人胚视网膜细胞培养研究

建立了人胚视网膜神经层分散细胞培养方法，并应用相差显微镜和免疫组织化学方法对视网膜神经地的体外发育进行了初步研究。结果表明：（１）选择３－４个月的人胚胎视网膜为培养材料比较适合；（２）体外培养的视网膜神经元经历了贴壁、重聚、迁移、分化和相互接触的过程；（３）免疫组化显示神经元呈ＮＳＥ阳性，非神经元细胞呈阴性。     

人胚胎视网膜细胞的体外培养及其生长形态的初步研究

为研究神经元的生长与分化，作者建立了人胚胎神经视网膜作材料的体外培养系统，初步研究视网膜神经元在体外的生长、分化状态。结果显示神经元在体外有突起样生长。为今后研究视网膜、脑神经元的再生和功能重建提供实验基础。     

体外培养新生大鼠海马神经元突起的形态学

目的研究体外培养过程中，神经元的形态学。方法用扫描电镜观察体外培养的新生大鼠海马神经元在体外发育过程中，神经突起的形态学。结果培养海马神经元呈现梭形、三角形、锥体形等多种形态；神经突起随着培养时间延长，逐渐出现长度增加，突起分支增多；扫描电镜下，突起上显示串珠样膨大的膨体样结构，垂直分出呈鼓槌状的树突棘，突起来梢有矛头样和泪滴状生长锥，并见突起之间有连接存在。结论体外培养的海马神经元，在形态学上经历了与在体相似的变化，并且具有执行神经元功能的基本形态学基础。     

新生大鼠视网膜神经元的改良培养及鉴定

目的探讨新生大鼠视网膜神经元获取和体外培养的方法,为视网膜神经细胞的基础研究提供合适的模型。方法采用酶消化法获取生后7—8天的SD（Sprague—Dawley）乳鼠的视网膜神经细胞,使用DMEM／F12培养液在体外培养,倒置显微镜观察细胞形态,免疫组织化学鉴定细胞类型。结果0．25％胰蛋白酶和0．02％EDTA联合消化能使视网膜神经细胞分散,获得单细胞悬液;多聚赖氨酸包被培养皿有助于神经细胞贴壁生长;体外培养第7天的视网膜神经细胞中,视网膜神经元占80％左右,其中50％以上为光感受器细胞。结论酶消化法原代培养视网膜混合神经细胞简单、可行,为视网膜神经细胞的基础研究提供良好的体外模型。     

体外培养视网膜神经元电压门控钾离子通道特性的研究

目的探讨体外培养不同时期新生小牛视网膜神经元电压门控钾离子通道的特性。方法分别取培养2、4、6周的视网膜神经元,进行全细胞膜片钳记录,并进行统计学分析。结果去极化刺激可诱导3组细胞产生IK电流,各组检出率差异无统计学意义(P>0.05);随培养时间延长,IK电流平均峰值增高(P<0.05)。超极化刺激可诱导培养6周的部分细胞产生内向整流钾电流。结论体外培养新生小牛视网膜神经元表达不同电压门控钾电流。某些神经元的电生理学特性在不同培养阶段发生变化。     

大鼠胚胎隔区神经元体外原代培养

目的：探讨大鼠胚胎隔区神经元培养方法。方法：建立稳定的大鼠胚胎隔区神经元体外培养模型，并观察神经元形态变化即生长过程。结果：神经元培养２小时，大部分细胞已贴壁；１２小时大部分细胞有突起伸出；２４小时部分细胞呈双极型；３天细胞开始聚集，突起互相连接成稀疏的网络；１周细胞簇明显，突起随时间延长而增长；２周细胞体积继续增大，突起连接成致密网络；３周细胞突起聚集明显，细胞数开始减少；４周细胞明显减少，仍见     

