咀嚼肌卫星细胞的体外培养及生物学特性的研究

目的:探讨咀嚼肌卫星细胞的体外培养方法并研究其生物学特性。方法:取新生绿色荧光蛋白(green fluorescence protein,GFP)转基因小鼠面部咀嚼肌,采用酶消化法分离出咀嚼肌卫星细胞,体外进行原代和传代培养。采用倒置显微镜及荧光显微镜观察、测定生长曲线等指标,研究咀嚼肌肌卫星细胞的增殖与分化能力,采用骨骼肌肌动蛋白(-αsarcometric actin)单抗免疫细胞化学染色对所获得的细胞进行鉴定。结果:酶消化法适于咀嚼肌卫星细胞的分离,体外培养的咀嚼肌卫星细胞增殖速度快。免疫化学染色显示,体外培养的咀嚼肌肌卫星细胞呈阳性表达。结论:体外培养的咀嚼肌肌卫星细胞有强的增殖分化能力,能用于颌面部组织工程种子细胞库的构建。     

静电纺丝聚乳酸-乙醇酸聚合物膜与大鼠嗅鞘细胞的生物相容性研究

目的 探索静电纺丝技术制作的聚乳酸-乙醇酸聚合物（PLGA）膜与大鼠嗅鞘细胞（olfactory ensheathing cells,OECs）的生物相容性。方法 体外培养纯化成年大鼠OECs,将细胞接种于静电纺丝PLGA膜上,使用相差显微镜观察静电纺丝PLGA膜上OECs的细胞形态与分布;用CFDA SE荧光染色检测接种后1～5 d内静电纺丝PLGA膜上OECs的增殖情况;以多聚赖氨酸（PLL）包被细胞做对照。将静电纺丝PLGA膜植入大鼠体内观察其与大鼠的组织相容性。结果 体外培养纯化所得的OECs的细胞纯度大于90%。相差显微镜下示OECs在静电纺丝PLGA膜孔隙中的纳米纤维上黏附良好,细胞状态佳,且特异性地沿纳米纤维方向生长。接种后1～5 d内OECs在静电纺丝PLGA膜上均正常增殖,细胞数目与对照组相比差异无统计学意义。静电纺丝PLGA膜局部移植后大鼠无死亡,PLGA膜与周围组织融合并部分降解。结论 静电纺丝PLGA膜具有良好的生物相容性,OECs在其膜上的黏附和增殖状况良好,是有潜力的神经修复组织工程化神经移植物。     

基于信号通路网络交互研究复叶耳蕨总黄酮诱导BMSCs成骨分化的分子机制

【目的】 探究复叶耳蕨总黄酮对大鼠骨髓间充质干细胞(BMSCs)增殖、定向成骨分化的影响以及相关信号通路。 【方法】 采用差速贴壁法体外分离、纯化和培养大鼠骨髓间充质干细胞,取P3代细胞,采用MTT法检测细胞生长曲线、流式细胞术测定细胞表面标志。实验采用不同浓度的复叶耳蕨总黄酮培养基对大鼠BMSCs定向诱导分化,观察BMSCs诱导分化过程中诱导细胞的形态;测定诱导细胞碱性磷酸酶(ALP)活性和组织化学染色观察钙化结节形成能力;采用RT-PCR和ELISA法检测BMSCs诱导分化过程中的WNT和BMP通路转导体及其交汇靶点Runx2相关基因表达。 【结果】 体外培养的细胞表达BMSCs细胞表面标志;复叶耳蕨总黄酮对BMSCs增殖具有一定的抑制作用;促进BMSCs分化为成骨细胞;增强ALP活性和钙结节形成;上调WNT通路转导体β-catenin、cyclinD1和BMP通路转导体BMP2、BMP4、两条通路交互靶点Runx2以及骨桥蛋白、骨钙蛋白和Ⅰ型胶原基因表达。 【结论】 复叶耳蕨总黄酮能够上调BMP和WNT通路,促进网络靶点Runx2表达,从而促进大鼠BMSCs定向分化为成骨细胞。     

