人体脂肪干细胞培养及成骨诱导分化

目的 通过成人脂肪体外分离培养脂肪间充质干细胞（adipose-derived stem cells,ADSCs）研究其生理特性及在特定培养条件下向成骨分化,探讨脂肪源性干细胞作为骨组织工程的种子细胞的前景.方法 从成人脂肪中利用胶原酶消化法分离并体外培养干细胞,流式细胞仪检测细胞表面标记物,CCK8检测细胞活性,P3代脂肪干细胞通过成骨诱导液诱导向骨细胞分化,BCIP/NBT比色法染色检测碱性磷酸酶（AKP）,茜素红染色检测钙结节形成,RT-PCR检测碱性磷酸酶（ALP）、骨桥蛋白（OP）表达变化.结果 成人脂肪间质来源的ADSCs,能稳定传代,增殖能力旺盛;流式细胞检测证实其特异表达相关干细胞表面标记物;成骨诱导后表现出呈典型的成骨细胞形态;ALP染色阳性,茜素红染色阳性,诱导培养0、3、7、14、21、28天后RT-PCR定量检测证实细胞中ALP、OP阳性表达.结论 成人脂肪中可分离得到ADSCs,其稳定表达特异性的表面抗原,并且经过相应诱导培养后可向骨细胞分化,阳性表达OP、ALP,可作为优良的骨组织工程的种子细胞来源.     

大鼠脂肪干细胞冷冻复苏后的生物学特性及成骨细胞分化潜能的研究

目的 研究冻存复苏后大鼠脂肪干细胞（ADSCs）的生物学特性及其体外诱导成骨细胞分化的潜能。方法 冷冻保存6 个月的大鼠ADSCs 复苏后传代培养,显微镜下观察细胞形态;CCK8 检测细胞增殖,流式细胞仪检测CD44,CD105。第3 代细胞诱导成骨培养2 ～ 3 周后碱性磷酸酶（ALP）和茜素红染色,观察成骨情况。结果 复苏后ADSCs 形态类似成纤维细胞,增殖迅速。成骨诱导培养后表现出成骨细胞特性,ALP 染色活性增加,茜素红染色出现矿化结节。结论 冷冻保存复苏后的ADSCs 细胞生物性能稳定,定向诱导后可分化为 成骨细胞,可作为骨组织工程的种子细胞。     

诱导骨髓间充质干细胞向成骨细胞分化的实验研究

目的 明确大鼠骨髓间充质干细胞(bone mensenchymal stem cells,BMSCs)在体外全骨髓培养条件下的生物学特性及其向成骨细胞分化的能力,探索更为简便有效的BMSCs体外培养方法.方法 提取大鼠原代BMSCs,用酠EM完全培养基和成骨诱导条件培养基体外培养,倒置相差显微镜和HE染色观察细胞形态;台盼蓝活细胞计数绘制细胞生长曲线;ALP检测试剂盒检测ALP含量变化和von Kossa染色检测细胞矿化结节,以判断细胞是否向成骨细胞分化.结果 全骨髓培养法在体外培养的大鼠原代BMSCs贴壁生长,呈梭形或多角形;使用Dex-SaMEM向成骨诱导后的BMSCs具有与成骨细胞在体外培养时相似的特征,倍增时间延长;合成ALP能力显著增强(P<0.05),von Kossa染色出现阳性的棕褐色或棕黑色团块的钙化结节.结论 全骨髓培养法取材方便,经成骨诱导培养的BMSCs表现出成骨细胞的形态特征和生物学特性,该方法可作为骨组织工程种子细胞培养的常规方法.     

大鼠骨髓间质干细胞体外分化为成骨细胞的实验研究

目的：建立大鼠骨髓间质干细胞(mesenchymal stem cells，MSCs)体外诱导分化为成骨细胞的模型。方法：分离大鼠骨髓间质干细胞进行体外培养，观察其生物学特性。并选用一定的诱导剂诱导成骨细胞，通过形态学变化、碱性磷酸酶染色及钙沉积对成骨细胞进行鉴定。结果：大鼠MSCs细胞形态呈长梭形，成骨细胞诱导后MSC细胞形态由长梭形向多边形转变，ALP染色阳性，Von Kossa染色阳性，呈现典型的成骨细胞形态和生物学特征。结论：大鼠骨髓MSCs体外能被诱导分化为成骨细胞，为骨组织工程研究的优良种子细胞。     

