人骨髓间充质干细胞的分离、培养和体外定向诱导成类软骨细胞的实验研究

目的建立一种体外分离培养、纯化扩增人骨髓间充质干细胞(MSCs)的方法,一定条件下诱导人MSCs定向分化成软骨细胞,为组织工程提供软骨种子细胞。方法用单纯的贴壁培养法与密度梯度离心法结合贴壁培养法分离纯化成年人MSCs;体外加自行配制软骨诱导剂,诱导2、4、6代MSCs细胞14d做免疫细胞化学比较;取2代MSCs不同浓度TGF-β1(5ng/ml、10ng/ml、20ng/ml),诱导14d做免疫细胞化学比较。结果软骨诱导后2、4、6代MSCs均表达Ⅱ型胶原,无显著性差异;不同浓度TGF-β1诱导2代MSCs,均表达Ⅱ型胶原,其中10ng/ml组和20ng/ml组Ⅱ型胶原呈强阳性。结论不同代的MSCs诱导率无明显差异;TGF-β1浓度为5ng/ml组Ⅱ型胶原弱阳性,不适合作为组织工程种子细胞;诱导软骨TGF-β1最适浓度为10ng/ml、20ng/ml。     

软骨形态发生蛋白1诱导真皮成纤维细胞向软骨细胞表型分化的影响因素

目的探讨软骨形态发生蛋白1（CDMP1）生长因子体外诱导真皮成纤维细胞向成软骨细胞表型分化的影响因素。方法取包皮环切术后丢弃的真皮组织共3例,分离成纤维细胞体外培养扩增,以含10％胎牛血清的F—12培养液加入CDMP1（终浓度为100ng／m1）进行诱导,未加CDMP1的作为对照。观察细胞多次传代后（P2、P5、P10）细胞表型分化情况;调整CDMP1浓度分别为10、30、100、300ng／ml,观察诱导后软骨细胞表型的变化;单层培养与微团块培养成纤维细胞分别体外诱导7、14d,逆转录-聚合酶链反应（RT—PCR）检测细胞诱导前后骨形态发生蛋白受体（BMPR）、Ⅱ、Ⅳ、Ⅹ型胶原表达;Western印迹检测细胞诱导前后Ⅱ型胶原、SOX9、蛋白聚糖的表达;流式细胞术检测表面标志CD29、CDl05、CDl06、CDl66及细胞诱导后Ⅰ、Ⅱ型胶原表达。结果细胞多次传代后（P5、P10）仍可表达特异软骨基质Ⅱ型胶原与蛋白聚糖,Ⅱ型胶原阳性细胞率与P2诱导后差异无统计学意义（P〉0．05）。CDMP1浓度为10、30ng／ml诱导7、14d,均未见Ⅱ型胶原与蛋白聚糖表达;CDMP1浓度为100、300ng／ml时,Ⅱ型胶原与蛋白聚糖表达强度差异无统计学意义。单层培养成纤维细胞诱导14d后,软骨基质表达消失;微团块三维培养诱导14d,软骨基质仍保持表达。RT—PCR检测诱导后ActR—Ⅰ／ALK-2,BMPR—IA／ALK-3,BMPR—IB／ALK-6基因表达明显增强。结论在CDMP1生长因子诱导下,人真皮成纤维细胞体外扩增后可保持向成软骨细胞表型分化能力,细胞分化与CDMP1浓度、细胞三维培养条件及BMPR介导有关。     

软骨分化形成蛋白-2体外诱导小鼠骨髓基质干细胞向软骨分化

目的 研究软骨分化形成蛋白-2(CDMP-2)对小鼠骨髓基质干细胞软骨分化的作用.方法 体外培养小鼠骨髓基质干细胞(MSCs),贴壁细胞传代,取第3代细胞,以不同浓度(0、10、20、50、100 ng/ml)CDMP-2因子干预培养14d后,倒置相差显微镜观察细胞形态,RT-PCR、Western blot和免疫细胞化学法检测不同浓度CDMP-2对Ⅱ型胶原表达的影响,阿尔辛蓝(Alcian)染色蛋白多糖.结果 RT-PCR、Western blot和免疫细胞化学显示经CDMP-2因子干预后,MSCs有Ⅱ型胶原mRNA和蛋白的表达,呈剂量依赖性;Alcian染色结果显示CDMP-2诱导细胞分泌蛋白多糖基质,高浓度组可见细胞小结区域呈明显的异染性.结论 CDMP-2能够在体外定向诱导小鼠骨髓基质于细胞向软骨方向分化,50～100 ng/ml为最佳剂量,为进一步探讨其在软骨发育机制中的作用提供了实验基础.     

