常染色体显性多囊肾疾病行胚胎植入前遗传学诊断的实验研究

目的：建立由PKD1突变所致常染色体显性多囊肾疾病（autosomal dominant polycystic kidney disease,ADPKD）的胚胎植入前遗传学诊断（preimplantation genetic diagnosis,PGD）方法。方法：①通过微卫星连锁分析确定2个多囊肾家系的ADPKD致病基因。检测的微卫星包括为与PKDI连锁的KG8、SM6、CW4和CW2以及与PKD2连锁的D4S1534、D4S1563、D4S414和D4S423。②对18个淋巴细胞和1个PKD1突变所致ADPKD成员行常规体外受精胚胎移植后的5个废弃胚胎15个卵裂球行多重巢式PCR和毛细管电泳检测与PKD1连锁的微卫星分型。结果：①KG8、CW4和CW2可作为连锁微卫星分析外周血和单个细胞的PKD1突变;②2个家系的致病基因均为PKD1;③单个卵裂球扩增成功率为86.67％（13／15）,单个淋巴细胞扩增成功率为88.89％（16／18）,CW4等位基因脱扣率为25％（4／16）,CW2未发现等位基因脱扣,均未发现污染,2个胚胎携带致病基因。结论：PKD1连锁的微卫星分型可作为PKD1突变所致ADPKD的PGD诊断方法。     

胚胎植入前遗传性疾病筛查的方法学研究

目的探索一种准确可行的胚胎植入前用性别检测方法来筛查X-连锁遗传性疾病.方法采用巢式PCR和引物延伸预扩增后续PCR检测单个淋巴细胞和单个胚胎细胞的性别决定基因(sex determining region on Y,SRY).结果两种方法在检测单个淋巴细胞和单个胚胎细胞的性别决定基因时都具有较高的特异性和敏感性,统计学分析无显著性差异.结论上述两种检测方法均可用于临床胚胎植入前遗传学诊断的性别检测.     

多重巢式聚合酶链反应在β地中海贫血植入前遗传学诊断中的应用

目的探讨应用多重巢式聚合酶链反应(PCR)技术在β地中海贫血(简称β地贫)植入前遗传学诊断(PGD)中的应用。方法获取β地贫基因携带者单个淋巴细胞,建立了稳定的单细胞多重巢式PCR检测技术,可同时检测β珠蛋白基因及与β珠蛋白基因紧密连锁的HumTH01基因,并对4例已出生的重型β地贫患儿及双方均为β地贫基因携带者的夫妇应用多重巢式PCR进行了β地贫的PGD。结果利用单细胞多重巢式PCR,可以同时检测中国人常见的16种β地贫突变类型,单个淋巴细胞平均扩增效率为91．3％,平均等位基因脱扣(ADO)率为17．0％。对4对夫妇进行4个周期PGD,共活检33个胚胎,获得33个卵裂球,其中30个卵裂球扩增成功,扩增效率为90．9％,ADO率为13．3％。26个胚胎经PCR分析后获得明确诊断,移植了8个胚胎,获得1例临床妊娠。孕17周时经脐带血穿刺,证实为完全正常胚胎,现已出生1名正常女婴。结论应用单细胞多重巢式PCR技术可对β地贫进行植入前遗传学诊断,达到优生目的。     

运用单细胞聚合酶链式反应进行植入前诊断的性别鉴定

目的：建立一种高效、快速、可靠的植入前诊断的性别鉴定方法。方法：取成人外血血单个淋巴细胞和人胚胎单卵裂球，采用多重荧光PCR和多重巢式PCR同时扩增性别识别位点Amelogenin和5号染色体上的基因IL－3。结果：男性单个淋巴细胞荧光PCR扩增效率为72％，巢式PCR扩增效率为64.7%；女性单个淋巴细胞荧光PCR扩增效率为76％，巢式PCR扩增效率为65％，两者与供体外周血gDNA扩增结果的一致性均达100％。单卵裂球荧光PCR扩增效率为86.9%，巢式PCR扩增效率为85.7%，来源于同一胚胎的不同单卵裂球扩增结果的一致性达100%。巢式PCR与荧光PCR的扩增效率比较无显著性差异（P＞0.05）。结论：巢式PCR和荧光PCR各有优劣。但荧光PCR操作简便、耗时短、灵敏度高，为今后的发展方向。     

单细胞PCR对种植前胚胎的性别鉴定

目的：用单一胚胎细胞进行人类种植前胚胎的性别鉴定。方法：巢式ＰＣＲ技术同时扩增单个活检细胞Ｙ染色体上的ＳＲＹ序列及位于７号染色体上的ＺＰ３基因。结果：卵裂期胚胎活检所得的２４个单细胞中５个已变性，无ＤＮＡ扩增，其余１９个中１１个显示ＳＲＹ和ＺＰ３基因阳性，８个显示ＺＰ３基因阳性而ＳＲＹ阴性。结论：选择ＳＲＹ和ＺＰ３基因引物对单个细胞进行巢式ＰＣＲ扩增是种植前胚胎性别鉴定的一种可靠的方法     

