小鼠胚胎干细胞克隆形成和传代的影响因素

目的:探讨昆明小鼠胚胎干细胞(ESC)分离后克隆形成和细胞传代的几种影响因素,对其特性作初步鉴定.方法:对比超排卵和自然受孕2种囊胚获取方法、囊胚培养液和高糖DMEM培养液2种培养液对其克隆形成和细胞传代的影响.对贴壁后形成的原代ESC克隆进行评分.稳定传代的一株ESC进行鉴定.结果:超排卵组、自然受孕组的内细胞团形成率(64.9% vs 68.2%)和原代ECS克隆形成率(10.1% vs 14.5%)比较,差异均无统计学意义(χ2=0.345和1.385,P=0.557和0.239).囊胚培养液组24 h囊胚孵出率明显高于DMEM培养液组(56.8% vs 17.1%)(χ2=33.026,P=0.001),原代ESC克隆形成率明显低于DMEM培养液组(35.2% vs 15.9%)(χ2=9.021,P=0.002).ICM的评分与ESC克隆传代能力相关(r=0.531,P＜0.001),直径70～100 μm、周边规则、隆起明显的ICM,ESC克隆传代持久.结论:高糖DMEM培养液对克隆的分离和传代有利.对ICM贴壁后形成的ESC克隆进行评分有助于确定挑选克隆传代的时机.     

激光辅助孵化技术对囊胚期冻融胚胎移植患者妊娠结局的影响

目的:探讨激光辅助孵化技术对囊胚期冻融胚胎移植患者妊娠结局的影响。方法:选取2017年1月-2018年12月在本院行冻融囊胚解冻移植技术助孕的339例患者。按激光辅助方式不同分为A组190例和B组149例,A组行透明带削薄,B组行透明带打孔。比较两组胚胎复苏率、胚胎着床率、移植周期妊娠率以及子代出生情况。结果:两组移植胚胎数、优质囊胚率、胚胎解冻复苏率、移植周期妊娠率、胚胎着床率、流产率、活产率、出生缺陷率、早产率比较,差异均无统计学意义(P0.05)。结论:透明带削薄、透明带打孔两种激光辅助在囊胚期冻融胚胎移植中效果相近,临床可根据实际情况选择适宜的辅助孵化技术。     

免疫外科法分离克隆BALB/c小鼠胚胎干细胞

目的　使用免疫外科法分离克隆BALB c小鼠胚胎干细胞 (embryonicstemcells,ES细胞 ) ,为进一步建立BALB c小鼠ES细胞系打下基础。方法　用免疫外科法从 4 5枚BALB c小鼠囊胚中分离得到 2 0枚去除滋养层细胞的ICM ,接种在MEF饲养层上 ,使用DMEM(高糖 ) 15 ?S 0 1mmol Lβ 巯基乙醇 0 0 1mmol L非必需氨基酸 10 0 0IU mlLIF 10 0IU mL青霉素 10 0IU ml链霉素培养液 ,17枚形成典型的ICM集落 (85 0 % ) ,有一枚胚胎传至第 10代。用于全胚培养的BALB c小鼠胚胎共 10 2枚 ,使用与免疫外科相同的培养方法 ,6 6枚形成典型的ICM集落 (6 4 7% ) ,其中一枚胚胎传至第 8代。结果　免疫外科法较全胚培养法有利于小鼠ES细胞的分离与克隆 ;添加LIF(10 0 0IU ml)有利于小鼠ES细胞的分离与传代 (P <0 0 5 ) ;0 0 5 %胰酶 0 0 0 8?TA是较好的ES细胞消化液 ,对细胞综合损伤力小 ,且传代后ES细胞集落形成能力也较高 (P <0 0 5 )。结论　分离得到的ES细胞经形态学观察 ,AKP染色 ,体外分化实验 ,核型分析等证明其具有胚胎干细胞的诸多特性。     