CDMP1诱导成纤维细胞向成软骨细胞表型分化的实验研究

目的 应用软骨形态发生蛋白-1(CDMP1)生长因子，体外诱导真皮成纤维细胞向成软骨细胞表型分化，探讨其作为组织工程化软骨种子细胞的可行性方法取成人真皮成纤维细胞，体外扩增至第2代，以加入cDMP1生长因子(100ng／mL)的含10％胎牛血清的F-12培养液诱导，每3d换液1次，进行单层培养，对照组为不加CDMP1培养的成纤维细胞-7d后，免疫荧光检测Ⅱ型胶原分泌，RT-PCR检测Aggrecan、Ⅱ型胶原mRNA表达。结果 诱导后成纤维细胞细胞形态由梭形向软骨细胞样多角形、多边形转变。Ⅱ型胶原免疫荧光检测表达阳性，RT-PCR检测Aggrecan、Ⅱ型胶原mRNA表达阳性。结论 成纤维细胞在CDMP1生长因子诱导下能向成软骨细胞表型分化，并能分泌软骨细胞特异性基质，有可能成为软骨组织工程新的种子细胞来源。     

软骨源性形态发生蛋白-1对后纵韧带骨化作用

目的 探讨软骨源性形态发生蛋白-1(CDMP-1)对体外培养的SD大鼠后纵韧带细胞的骨化作用.方法 切取SD大鼠的后纵韧带体外培养后总人带细胞,观察体外培养条件下后纵韧带的形态学特征和表型.分别用不同浓度的软骨源性形态发生蛋白-1诱导后纵韧带细胞.对照组:CDMP-1:0ng/ml;诱导组(ng/ml):10、20、50、75、100.检测后纵韧带细胞诱导后的碱性磷酸酶活性,免疫组化染色检测Ⅰ、Ⅱ型胶原表达情况.结果 后纵韧带细胞表达成骨细胞表型,具有高碱性磷酸酶活性.软骨源性形态发生蛋白-1对后纵韧带细胞的骨化作用与浓度密切相关,免疫组织化学染色显示:软骨源性形态发生蛋白-1诱导后纵韧带细胞有Ⅱ胶原表达并随着软骨源性形态发生蛋白-1诱导剂量增加而增多,诱导组与对照组有显著性差异;诱导组Ⅰ型胶原染色深度和强度比对照组减弱,诱导组与对照组有显著性差异.结论 大鼠后纵韧带细胞体外培养可诱导向成骨细胞分化,CDMP-1在后纵韧带骨化的发生中起着重要作用.     

骨形态发生蛋白-7对椎间盘细胞Sox9和Ⅱ型胶原基因的调节作用

目的探讨骨形态发生蛋白-7（BMP-7）对椎间盘细胞Sox9和Ⅱ型胶原基因表达的调节作用。方法应用逆转录聚合酶链反应检测不同浓度BMP-7对培养的人椎间盘细胞中软骨特异性基因Sox9和Ⅱ型胶原基因表达的影响。结果在10ng/ml、100ng/ml、1000ng/mlBMP-7作用下,其对椎间盘细胞Sox9和Ⅱ型胶原基因mRNA可起到显著的正向调控作用。结论BMP-7可以按照剂量依赖方式正向调节椎间盘细胞Sox9和Ⅱ型胶原基因的表达。提示BMP-7对退变早期的椎间盘具有一定的修复功能。     

骨形态发生蛋白-7对椎间盘细胞Sox9和Ⅱ型胶原基因的调节作用

目的 探讨骨形态发生蛋白-7(BMP-7)对椎间盘细胞Sox9和Ⅱ型胶原基因表达的调节作用.方法 应用逆转录聚合酶链反应检测不同浓度BMP-7对培养的人椎间盘细胞中软骨特异性基因Sox9和Ⅱ型胶原基因表达的影响.结果 在10ng/ml、100ng/ml、1000ng/mlBMP-7作用下,其对椎间盘细胞Sox9和Ⅱ型胶原基因mRNA可起到显著的正向调控作用.结论 BMP-7可以按照剂量依赖方式正向调节椎间盘细胞Sox9和Ⅱ型胶原基因的表达.提示BMP-7对退变早期的椎间盘具有一定的修复功能.     

