肾小球系膜细胞的分离和培养

目的 分离、培养和鉴定正常人肾小球系膜细胞。方法 筛网滤过分离正常人肾小球系膜细胞，同时采用免疫荧光技术，利用异硫氰酸荧光素(FITC)间接标记单克隆抗体；抗Ⅷ因子相关抗原抗体、抗结蛋白抗体、抗角蛋白抗体、抗细胞角蛋白抗体、抗Ⅳ型胶原抗体、抗纤维连接蛋白抗体、抗层粘连蛋白抗体对系膜细胞特有的中间微丝进行鉴定。并采用同期培养的同一来源的肾小球内皮细胞作对照。结果 抗Ⅷ因子相关抗原、抗角蛋白和抗细胞角蛋白抗体表达均为阴性，而抗结蛋白抗体、抗Ⅳ型胶原抗体、抗纤维连接蛋白抗体、抗层粘连蛋白抗体为阳性，说明培养的细胞为系膜细胞。结论 该种方法分离培养的细胞经过免疫荧光染色鉴定为肾小球系膜细胞。     

大鼠肾小球系膜细胞原代培养与鉴定

目的: 原代培养及鉴定大鼠肾小球系膜细胞(GMC),探索分离、培养GMC的最佳条件.方法: 对大鼠双肾灌洗后,筛网滤过分离肾小球,并用Ⅳ型胶原酶消化处理,然后种植法体外培养大鼠肾小球.同时采用免疫细胞化学和免疫荧光技术,检测亚代GMC的α-平滑肌肌动蛋白(α-SMA)、波形蛋白(Vimentin)、细胞角蛋白(Cytokeratin)、Ⅷ因子相关抗原抗体的表达,鉴定GMC,并绘制其生长曲线.结果: 种植4 d后,约80%肾小球贴壁,可见卵圆形上皮细胞生长,18 d左右镜下细胞多呈星状或三角形并伴有不规则的突起,21 d后细胞生长密集, 细胞团簇之间形成网络.免疫细胞化学和免疫荧光证实抗α-SMA及抗Vimentin染色阳性,而抗Cytokeratin、抗Ⅷ因子抗体染色为阴性.GMC亚代倍增时间为4 d. 结论: 结果表明,用改良的培养方法成功率高,培养的细胞为大鼠肾小球系膜细胞.     

肾小球内皮细胞培养及产生细胞外基质的实验研究

作者成功地培养了肥儿及牛肾小球内皮细胞，将内皮细胞克隆后传代培养了７代，并利用间接免疫荧光方法进行鉴定，观察胎儿及牛肾小球内皮细胞都可以表达第ＶＩＩＩ因子相关抗原，纤维连接蛋白，ＩＶ型胶原及层粘连蛋白。测定了不同时胎儿肾小球内皮细胞培养上清中细胞外基质的水平。结果证实不加任何刺激因子，肾小球内皮细胞可以稳定地产生细胞外基质。     

人肾小球系膜细胞原代培养及鉴定方法的研究

目的：建立一种重复性好、传代次数多的人肾小球系膜细胞培养方法。方法：取肾脏肿瘤手术切除后远离患病部位的正常肾脏组织、水囊引产的胎儿肾,三层筛网滤过分离,取最下层筛网上组织,用胶原酶消化,培养正常人肾小球系膜细胞。同时采用形态学观察和免疫荧光技术,对鼠抗人结蛋白,鼠抗人角蛋白进行鉴定,采用MTT、 流式细胞仪技术对不同来源肾脏的系膜细胞生长状况进行比较。结果：形态学、免疫荧光法鉴定表明培养细胞为肾小球系膜细胞,胎肾原代培养后8～10d即可传代,而成年肾脏原代培养后需15～16d方可传代。成年肾脏培养细胞至第4代细胞生长缓慢,贴壁能力减弱,出现死亡。而胎肾培养细胞则可继续培养至第7～10代。结论:三层滤网法分离人肾小球系膜细胞, 方法简单、高效,培养的原代肾小球系膜细胞符合肾小球系膜细胞的生物学特性,且进一步证明胚胎组织比成年个体肾脏原代细胞培养容易成功。     

L158,809和西拉普利对体外培养系膜细胞TGF-β1表达和细胞外基质蛋白分泌的影响

目的：观察血管紧张素受体拮抗剂(ARBs)L158，809和血管紧张素转换酶(ACE)抑制剂西拉普利对体外培养人肾小球系膜细胞转化生成因子(TGF—β1)表达和纤维连接蛋白、层粘连蛋白和Ⅳ型胶原分泌的影响。方法：分别在不同葡萄糖浓度(5.6mmol／L和30mmol／L)和药物浓度(1、10、100和500μmol／L)下体外培养人肾小球系膜细胞，分别于24、48和72h后测定细胞增殖。然后将系膜细胞分为低糖(5．6mmol／L)对照组(LG)、高糖(30mmol／L)对照组(HG)、L158，809(10μmol／L)组和西拉普利(10μmmol／L)组，48h后，分别用RT-PCR法测定TGF-β1表达，ELISA和放射免疫法测定细胞上清液中TGF-β1、纤维连接蛋白、层粘连蛋白和Ⅳ胶原浓度。结果：与低糖对照组相比，高糖对照组系膜细胞过度增殖，细胞上清液中TGF-βl、纤维连接蛋白、层粘连蛋白和Ⅳ胶原浓度明显升高，TGF-βlmRNA表达也显升高；而L158，809组和西拉普利组TGF-β1和细胞外基质(ECM)蛋白水平明显低于高糖对照组，且TGF-β1mRNA水平亦表达明显降低。结论：高糖可刺激体外培养系膜细胞过度增殖，TGF-β1表达增高，ECM蛋白分泌明显增加，而L158，809和西拉普利均可抑制高糖环境下上述现象。     