体外培养大鼠脑皮层神经元机械性损伤后mGluR1a及Homer蛋白的表达及其意义

目的：研究体外培养大鼠脑皮层神经元机械性损伤后mGluRla及Homer1a蛋白表达及其意义．方法：实验分为损伤与对照组，将SD胎鼠脑皮层神经元体外培养7d．损伤组以微量移液器塑料滴头在培养孔内“十”字形划割，造成机械性损伤，伤后不同时间点(10，30min，1，3，6，12，24h)行神经元免疫组化染色；对照组除不进行“十”字形划伤培养神经元外，其他处理同损伤组．结果：损伤组神经元mGluRla与Homerla蛋白免疫组化染色均呈阳性，自伤后10min持续至伤后24h，阳性染色颗粒分布于胞质、胞膜及突起；对照组mGluRla与Homerla蛋白免疫组化染色均呈弱阳性．结论：体外培养大鼠脑皮层神经元机械性损伤后mGluRla及Homerla蛋白表达时间与分布规律基本一致，提示Homerla与mGluRla可能共同参与了神经元损伤过程。     

新生大鼠视网膜神经元的原代培养与观察

目的：建立新生大鼠视网膜神经元体外培养的有效方法。方法：取出生后1～3?d的Wistar大鼠视网膜,胰蛋白酶+依地(EDTA)消化制成单细胞悬液,接种于置有包被多聚赖氨酸(poly L lysine)玻片的24孔培养板中培养,利用相差显微镜对培养的细胞进行动态观察;并以免疫细胞化学技术对视网膜神经元进行染色。结果：在多聚赖氨酸上培养的视网膜神经元生长良好,部分细胞伸出突起,且有些突起相互连接。免疫细胞化学染色显示,培养的细胞大多数抗神经丝蛋白（NF）抗体反应阳性。胰蛋白酶+EDTA联合消化能使视网膜细胞很好得分离,获得视网膜单细胞悬液。结论：酶消化法原代培养视网膜神经元有较好的视网膜神经元生长率;胰蛋白酶+EDTA联合消化较单独用胰蛋白酶消化效果好。     

大鼠视网膜Müller细胞的培养与鉴定

目的：仿照前人建立的方法，优化SD大鼠视网膜Müller细胞体外培养的技术和方法，为后续相关研究建立实验室条件。方法：使用胰蛋白酶消化的方法分离新生SD大鼠视网膜Müller细胞，以DMEM为培养基体外培养，光镜观察。采用电镜观察及荧光免疫组织化学染色的方法进行鉴定。结果：培养的细胞贴壁生长，原代细胞胞体狭长，细胞质丰富，折光性好。电镜下可见特征性的中间丝（直径8～10nm），细胞器丰富；荧光免疫组织化学显示细胞呈神经胶质纤维酸性蛋白（GFAP）和谷氨酰胺合成酶（GS）阳性。结论：酶消化法培养SD大鼠视网膜Müller细胞是一种稳定、可靠的方法。     

慢性粒细胞性白血病患者间充质干细胞的分离、纯化及向神经元样细胞分化的研究

目的研究慢性粒细胞性白血病（CML）骨髓间充质干细胞（MSCs）的分离、纯化、扩增以及在体外向神经元样细胞定向分化的能力。方法获取CML患者的骨髓MSCs，采用低血清培养液培养，瑞士染色观察其形态；流式细胞仪检测其免疫表型和细胞周期；逆转录-聚合酶链反应（RT—PCR）检测CML特异性表达的bcr／abl基因；分析骨髓MSCs染色体核型。全反式维甲酸（ATRA）诱导MSCs在体外分化为神经元样细胞，倒置相差显微镜观察神经元样细胞的形态，免疫组织化学染色法和Western blot法检测神经丝蛋白（NF）和神经元烯醇化酶（NSE）的表达。结果CML来源的骨髓MSCs呈纤维样贴壁生长，体外培养易扩增；表达相关抗原CD29、CD44、CD105，不表达CD31、CD13、CD34、CD45、HLA—DR；不表达bcr／abl融合基因，且具有正常的核型；不同扩增代数的MSCs在诱导剂的作用下呈现典型的神经元样细胞形态，NF、NSE呈阳性反应。结论CML患者骨髓中可以分离培养MSCs，它具有体外大量扩增并保持低分化状态的特性和定向分化为神经元样细胞的能力，是一种可用于神经系统疾病治疗的种子细胞。     

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.     

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.