人骨形态发生蛋白2促兔椎间盘细胞aggrecan和Ⅱ型胶原的合成作用

目的:研究人骨形态发生蛋白2(BMP-2)对体外培养兔腰椎间盘髓核细胞Ⅱ型胶原和软骨聚集蛋白多糖(aggrecan)的影响。方法:兔髓核细胞体外分离培养,分别利用Antonopulos和Miamine法检测对照组和不同剂量hBMP-2(10,100和1000ng/ml)组胶原和蛋白多糖表达水平。RT-PCR检测细胞Ⅱ型胶原和aggrecan mRNA的表达水平。结果:加入BMP-2后可见随剂量增加胶原和蛋白多糖表达量逐渐增加,并存在剂量依赖关系(P<0.01)。在6dRT-PCR结果显示BMP-2组aggrecan和Ⅱ型胶原mRNA的表达高于对照组,并且随剂量增高aggrecan和Ⅱ型胶原mRNA的表达量逐渐增加。结论:BMP-2可促进体外培养兔腰椎间盘细胞合成代谢,随剂量增加胶原和蛋白多糖表达量逐渐增加,并存在剂量依赖关系,提示hBMP-2可能对退变椎间盘具有修复功能。     

软骨形态发生蛋白1诱导真皮成纤维细胞表达软骨细胞表型的实验研究

目的应用软骨形态发生蛋白(CDMP)生长因子,体外诱导真皮成纤维细胞向成软骨细胞表型分化,探讨其作为组织工程化软骨种子细胞的可行性.方法取包皮环切术后丢弃真皮组织共3例,成纤维细胞体外培养扩增至第二代,以1×104/cm2密度接种,12 h后加入细胞诱导液(10%胎牛血清F-12培养液,CDMP1终浓度为100 ng/ml)单层培养,未加的成纤维细胞培养用CDMP1为对照.诱导7 d后观察细胞形态变化,Image Plus图像分析软件计算细胞长宽比例.激光共聚焦显微镜分别观察诱导前后细胞内Ⅰ、Ⅱ、Ⅲ型胶原表达及分布,Western印迹检测诱导前后细胞Ⅱ型胶原蛋白表达.反转录聚合酶链式反应(RT-PCR)检测诱导后成软骨相关的Sox9、聚集蛋白聚糖与Ⅱ、Ⅸ型胶原mRNA表达;同时检测与成骨相关的X型胶原、碱性磷酸酶(AKP)mRNA表达.培养第二代细胞以2×107个细胞/ml细胞密度进行离心管法培养,诱导14 d后行阿辛蓝与Ⅱ型胶原免疫组化染色.结果诱导后可见单层培养的成纤维细胞形态由长梭形向软骨细胞样多角形转变,Image Plus图像分析软件计算细胞诱导后的长宽比例为(1.40±0.15),较诱导前(7.40±1.30)差异有显著意义(P＜0.05);与正常软骨细胞(1.29±0.24)差异无显著意义.免疫荧光激光共聚焦观察发现诱导后细胞内出现Ⅱ型胶原表达,Ⅰ、Ⅲ型胶原表达呈阳性.RT-PCR检测成软骨相关的Sox9,聚集蛋白聚糖,Ⅱ、Ⅸ型胶原mRNA表达阳性;X型胶原、AKP未检测到mRNA阳性表达.Western印迹检测细胞诱导后Ⅱ型胶原蛋白表达阳性.离心管培养诱导7 d后阿辛蓝染色示糖胺聚糖(GAG)均匀分布于基质中,免疫组化染色示基质中Ⅱ型胶原表达阳性.结论第二代成人真皮成纤维细胞在CDMP1生长因子诱导下可向成软骨细胞表型分化,并能分泌软骨细胞特异性基质,有望成为软骨组织工程新的种子细胞来源.     

IL-6对体外培养的兔软骨终板细胞生物学行为的影响

目的 探讨IL-6对体外培养的兔软骨终板细胞生物学行为的影响.方法 分离培养兔软骨终板细胞,通过甲苯胺兰染色等方法鉴定后,分别加入不同浓度的IL-6,MTT法测定不同时间点软骨终板细胞增殖活性的变化;流式细胞仪检测软骨终板细胞生长周期的变化;RT-PCR检测蛋白聚糖、Ⅱ型胶原mRNA的表达变化.结果 10、50、100 ng/ml IL-6对软骨终板细胞的增殖无影响,50 ng/ml IL-6对软骨终板细胞细胞周期无影响,10、50 ng/ml IL-6均对Aggrecan mRNA的表达无影响,仅50 ng/ml浓度时IL-6可降低Collagen Ⅱa mRNA的表达.结论 较大剂量IL-6时可抑制软骨终板基质合成,从而促进椎间盘的退变.     

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.