狼疮肾炎中新发现的抗肾小球系膜细胞抗体靶抗原的鉴定

目的 研究狼疮肾炎血清中新发现的抗肾小球系膜细胞抗体与抗DNA抗体的关系,及其靶抗原是否存在于细胞膜上。方法 DNA纤维素柱亲和层析分离狼疮肾炎患者血清中的抗DNA抗体,Western印迹法检测祛除抗DNA抗体的血清和亲和层析纯化的抗DNA抗体与系膜细胞靶抗原的结合。从体外培养的人系膜细胞系分离细胞膜并提取膜蛋白,以已知抗肾小球系膜细胞抗体阳性血清为探针应用Western印迹法鉴定膜蛋白中的靶抗原。结果系膜细胞中相对分子质量为74000、36000的蛋白可以被祛除抗DNA抗体的血清识别。而相对分子质量为63000、91000、125000的蛋白被亲和层析的抗DNA抗体识别。系膜细胞膜上至少相对分子质量为101000、91000、74000和31000的蛋白可以被狼疮肾炎患者的血清识别。结论部分抗肾小球系膜细胞抗体与抗DNA抗体无关。人系膜细胞系的膜蛋白中存在狼疮肾炎血清可识别的抗原物质,提示抗DNA抗体以外的直接针对肾小球系膜细胞的自身抗体在狼疮性肾炎的发病机制中可能起重要作用。     

免疫组织化学研究流行性出血热肾组织中细胞骨架蛋白和Ⅳ型…

研究流行性出血热尸检肾脏中细胞骨架蛋白和Ⅳ型胶原的变化以客观反映肾脏的损伤。应用免疫组化方法，以抗平滑肌特异性肌动蛋白α链，结蛋白，波形蛋白，Ⅳ型胶原单克隆抗体评价肾小球的损伤，以抗Ⅳ型胶原，Ｖｉｍｅｎｔｉｎ单克隆抗体显示肾小管基底膜，血管和间质的改变；以抗细胞角蛋白，角蛋白，上皮细胞膜抗原单克隆抗体判断肾小管的损伤；ＰＣＮＡ和Ｖｉｍｅｎｔｉｎ显示肾小管的增生和分化。结果：ＥＨＦ肾脏肾小球固有细胞     

肾小球肾炎系膜增殖和基底膜组成变化的相关研究

应用免疫组织化学方法检测了随机选择的９５例肾小球肾炎的Ⅳ型胶原和纤维连接蛋白。结果表明，两种抗原的表达和定位可分为单纯基底膜型和系膜、基底膜复合型，系膜增殖明显的肾小球肾炎的Ⅳ型胶原和纤维连接蛋白阳性表达明显多于系膜无增殖和轻微增殖的肾小球肾炎（Ｐ＜０．０１）。Ⅳ型胶原和纤维连接蛋白在免疫球蛋白和补体沉积阳性、阴性肾炎之间无明显区别（Ｐ＞０．０５）。提示系膜增殖有助于合成参与基底膜炎症损伤修复的大分子物质。     

肾小球肾炎系膜增殖和基底膜组成变化的相关研究

应用免疫组织化学方法检测了随机选择的９５例肾小球肾炎的Ⅳ型胶原和纤维连接蛋白。结果表明，两种抗原的表达和定位可分为单纯基底膜型和系膜，基底膜复合型，墓产殖明显的肾小球肾炎的Ⅳ型胶原和纤维连接蛋白阳性表达明显多于系统膜无增殖和轻微增殖的肾小球肾炎。     

苯那普利对抗Thy1.1大鼠系膜基质积聚的影响

目的 观察血管紧张素转换酶抑制剂苯那普利对系膜增殖性肾炎（MsPGN）大鼠系膜基质的影响。方法 用抗胸腺细胞血清（ATS）诱导SD大鼠产生MsPGN后，用苯那普利治疗24d，处死大鼠并用ELISA方法测定大鼠肾小球系膜中胶原Ⅳ（ColⅣ）、胶原Ⅲ（ColⅢ）、层粘连蛋白（LN）、纤维连接蛋白（FN）的含量。结果 治疗组大鼠系膜中ColⅣ、ColⅢ、LN、FN的含量明显低于对照组大鼠系膜中各相应成分的含量，各对应成分之间两两比较差异均有统计学意义（P〈0．01）。结论 苯那普利对MsPGN大鼠系膜基质的积聚有明显的抑制作用。     

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.