大鼠脂肪干细胞体外定向诱导分化为脂肪细胞和成骨细胞的研究

目的研究大鼠脂肪干细胞的基本生物学特性及其向脂肪细胞和成骨细胞诱导分化潜能。方法取6周龄SD大鼠腹股沟处脂肪组织,胶原酶消化法分离出脂肪干细胞,体外培养。取第3代细胞,免疫细胞化学测定其CD44、CD34抗原表达,分别用成脂和成骨诱导培养液诱导其向脂肪细胞及成骨细胞分化。油红染色、碱性磷酸酶染色和vonKossa染色鉴定其分化能力。结果大鼠脂肪干细胞生长能力旺盛,表达CD44,而不表达CD34;定向诱导后,油红染色、碱性磷酸酶染色和vonKossa染色阳性。结论大鼠脂肪组织可分离培养出脂肪干细胞,能向脂肪细胞和成骨细胞诱导分化,有可能成为组织工程理想的种子细胞来源之一。     

人脂肪干细胞体外培养及向软骨细胞诱导分化的实验研究

目的:观察人脂肪基质干细胞(ADSCs)体外分离培养及成脂、成软骨诱导分化的生物学性状,探讨其作为软骨组织工程种子细胞的可行性.方法:(1)人脂肪组织来源于健康成年女性腹部吸脂术,酶消化法分离出ADSCs,体外培养扩增.(2)取第3代ADSCs,测细胞生长曲线,流式细胞仪测原代细胞表面标记;成脂和成软骨诱导分化.(3)倒置显微镜观察油红O、阿尔辛蓝(AB-PSA)染色和Ⅱ型胶原免疫细胞化学染色检测分化情况;RT-PCR检测相关标志基因表达.结果:(1)体外培养的ADSCs细胞形态均一,传代稳定.(2)经成脂诱导,细胞内出现空泡,油红O染色呈红色,RT-PCR检测到有Leptin、PPAR-γ表达;经成软骨诱导,AB-PSA染色呈紫红色,Ⅱ型胶原免疫细胞化学染色胞浆呈棕黄色,RT-PCR检测COLLⅡ、SOX9和aggrecan表达.结论:脂肪干细胞能向软骨细胞方向诱导分化,可作为软骨组织工程种子细胞.     

兔脂肪基质细胞的分离培养及其成骨活性的研究

目的探讨兔脂肪组织来源脂肪基质细胞的分离培养方法及其成骨活性．方法取成年新西兰大白兔腹股沟皮下脂肪组织1～2mL,采用机械切割及Ⅰ型胶原酶消化法从脂肪组织中分离获得脂肪基质细胞,原代培养及传代以后,以诱导培养基（内含10mmol／Lβ-甘油磷酸钠、10～8mol／L地塞米松、50mg／L维生素C）进行诱导培养．实验评估：应用形态学观察、碱性磷酸酶钙-钴法染色检测碱性磷酸酶,Von Kossa钙化结节染色检测钙化结节的形成等方法来鉴定诱导分化所得细胞的成骨活性．结果行诱导培养的脂肪基质细胞呈多层成长,形态多为梭形,细胞外基质有白色钙化结节形成;细胞增殖速度减慢,生长周期变长．碱性磷酸酶钙钻法染色及Von Kossa钙化结节染色后均表现为阳性,未诱导培养的脂肪基质细胞则表现为阴性．结论初步建立了一套南脂肪组织分离培养脂肪基质细胞的方法,并证明该细胞在体外诱导培养条件下具备向成骨细胞分化的能力．     

