曲古抑菌素干预骨髓间充质干细胞向心肌细胞特化体外实验

目的:在体外以新生大鼠心肌细胞(Cardiomyocytes,CM)与骨髓间充质干细胞((Mesenchymal stem cells,MSCs)共同培养的方式模拟体外心肌微环境,研究间质干细胞在HDAC酶抑制剂曲古抑菌素(Trichostatin A,TSA)干预后乙酰化酶活性与MSCs向心肌细胞特化之间的联系.方法:分离大鼠MSCs将已标记的MSCs与心肌细胞、分别加入不同浓度TSA干预后的MSCs与心肌细胞混合培养.贴壁法分离大鼠MSCs在体外培养纯化后进行细胞标记.培养至第4代.根据实验需要分为3组:阳性对照组,实验组,阴性对照组.用4.6-二乙酰基-2-苯基吲哚(DAPI)和5-溴脱氧尿嘧啶核苷(Brdu)对核进行双标.分别在共培养1周后用免疫荧光染色检测MSCs细胞中的心肌结构功能蛋白:肌球蛋白重链(Myosin heavy chain,MHC)、心肌组织缝隙连接的特异性组成蛋白质Connexin43(Cx43)的表达情况.结果:①MSCs与搏动的心肌细胞共培养1周,MSCs开始出现心肌样分化说明建立了心肌微环境;②阳性对照组中,MSCs经TSA干预48 h后与心肌细胞混合培养.出现心肌样分化并表达心肌结构功能蛋白表达.③阴性对照组中的部分MSCs可少量表达心肌结构功能蛋白.在共培养的7 d,经TSA诱导后经Brdu-DAPI双标MSCs的核,MHC和Cx43荧光显示分化率与阳性对照组的分化率有显著差别.结论:MSCs与经HDAC酶抑制剂干预后的MSCs在心肌微环境中,HDAC酶抑制剂对MSCs向心肌样细胞的特化有促进作用,提示乙酰化酶活性与细胞特化之间的存在联系.     

条件培养液体外诱导骨髓间充质干细胞向心肌样细胞分化的实验研究*

[目的]观察乳鼠心室肌成纤维细胞条件培养液干预体外诱导骨髓间充质干细胞(MSCs)分化为心肌样细胞的作用及其机制.[方法]分离培养及鉴定大鼠MSCs,对其进行分组诱导:单纯条件培养液组(TJ)、条件培养液1组(TJ-ZY)、条件培养液2(TJ-XFK),诱导,24h后继续培养4周,免疫细胞化学法鉴定心肌特异性肌钙蛋白T(cTnT)的表达,实时荧光定量逆转录-聚合酶链反应(RT-PCR)法检测心肌早期基因NKX2.5、GATA-4、Desmin和α-actin mRNA的表达.[结果]免疫细胞化学结果显示单纯条件培养液诱导后的MSCs不表达心肌特异性蛋白cTnT,心复康参与组则表达.实时荧光定量(RT-PCR)结果显示各组均表达心肌分化早期基因,心复康参与组诱导后的MSCs的心肌分化早期基因表达明显高于单纯条件培养液组.[结论]微环境在MSCs向心肌样细胞的分化过程中起了重要作用,心复康能够提高MSCs向心肌样细胞的分化和特化作用.     

心肌细胞培养环境对骨髓间充质干细胞定向分化成心肌样细胞有促进作用

目的:研究心肌细胞培养微环境对骨髓间充质干细胞(MSCs)定向分化成心肌样细胞的影响.方法:取SD大鼠骨髓,通过贴壁法进行骨髓间充质干细胞培养,经多次传代后获得较纯的MSCs.用5-氮胞苷(5-aza)进行诱导分化,实验组为在玻片上的5-aza干预的MSCs移入到心肌细胞培养环境中;对照组为5-aza干预的MSCs.显微镜下观察两组形态变化,并通过免疫组化鉴别心肌心肌特异蛋白Desmin和肌钙蛋白Ⅰ(cTnI)表达及其出现时间的早晚.结果:对照组MSCs诱导后10天可见细胞由原来扁平多角形变成长梭形,15天后部分细胞体明显增粗,可见到有些细胞间有融合样情况发生,20天左右类似肌管样细胞出现.而实验组在17天左右可以看到类似心肌管样细胞.免疫组化表明实验组的阳性结果出现的时间比对照组要早.结论:心肌细胞培养环境对骨髓间充质干细胞定向分化成心肌样细胞有促进作用.     