成年大鼠骨骼肌卫星细胞原代培养及鉴定

目的：探讨骨骼肌卫星细胞体外培养，鉴定的方法。方法：采用I型胶原酶和胰蛋白酶两步消化法分离大鼠骨骼肌卫星细胞，经差速贴壁法纯化后，在培养液中进行培养，观察不同血清浓度下，卫星细胞生长融合的情况，倒置显微镜观察细胞形态，利用结蛋白免疫细胞化学染色方法鉴定肌卫星细胞。结果：细胞经过两步消化和差速贴壁后，纯度在90%以上，在生长培基中，细胞分裂增生；在融合培基中，细胞发生融合，形成肌管。结论：成年大鼠肌卫星细胞在不同的培养条件下可增生或分化，结蛋白免疫细胞化学染色可以早期鉴定骨骼肌卫星细胞。     

成年大鼠骨骼肌卫星细胞的原代培养、鉴定及体外增殖

目的探讨成年大鼠原代骨骼肌卫星细胞体外培养方法,观察细胞的增殖、成肌分化特性。方法采用改进的两步酶消化法结合差速贴壁技术,获得较高纯度的骨骼肌卫星细胞。免疫组化鉴定所获取细胞的肌源性标志desmin。MTT法观察骨骼肌卫星细胞的体外增殖特性并绘制生长曲线图。同时观察不同培养条件下骨骼肌卫星细胞的成肌分化特性。结果所分离的原代骨骼肌卫星细胞纯度在90%以上。细胞体外培养时在第3天进入对数生长期,5～6d进入增殖平台期。细胞体外培养时无须特殊诱导可以自发出现成肌定向分化,相互融合形成具有自发收缩特性的肌管细胞。结论体外培养获取的骨骼肌卫星细胞数量可以满足组织工程研究的需要。     

大鼠骨骼肌卫星细胞体外培养生长和成肌特性

目的建立大鼠骨骼肌卫星细胞分离和纯化方法,并观察骨骼肌卫星细胞的体外增殖和成肌特性。方法采用改良两步酶消化法结合差速贴壁技术,获得高纯度的骨骼肌卫星细胞。免疫组化染色鉴定体外培养的骨骼肌卫星细胞;MTT法检测体外培养骨骼肌卫星细胞的生长情况;倒置显微镜观察不同培养条件下骨骼肌卫星细胞的分化特性。结果获取的骨骼肌卫星细胞纯度高,细胞中肌源性标志中间丝蛋白desm in呈强阳性表达。体外培养时,骨骼肌卫星细胞有1~2 d的潜伏期,5~6 d进入平台期。细胞汇合至60%~70%或降低培养基血清浓度时,开始相互融合形成肌管细胞。结论酶两步消化法和差速贴壁技术是一种简便易行、获得较高纯度的骨骼肌卫星细胞的方法。骨骼肌卫星细胞体外培养时无需特殊诱导即可相互融合,形成具有骨骼肌收缩特性的肌管细胞。     

大鼠骨骼肌卫星细胞体外培养生长和成肌特性

目的 建立大鼠骨骼肌卫星细胞分离和纯化方法,并观察骨骼肌卫星细胞的体外增殖和成肌特性.方法 采用改良两步酶消化法结合差速贴壁技术,获得高纯度的骨骼肌卫星细胞.免疫组化染色鉴定体外培养的骨骼肌卫星细胞;MIT法检测体外培养骨骼肌卫星细胞的生长情况;倒置显微镜观察不同培养条件下骨骼肌卫星细胞的分化特性.结果 获取的骨骼肌卫星细胞纯度高,细胞中肌源性标志中间丝蛋白desmin呈强阳性表达.体外培养时,骨骼肌卫星细胞有1～2 d的潜伏期,5～6 d进入平台期.细胞汇合至60%～70%或降低培养基血清浓度时,开始相互融合形成肌管细胞.结论 酶两步消化法和差速贴壁技术是一种简便易行、获得较高纯度的骨骼肌卫星细胞的方法.骨骼肌卫星细胞体外培养时无需特殊诱导即可相互融合,形成具有骨骼肌收缩特性的肌管细胞.     