脂肪来源干细胞体外成骨诱导和成脂诱导分化

目的 探讨脂肪组织来源的多能干细胞(adipose tissue-derived stromal ceHs,ADSCs)的多向分化潜能.方法 取小香猪腹部皮下脂肪组织,通过Ⅰ型胶原酶消化、离心等步骤分离培养ADSCs,经过原代培养和传代培养.分别加入成骨诱导剂和成脂诱导培养.经过倒置显微镜观察诱导后细胞形态变化,并通过Gomori改良钙钴法、yon Kossa染色和油红O染色对其成骨细胞和脂肪细胞表型进行鉴定,进一步比较细胞的转化率.结果 ADSCs呈成纤维细胞样贴壁生长,其经成骨、成脂诱导培养2周后形态、体积发生明显改变.Gomori改良钙钴法染色显示其细胞质内富含碱性磷酸酶(alkalinephosphatase,ALP)颗粒,诱导后15 d和19 d,ALP阳性细胞分别为(25.0±7.5)%和(49.7±6.9)%,von Kossa染色表明聚集的细胞团中央能形成钙化结节;倒置显微镜,成脂诱导3、7、14 d后脂肪细胞转化率分别为(22.3±12.5)%、(49.6±7.1)%和(89.4±9.3)%.油红O染色可见细胞质内出现橙红色脂滴,14 d油红O染色阳性率高达(92.5±9.1)%.结论 ADSCs经体外诱导培养后可向成骨细胞和脂肪细胞分化,并具有明显的成骨和成脂表型,表明ADSCs具有多向分化潜能.     

培养的人骨髓间充质干细胞向成骨细胞、脂肪细胞分化

目的探讨体外培养的人骨髓间充质干细胞(MSCs)向成骨细胞、脂肪细胞分化的方法,以及钙结节形成、Ⅰ型胶原表达等情况. 方法分离人骨髓间充质干细胞,经过原代培养和传代培养,分别加入成骨诱导、脂肪诱导培养体系,经过倒置显微镜观测与HE染色了解诱导后细胞形态变化,并通过von Kossa染色、Ⅰ型胶原免疫组化染色、苏丹黑染色与油红O法染色鉴定细胞的性质.另取2代细胞体外培养,加入成骨诱导分化试剂,以碱性磷酸酶(ALP)为检测指标计数细胞阳性率,比较成骨诱导1、2、3、4周时细胞碱性磷酸酶的变化.结果体外培养的MSCs在合适的条件下能够向成骨细胞、脂肪细胞分化.细胞经成骨诱导2～3周时细胞碱性磷酸酶阳性率最高,达到85%.结论 MSCs体外培养在一定条件下向成骨细胞、软骨细胞与脂肪细胞分化,体外培养时形成钙结节,并表达Ⅰ型胶原.培养2～3周后细胞经成骨诱导分化细胞ALP阳性率较高.     

骨髓基质细胞体外培养的生物学特性和成骨能力初步鉴定

目的体外分离培养狗的骨髓基质细胞（BMSCs），诱导其向成骨细胞分化，初步鉴定其成骨潜能和生物学特性。方法抽取毕格犬髂骨骨髓体外分离培养获得BMSCs，DMEM、新生牛血清培养基进行原代培养，部分传代细胞以含10mmol／L地塞米松、50μg／ml抗坏血酸和10mmol／L β-甘油磷酸钠的矿化培养液诱导其向成骨细胞增殖分化。倒置相差显微镜进行细胞形态学和细胞生长增殖观察，VonKossa法染色检测体外矿化结节形成，细胞碱性磷酸酶染色检测BMSCs的成骨活性。结果体外分离培养的BMSCs经条件培养基诱导后表现出明显的成骨活性，体外矿化（骨样）结节的Von Kossa染色阳性；传代BMSCs碱性磷酸酶染色阳性。结论体外分离培养的BMSCs中含有骨源性前体细胞．传代细胞具有较强的成骨潜能。     

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.     

APPLICATION OF LORNOXICAM TO PATIENT-CONTROLLED ANALGESIA IN PATIENTS UNDERGOING ABDOMINAL SURGERIES

FOR anesthesiologis s ,treatingpostoperativepainhas alwaysbeen a problem.Althoughopioidshave been provedtobe effective,theirsideeffectscouldnotbeignored.With thedevelopmentofscienceand pharmacology,many drugs with aspectsof satisfactoryanalgesicefficacyand couldbe welltoleratedby patientshave been developed.And lornoxicamisone of them, which isa non-steroidalanti-inflammatorydrug (NSAID ), with analgesic, anti-infl-ammatory,andantipyreticproperties.Itseliminationhalf-time(3 to 5 hours) isle…     

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.     

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.