心肌细胞介导骨髓间充质干细胞的心肌样分化

目的探讨骨髓间充质干细胞(mesenchymal stem cells,MSCs)在不同培养条件下定向分化过程中的生物学特性变化及其机制.方法贴壁法分离大鼠MSCs,培养至第6代.实验分3组:条件培养基组,用新生鼠心肌细胞培养上清液制成的条件培养基处理MSCs48 h 2次,其间间隔48 h;混合培养组,新生鼠心肌细胞和MSCs按1:1的比例混合培养1周;对照组,MSCs常规培养.检测各组MSCs中心肌细胞结构功能蛋白:心肌肌联蛋白(titin)、肌球蛋白重链(myosin heavy chain,MHC)、心肌组织缝隙连接的特异性组成蛋白质Connexin43(Cx43)的表达情况.结果①条件培养基可诱导MSCs心肌样分化,表达心肌细胞骨架蛋白titin的Z带及A带部分,Cx43大量表达,但在检测期内未观察到MHC的表达;②MSCs在混合培养中与心肌细胞形成连接,表达titin,部分细胞内可检测到类似肌小节的结构,Cx43集中分布于细胞相邻的界面;③对照组中的部分MSCs可少量表达Cx43及titin.结论源于心肌细胞的各种因素可促使MSCs心肌样分化,分化的程度取决于作用信号的性质.     

直接与间接接触对诱导骨髓间充质干细胞成心肌样分化的影响

目的　观察心肌细胞 (cardiomyocytes ,CM)与骨髓间充质干细胞 (bonemarrowstromalcells ,MSCs)直接接触和非直接接触对诱导MSCs成心肌样分化的影响。方法　体外以新生大鼠CM与MSCs直接接触和非直接接触的培养方式模拟心肌微环境 ,分析MSCs形态和结构蛋白表达等方面的变化。结果　直接接触共培养的MSCs与CM同步搏动 ,且心肌特异性肌钙蛋白T染色阳性 ,阳性率为 1.3 % ;而心肌细胞条件培养液无法单独诱导MSCs的横向分化。结论　与CM细胞间的直接接触是诱导MSCs分化为心肌细胞的必须条件 ,条件培养液则非关键因素。     

骨髓间充质干细胞体外诱导分化为心肌细胞的初步实验观察

目的体外培养并诱导大鼠骨髓间充质干细胞(MSCs)分化为心肌细胞。方法采用密度梯度离心法和贴壁培养法分离、纯化大鼠骨髓MSCs,在第3代细胞以5-氮杂胞苷进行诱导,用免疫细胞化学的方法鉴定肌细胞及心肌细胞特异蛋白的表达。结果诱导后细胞体积较诱导前增大,而且更加细长,呈长梭形。14d细胞之间出现连接,排列方向渐趋一致。免疫细胞化学染色显示Desmin,α-SarcomericActin和心肌特异性cTnI呈阳性反应。结论MSCs可能具有表达心肌细胞的特异性启动或分化调控基因,5-氮杂胞苷能在体外诱导MSCs向心肌细胞分化。     

与胎儿心肌细胞共同培养诱导人胚胎干细胞发生心肌样分化

目的 了解与胎儿心肌细胞共同培养对人胚胎干细胞(embryonic stem cells,ESCs)向心肌方向分化的影响.方法 通过酶消化法从流产胎儿中分离培养出胎儿心肌细胞及成纤维细胞,将后者用丝裂霉素处理后制备成滋养层;收集辅助生育中心施行试管婴儿后所剩余的健康胚胎,取内细胞团接种在滋养层上分离培养出人ESCs.用荧光原位杂交(fluorescent in situ hybridzation,FISH)方法来检测上述两种细胞的XY染色体,选用不同性别的胎儿心肌细胞与2～5代人ESCs以1∶2的比例混合,然后以5×104/cm2的密度接种于培养皿中进行共同培养,诱导其向心肌方向的分化.将未与胎儿心肌细胞混合的人ESCs以相同密度接种于培养皿中作为阴性对照.至第5天收集共同培养的细胞,进行双重染色,即 FISH染色及心肌特异性蛋白Desmin或cTnI免疫组化染色.结果 在与胎儿心肌细胞共同培养5 d后,40%～50%的人ESCs在心肌细胞的诱导下表达心肌特异性蛋白Desmin或cTnI,而没有进行共同培养的人ESCs不表达上述蛋白.结论 与胎儿心肌细胞的共同培养能诱导人ESCs发生向心肌方向的分化,人ESCs有望成为心肌干细胞移植的细胞材料.     