应用卵裂期废弃胚胎培养建立人胚胎干细胞系的方法探索

目的：探讨应用卵裂期废弃胚胎,采用两种不同培养法建立人胚胎干细胞（hESC）系的方法。方法：将临床IVF及ICSI周期中剩余废弃的卵裂期胚胎继续培养至扩张囊胚,分别用免疫切割法及全囊胚培养法分离出内细胞团（ICM）,接种在用丝裂霉素处理过的小鼠胚胎成纤维细胞饲养层上,用hESC培养液培养、传代,建立胚胎干细胞系。通过胚胎干细胞生物学特性、特异性表面标志物、核型分析及体内外分化全能性等传统方法进行鉴定。结果：132枚废弃的卵裂期胚胎在体外继续培养后,有28枚发育为囊胚,随机分组14枚囊胚采用免疫切割法分离ICM,未得到干细胞系;另14枚采用全囊胚培养法,最终得到1个干细胞系,经鉴定,这些细胞都具有hESC的生物学特性。结论：①部分废弃卵裂期胚胎能够发育至囊胚期,可作为建立hESC系的材料来源之一;②全囊胚培养法建系成功率高于免疫切割法,尤其适用于评分低的囊胚。     

冻融周期囊胚移植的临床价值

目的：探讨冻融囊胚移植、冻融卵裂期胚胎复苏后行囊胚移植的临床价值。方法回顾性分析2012年9月至2013年8月在该院生殖医学中心接受冻融胚胎移植治疗患者共518个周期的临床资料,按移植冻融胚胎类型分为3组。A组：冻融囊胚移植,共129个周期;B组：卵裂期胚胎复苏后行囊胚培养后移植,共123个周期;C组（对照）：冻融卵裂胚移植,共266个周期。比较各组的临床结局及 A、B两组间新鲜周期胚胎和冻融周期的囊胚形成率和取消率。结果 A、B组的生化妊娠率、临床妊娠率和胚胎种植率（70．5％、61．2％、42．3％;67．5％、58．2％、40．2％）显著高于 C组（53．0％、42．5％、23．1％）（P＜0．05）;3组间流产率、异位妊娠率、多胎妊娠率差异无统计学意义（P＞0．05）。新鲜周期第3天优质胚胎与冻融胚胎复苏后的囊胚形成率（62．5％vs．57．7％）差异无统计学意义（P＞0．05）,均显著高于新鲜周期第3天非优质胚胎的囊胚形成率（20．3％）（P＜0．05）。结论冻融周期行囊胚移植可以获得较满意的临床结局;冻融卵裂期胚胎行囊胚培养后可获得较高的囊胚形成率和临床结局。     

表皮生长因子对小鼠早期胚胎体外发育影响的研究

目的：探讨表皮生长因子对小鼠植入前胚胎体外发育的影响。方法：用添加不同浓度EGF(epidermal growth factor)的CZB培养液对小鼠早胚进行体外培养，观察囊胚发育率和胚胎细胞数的变化。结果：HCG(human chorionic gonadotropin)注射后138h，0．1ng／ml EGF添加组扩张囊胚率、孵化囊胚率显著高于其他各组。100．0ng／ml EGF添加组扩张囊胚率和孵化囊胚率显著低于其余各组，且囊胚细胞死亡较多。结论：EGF通过刺激囊胚期细胞分化而促进胚胎体外发育。但高浓度EGF抑制胚胎体外发育并使早期胚胎受损。     

冷冻胚胎复苏移植周期不同质量和发育速度的单囊胚移植的临床结局

目的:探讨冷冻胚胎复苏移植周期中单囊胚移植时不同质量和发育速度囊胚的优先移植顺序。方法:对2015年9月至2019年12月期间就诊于郑州大学第三附属医院生殖医学中心的不孕症患者(7 195个冷冻胚胎移植周期)的临床资料进行回顾性分析。根据移植囊胚质量和发育速度的不同分为4组：D5优质囊胚组(第1组)、D5非优质囊胚组(第2组)、D6优质囊胚组(第3组)、D6非优质囊胚组(第4组);选择第2组的4期囊胚为2B组,第3组4期囊胚内细胞团(ICM)或滋养外胚层细胞(TE)为A级的为3A组,ICM和TE均为B级的为3B组。比较各组患者的活产率、临床妊娠率、胚胎种植率、流产率、异位妊娠率和多胎妊娠率等临床结局。对4组临床结局进行Logistic回归分析。结果:与第1组比较,其他组临床妊娠率、胚胎种植率、活产率降低;与第2组比较,第3组的多胎妊娠率升高;与第2和3组比较,第4组的临床妊娠率、胚胎种植率、活产率降低(P<0.05)。排除女方年龄、不孕时间、BMI、基础卵泡刺激素、移植日子宫内膜厚度等混杂因素后,以第3组为参照,第1组的临床妊娠率、胚胎种植率和活产率增加[OR(95%CI)为1....     