对孕妇外周血中的单个有核红细胞及游离DNA来源的鉴定

目的：探讨孕妇外周血中单个胎儿有核红细胞及游离DNA在非创伤性产前诊断的可行性。方法：对116个孕妇外周血进行检测。（1）对51例14－26孕周的妇女外周血经密度梯度离心后用显微操作分离单个有核红细胞。（2）提取65例孕妇（5－40孕周）外周血血浆DNA。（3）应用巢式聚合酶链反应（PCR）扩增单个有核红细胞的男性SRY基因,应用引物延伸预扩增（PEP）法及巢式PCR扩增孕妇血浆游离DNA的SRY基因。结果（1）分选单个有核红细胞的成功率为90．20％（46／51）。（2）单细胞SRY基因的结果与胎儿实际性别的符合率为82．61％（38／46）,敏感性为80．00％（24／30）,特异性为87．50％（14／16）。（3）游离DNA的SRY基因的结果与胎儿实际性别的符合率为90．77％（59／65）,敏感性为89．13％（41／46）,特异性为94．74％（18／19）。结论（1）孕妇外周单个有核红细胞及游离的DNA可来自胎儿,它可成为产前诊断的胎儿物质来源。（2）单细胞分离技术使孕妇外周血中的胎儿有核红细胞的分选纯度几乎达到100％。解决了母胎细胞混合的难题,为非创伤性产前诊断提供了一条新思路。     

孕妇外周血血浆胎儿DNA的检测

目的：建立可靠有效的检测孕妇血浆胎儿DNA的方法,探讨其在非创伤性产前诊断中的价值。方法：采用引物引伸预扩增（PEP）法及巢式PCR技术对65例孕妇外周血血浆DNA进行正常男性SRY基因检测。结果：单用巢式PCR技术和联合应用PEP,PCR技术对怀男胎的孕妇血中SRY基因检出率分别为65．2％（30／46）和89．1％（41／46）,两组差异有极显著性,对怀女胎孕妇血中SRY基因的未检出率为78．9％（15／19）和94．7％（18／19）,两组差异无显著性。结论：应用PEP法可对胎儿DNA进行全基因组扩增使DNA模板量增加,并使继后的PCR技术检测SRY基因的敏感性明显提高,PEP法是解决母血中胎儿细胞数量太少的途径之一,孕妇血浆中胎儿DNA可成为非创伤性产前诊断的胎儿物质来源。     

多重置换扩增在植入前胚胎性别诊断中的应用

目的 用多重置换扩增(MDA)方法,对植入前胚胎的单个卵裂球全基因组扩增后进行植入前胚胎性别的遗传学诊断.方法 用单细胞多重置换扩增方法对20个胚胎共20个单卵裂球进行全基因组扩增,用荧光PCR检测SRY、AMEL、XHPRT及X22 4个性别相关的基因座,检测胚胎性别,同时用同一胚胎另一卵裂球FISH结果作为对照.结果 20个卵裂球MDA均扩增成功,扩增成功率100％.20个胚胎均得到诊断,性别诊断成功率100％,其中12例为男性胚胎,8例为女性胚胎,与FISH结果一致率100％,诊断准确率100％.结论 可以用单卵裂球MDA后荧光PCR检测多个基因座的方法对胚胎性别进行快速、准确的植入前诊断.     

杜氏肌营养不良症(DMD)的植入前遗传学诊断

目的 对杜氏肌营养不良症(ducherule muscular dystrophy,DMD)进行植入前遗传学诊断(preim-plantafton genetic diagnosis,PGD),阻断DMD患儿的出生.方法 针对患者的DMD基因的48号外显子缺失位点采用巢式PCR分别对患者和携带者的单个淋巴细胞、行体外授精一胚胎移植治疗的健康志愿捐献者的单个卵裂球细胞进行扩增,建立稳定的经单细胞基因诊断DMD的方法 .再对在该中心进行超排和体外授精一胚胎移植治疗的DMD携带者的胚胎活检后完成PGD,根据诊断结果 选择健康的优质的胚胎移植入子宫.结果 携带者的单个淋巴细胞的PCR扩增成功率为90.5%(95/105),健康志愿捐献者的单个卵裂球细胞PCR扩增成功率为85.7%(54/63),假阳性率为0(0/28).分别对3例DMD携带者施行了植入前遗传学诊断,病例1移植了2枚未受累的优质胚胎,且成功妊娠单胎并已于2005年4月分娩了1个健康的女婴;病例2移植了2枚优良胚胎,不幸的是未能妊娠.病例3诊断为未受累的仅有的2枚胚胎因胚胎质量差而未能移植.结论 该组采用的方案可对DMD基因的48号外显子缺失突变的DMD家庭进行PGD,达到了阻断DMD惠儿出生的目的 .     

单细胞水平β地中海贫血诊断膜芯片检测技术的探讨

目的 对在单细胞水平β地中海贫血诊断膜芯片检测技术进行探讨。方法 采用15个碱基的随机引物对单个细胞的基因组进行引物延伸预扩增(Primer extension preamplification,PEP)，取其产物5μl分别对β地中海贫血的目的基因片段进行巢式或半巢式扩增，然后来用反向斑点杂交(Reverse dot blot,RDB)技术检测β地贫突变型。结果 将这些技术应用于4例已经确定突变基因型的β地贫患者及1例正常女性，检测结果与预料的相符。结论 结合我们的实验结果，提示在单个细胞或微量DNA模板水平运用基因芯片技术进行遗传病检测是可行的，而且将来在胚胎植入前基因诊断和无创性产前诊断领域可能有一定的实用价值。     

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.