人参总皂甙诱导骨髓间充质干细胞分化为心肌样细胞的实验研究

【目的】观察人参总皂甙(GS)对骨髓间充质干细胞(MSCs)分化为心肌样细胞的诱导作用。【方法】取成年SD大鼠骨髓细胞,采用密度梯度分离法与贴壁法结合获取MSCs,进行原代培养和克隆培养;采用流式细胞仪检测细胞表面抗原 CD34和CD44以鉴定该法所获细胞是否为MSCs;对传至第8代的MSCs进行分组诱导,分别为GS组(终末浓度为 250 mg／L)、5-氮胞苷(5-aza)组(终末浓度为10μmoL／L)、GS 5-aza组(终末浓度分别为250 mg／L、10μmol／L),每组再分 3个亚组,分别对MSCs进行24、48、72 h诱导,并设空白对照组;采用共聚焦荧光显微镜观察细胞数及阳性细胞数,计算心肌样细胞转化率;采用免疫组化法检测分化的心肌样细胞的特异性蛋白结蛋白(Desmin)和肌钙蛋白(cTnI),采用逆转录聚合酶链反应(RT-PCR)检测诱导前后心肌细胞特征性基因α-心肌肌球蛋白重链(α-MHC)和β-心肌肌球蛋白重链(β- MHC)的表达。【结果】分离培养后的细胞生长密集,形态呈纺锤状,表面抗原鉴定为CD44表达阳性,表明所获细胞为 MSCs;经GS、5-aza诱导后,MSCs分化成类心肌细胞,3组的心肌样细胞转化率无显著性差异(分别为38％、35％、39％); 免疫组化和RT-PCR检测结果显示Desmin、cTnI和MHC表达均阳性,阳性细胞呈梭形和成纤维细胞样形态,表明MSCs在 GS体外诱导下已向心肌样细胞转化,并具有心肌样细胞的特性。【结论】GS可体外诱导MSCs分化为心肌样细胞,为细胞移植治疗心肌梗死提供了实验依据。     

心肌直接接触诱导骨髓间充质干细胞成心肌样分化

目的 应用新生大鼠心肌细胞(CM)与骨髓间充质干细胞(MSCs)体外共培养的方法模拟心肌内环境,研究模拟环境下MSCs分化为CM的相关因素及其适宜条件。方法利用CM与MSCs不同比例直接接触和非直接接触共培养的方法模拟心肌微环境,分析MSCs形态和结构蛋白表达等方面的变化。结果直接接触共培养的MSCs与(2M同步搏动,表达心肌特异性肌钙蛋白T,且MSCs与CM等比例培养时分化率最高;而在缺乏直接接触条件下,单纯的CM条件培养液无法单独诱导MSCs的横向分化。结论与CM间的直接接触为诱导MSCs分化为CM的必需条件,共培养的细胞密度对于诱导分化率有影响,条件培养液则非关键因素。     

在心肌细胞/MSCs共培养体系中MSCs出现心肌分化表型

目的:观察MSCs在心肌细胞/MSCs共培养体系中向心肌细胞的分化情况.方法:体外分离培养大鼠心肌细胞,然后将传代后大鼠MSCs与心肌细胞混合培养,建立心肌细胞/MSCs共培养体系,同时以心肌细胞培养上清液及成纤维细胞培养上清液为诱导因素作对照.培养1周后行免疫细胞化学染色,鉴定MSCs中心肌特异性cTnT蛋白的表达.结果:在心肌细胞/MSCs共培养体系中,MSCs与心肌细胞交织成网状,部分MSCs出现cTnT的表达,而对照组MSCs未检出cTnT的表达.结论:体外模拟的心肌环境可以诱导MSCs向心肌细胞分化并表达心肌细胞特异性标志物,诱导MSCs向心肌分化的分子信号可能是心肌细胞膜蛋白.     

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.