体外受精时程和胚胎培养方式对小鼠早期胚胎发育潜能的影响

目的 探讨体外受精时程、培养基体积以及单胚胎或多胚胎培养对小鼠早期胚胎发育潜能的影响。方法 根据体外受精时间长短的不同，将受精时程分为2组，一组精子和卵子共孵育的时间为2h（A组），另一组精子和卵子共孵育的时间为18h（B组）；根据培养基体积及培养胚胎数目的不同，将胚胎培养方式分为6组：100μl培养基＋1枚胚胎（组1），100μl培养基＋3枚胚胎（组2），50μl培养基＋1枚胚胎（组3），50μl培养基＋3枚胚胎（组4），20μl培养基＋1枚胚胎（组5），20μl培养基＋3枚胚胎（组6）。结果 ①A组的优质胚胎率和囊胚形成率均明显高于B组（P〈0.05）；②组6的优质胚胎率和囊胚形成率显著高于组1、组2和组3（P〈0.01），组4的囊胚形成率明显高于组1和组2（P〈0.05），组5的囊胚形成率明显高于组1、组2和组3（分别为P〈0.01，P〈0.05）。结论 缩短受精时间、减少培养基体积以及多胚胎群体培养有利于小鼠早期胚胎的发育。     

体外受精治疗周期中剩余胚胎囊胚培养的临床价值

目的探讨体外受精治疗周期中d3移植和冷冻后剩余胚胎的体外发育潜能。方法对剩余胚胎继续培养至囊胚期,观察其囊胚发育率和质量,并分析治疗周期中剩余胚胎囊胚形成情况和妊娠结局的关系。结果收集115个IVF／ICSI-ET周期中的603枚剩余胚胎,囊胚培养后获得了222枚囊胚（36．82％）,其中优质囊胚49枚（8．13％）;36个周期（31．30％）共获得53枚冷冻囊胚（8．79％）;12例含有剩余胚胎来源囊胚的解冻移植周期,临床妊娠4例;≥6细胞、2PN来源胚胎囊胚形成率显著高于其他组（P〈0．05）;剩余胚胎有囊胚形成的周期临床妊娠率显著高于无囊胚形成组（P〈0．05）。结论根据d3胚胎形态学评分预测其发育潜能有一定局限性;通过囊胚培养筛选出剩余胚胎中具有发育潜能的并冷冻保存,是提高病人每个治疗周期可用胚胎数的可行方法;剩余胚胎囊胚形成情况可有效预测妊娠结局。     

冻融后2-细胞胚胎发育至囊胚建立人胚胎干细胞系

目的 用解冻后废弃胚胎及部分患者捐献的冷冻胚胎建立胚胎干细胞系.方法 用程序解冻法解冻2001-2007年来我生殖中心就诊的患者剩余冷冻胚胎,培养至囊胚,Pronase酶去除透明带后,全囊胚置于小鼠饲养层(MEF)中培养获得原代细胞,机械法结合Ⅳ型胶原酶消化法传代,并利用细胞形态观察、免疫荧光染色、核型分析、畸胎瘤切片胚层分析方法对所建胚胎干细胞系进行鉴定.结果 序贯培养23枚废弃胚胎,有4枚培养到囊胚阶段;去除透明带后有2枚在MEF上贴壁生长,但只有1株成功扩增稳定传代至第80代;细胞集落呈典型的鸟巢状,有稳定的46XY核型;细胞系表达SSEA-3、SSEA-4、TRA-1-60和TRA-1-81抗原,RT-PCR检测有Nanog、Oct-4、Sox2等基因表达;同时在体外能分化为内、中、外3个胚层.结论 解冻后2～3细胞Ⅲ～Ⅳ级的废弃胚胎仍具有发育形成囊胚的潜能,并利用所得囊胚采用酶去透明带全胚培养法能建立稳定增殖的hESC细胞系.     

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.