Muse细胞体外诱导为神经前体细胞的初步研究

【目的】 从成人骨髓中分离出Muse细胞,体外诱导成神经前体细胞,为细胞移植修复神经系统损伤提供种子细胞。 【方法】 利用密度梯度离心和差速贴壁法从健康成人骨髓中分离、培养骨髓基质干细胞,通过免疫荧光细胞化学技术和流式细胞仪技术对其进行鉴定;体外扩增、传代6次后利用Muse细胞特征性的SSEA-3/CD105分子表型阳性,通过流式细胞仪从骨髓基质干细胞中分选出Muse细胞并进行体外培养,观察其干细胞成球特性;对Muse细胞进行胰蛋白酶孵育,分析其应激耐受能力;利用免疫荧光细胞化学技术和RT-PCR技术检测Muse细胞的多能干细胞标记物表达情况;通过在培养基中添加成纤维细胞生长因子、表皮生长因子等对Muse细胞向神经前体细胞方向进行体外诱导,观察神经前体细胞的干细胞成球情况,利用免疫荧光细胞化学技术和RT-PCR技术对其进行表型鉴定并观察神经前体细胞标记物的表达情况。 【结果】 从成人骨髓中分离出骨髓基质干细胞,免疫荧光细胞化学检测和流式细胞仪检测结果显示CD90表达阳性、CD45和CD11b表达阴性;通过流式细胞仪从骨髓基质干细胞中分选出约0.5%的Muse细胞进行培养,细胞聚集成球,悬浮生长,表现为胰蛋白酶耐受;免疫荧光细胞化学检测和RT-PCR检测结果显示Muse细胞表达多能干细胞标记物Nanog、Oct4、Sox2阳性;体外诱导Muse细胞向神经前体细胞方向分化,观察到细胞成球现象,免疫荧光细胞化学检测和RT-PCR检测结果显示诱导后细胞表达神经前体细胞标记物nestin、βIII-tubulin。 【结论】 从成人骨髓中成功分离出Muse细胞,具有多能干细胞特性,经体外诱导形成神经前体细胞,为以后组织工程移植修复神经损伤提供新的种子细胞。     

兔骨骼肌卫星细胞的分离、培养及成肌特性

目的建立高纯度、高丰度的兔骨骼肌卫星细胞的分离、培养方法,并对培养的肌卫星细胞体外增殖和成肌特性进行观察。方法采用改良I型胶原酶和胰蛋白酶混合消化法结合差速贴壁培养以分离、纯化1周龄幼兔小腿三头肌来源的骨骼肌卫星细胞。镜下观察肌卫星细胞生长形态,结蛋白(Desmin)免疫组化染色鉴定体外培养的肌卫星细胞,MTT法绘制细胞生长曲线,并对分离培养的肌卫星细胞成肌特性进行诱导培养观察。结果差速贴壁后的悬浮肌卫星细胞折光性强,呈透亮球形,数量较多,贴壁生长后逐渐变为长梭形;免疫组化染色检测分离培养的细胞高表达Desmin,证明为肌卫星细胞,纯度高达95%;传代培养的肌卫星细胞24 h后开始增殖,第3~7天处于对数生长期,第8天后进入平台期;不经诱导分化培养基培养的肌卫星细胞也可融合形成肌管,但形成肌管速度不及诱导分化培养组快速。结论成功建立了具有操作简便、不易污染和纯度高的兔骨骼肌卫星细胞分离、培养方法,为采用肌卫星细胞为种子细胞的再生医学研究奠定实验基础。     

壮骨止痛胶囊含药血清对大鼠成骨样细胞增殖分化影响的实验研究

[目的]观察壮骨止痛胶囊含药血清对成骨细胞增殖、分化的影响,为明确壮骨止痛胶囊防治骨质疏松症的作用机制提供实验依据。[方法]取2代培养的大鼠成骨细胞,在壮骨止痛胶囊含药血清为40%的浓度中培养。观察细胞的生长及钙结节的形成,碱性磷酸酶(ALP)测定成骨细胞增殖和分化,噻唑蓝(MTT)法测成骨细胞生长曲线。[结果]成骨细胞在6d可铺满瓶壁,30d可形成钙结节,壮骨止痛胶囊含药血清可促进成骨细胞的增殖、ALP的分泌。[结论]壮骨止痛胶囊含药血清可促进大鼠成骨细胞的增殖、分化。壮骨止痛胶囊含药血清可能通过直接促进成骨细胞增殖、分化,从而防治骨质疏松症。     

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.