的试验材料。方法采用组织贴块法进行细胞原代培养,胰酶消化传代,液氮冻存,并应用倒置相差显微镜和SP法免疫组化染色对细胞进行鉴定。结果85％的组织块接种存活,传代后90％以上的平滑肌细胞重新贴壁生长,冻存后细胞再行1～2次传代后可恢复增生活力。显微镜下细胞呈典型的“谷峰状”生长,SP法免疫组化染色显示胞质内“平滑肌肌动蛋白表达阳性。结论组织块贴壁法简便易行,短期内可获大量符合后续试验要求的血管平滑肌细胞。     

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.     

The clinicopathological and immuohistochemical analysis of solid-pseudopapillary tumor of the pancreas： report of 9 cases

Objective：To investigate the clinical features, pathological characteristics and immunophenotype of solid-pseudopapillary tumor of the pancreas（SPTP）. Methods：Nine surgically treated cases of SPTP were retrospectively reviewed. Hematoxylin and Eosin（HE） staining and immunohistochemical staining were used to analyze all cases, and the general clinical data was collected. Results：Six patients were asymptomatic except for a palpable mass. Two patients complained of vague-epigastric pain. One patient appeared jaundice. The tumor was encapsulated and solid tissues alternately with cystic tissues. Histologically, the histological structure of solid portion was pseudopapillary with a fibrovascular core. Tumor cells were uniform and medium-sized which were arranged in sheets ets or nests or pseudopapillary patterns. Immunohistochemical studies demonstrated that SPTP proved positive in vimentin（9/9 cases）, AAT（9/9 cases）, NSE（9/9 cases）, ACT（7/9 cases）, CK20（2/9 cases）, CgA（1/9 cases）, S-100（3/gcases）, PR（4/gcases）, Syn（3/9 cases） and CD56（5/9cases）, negative in CEA and ER. Conclusion：SPTP is a tumor predominantly occurring in young women frequently without special symptoms. This tumor has various characteristical histological patterns with different immunophenotype.     

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.