体外培养的婴幼儿MSCs生物学特性研究

目的探讨体外分离和培养的婴幼儿骨髓间充质干细胞(MSCs)的基本生物学特性. 方法无菌条件下抽取先心病婴幼儿骨髓,经密度梯度离心、接种获得MSCs,观察其形态、贴壁率、克隆形成能力、超微结构和生长曲线,流式细胞仪检测细胞表面标记物.结果体外培养的MSCs 3 ～ 4 h后开始贴壁,贴壁率为65% ～ 80%;2 ～ 3 d可见集落形成,10 d左右融合90%以上,随着传代次数增加, 克隆形成率逐渐降低.透射电镜观察显示体外培养的MSCs具有MSCs典型的超微结构,其表面标记物CD29、CD44、CD71、CD90表达阳性.结论体外分离培养的先心病婴幼儿MSCs生长稳定且增殖较快,可作为体外构建工程化先心病修复材料的种子细胞来源.     

巴马香猪骨髓间充质干细胞的分离扩增及初步鉴定

目的建立巴马香猪骨髓问充质干细胞（mesenchymal stem cells,MSCs）分离、培养、扩增的方法,并进行鉴定。方法采用梯度离心法分离并结合贴壁筛选法传代纯化培养猪的MSCs;采用倒置相差显微镜观察MSCs的形态学特征;流式细胞仪检测第3代细胞表面标志抗原CD71、CD34的表达率。结果原代培养的MSCs于6h后贴壁,贴壁细胞呈集群生长趋势。培养7～9d后可见细胞融合,14～21d达到95％融合。第3代细胞表面标记物CD71阳性率为95％,CD34阳性率为O％。结论采用全骨髓梯度离心法分离并结合贴壁筛选法能够成功分离和培养巴马香猪的MSCs,在第3代可获得高纯度MSCs。     

人骨髓间充质干细胞体外培养及生物学特性观察

目的：观察体外培养的人骨髓间充质干细胞(MSCs)部分生物学特性。方法：自健康人骨髓中分离出MSCs,进行原代培养,第5d首次更换培养液,以去除未贴壁细胞,以后每周换液2次,细胞铺满整个培养瓶底90％时开始传代培养。倒置相差显微镜下观察原代及传代细胞形态及生长特性,并绘制传代细胞的生长曲线。流式细胞仪检测第3代MSCs细胞表面分子CD29、CD44,以进行MSCs鉴定。结果：MSCs体外培养为贴壁生长。MSCs原代培养约18～21d才达传代要求;传代培养细胞生长潜伏期仅24～36h,对数增长期约3～5d,之后为平台期,4～6d传1代。传至第8代,出现凋亡征象。第3代MSCs细胞表面CD29、CD44的表达率分别为90．6％和91．5％,MSCs均一性较高。结论：从人骨髓中分离出的MSCs在体外环境中具有很强的自我更新和分裂增殖能力。     

大鼠骨髓间充质干细胞的生物学特性研究

目的 建立大鼠骨髓间充质干细胞(MSCs)分离和培养扩增的方法。研究MSCs的生物学特性，为利用MSCs作为种子细胞治疗多种疾病提供实验基础。方法通过密度梯度离心、贴壁筛选法分离培养大鼠MSCs，测定其接种贴壁率；在倒置显微镜下连续观察细胞的形态变化，利用MTT法测定MSCs生长曲线；并应用流式细胞术测定细胞周期和表面标记物。结果MSCs体外培养生长状况良好，具有活跃的增殖能力，接种10h后贴壁率为96％以上；细胞周期显示有91．4％处于G0／Gl期；培养的MSCs阳性表达CD29、CD44，阴性表达CD34、CD45。结论在实验条件下，体外培养8代以前的MSCs生长稳定，增殖较快，细胞周期表现出较早期细胞特点，可作为组织工程的种子细胞。     

骨髓间充质干细胞的分离培养和生物学性状

目的建立一种体外分离纯化和培养扩增人骨髓间充质干细胞(mesenchymal stem cells,MSCs)的方法,并探讨其生物学特性,为MSCs的进一步应用研究提供数据.方法抽取人骨髓后用Percoll分离液经密度梯度离心法分离得到单个核细胞,以105个细胞/cm2密度接种,培养液为含10%胎牛血清的L-DMEM.倒置相差显微镜及吉姆萨-瑞氏染色观察其形态,MTT法测其生长曲线、流式细胞仪检测细胞周期及表面标记物.结果培养的MSCs 12h后换液,有部分细胞贴壁生长,4～5d可见有集落形成,14～20d左右可达到80%～90%融合,倒置相差显微镜及吉姆萨-瑞氏染色可见细胞形态为梭形、多角形及纺锤状等,生长曲线图显示MSCs增殖活跃,流式细胞仪检测显示约有86%的细胞处于G0、G1期,表面标记物中CD14、CD34、VLA-1、CD45和HLA-DR阴性,而CD44、CD105和CD29阳性.结论成功地建立了一种分离培养人骨髓MSCs的方法,获得的细胞形态多样、增殖稳定.     

小鼠骨髓间质干细胞的生物学特点及分化潜能鉴定

目的：建立一种体外分离，培养和鉴定小鼠骨髓间质干细胞的方法，探讨体外培养中间质干细胞的一些生物学特点，为利用MSCs促进创伤修复提供实验基础，方法：分离5－6周龄的小鼠胫骨，股骨，用预冷的IMDM培养基冲洗出骨髓，经密度梯度离心得到骨髓单个核细胞，接种后12－16d形成意志贴壁的成纤维状细胞，体外多向诱导分化鉴定分离的细胞，用传代的细胞进行生长曲线的测定，观察其接种贴壁率，检测细胞周期和超微结构。结果：体外传代培养的MSCs具有分化形成成骨和成脂细胞的能力；MSCs倍增时间约为38h，传代后12h贴壁达90％以上，细胞周期显示有80％细胞处于G0／G1期，并用电镜照片显示其超微结构。结论：在本实验条件下，体外培养的MSCs具有多向分化潜能,体外生长稳定，传代后的细胞适应性强，增殖较快，超微结构和细胞周期表现出较早期细胞特点，可望用于自体创伤促愈。     

人骨髓间充质干细胞的分离培养及部分生物学特性的实验研究

目的 建立一种体外分离和培养人骨髓间充质干细胞(MSCs)的方法，并探讨其部分生物学特性。方法 无菌条件下抽取正常健康人骨髓3-5ml，经密度梯度离心得到单个核细胞，接种入含10％胎牛血清的IMDM培养液，测定其生长曲线，并观察其贴壁率、细胞周期和超微结构变化。结果 培养的MSCs3-4h开始贴壁，贴壁率为60％-80％，2-3d即可见集落形成，14d左右融合90％以上，基本上为梭形成纤维细胞状形态，流式细胞检测显示有70．03％的细胞处于G0/G1期，电镜观察表现出早期细胞的特点。结论 成功地建立了一种分离培养人骨髓MSCs的方法，获得的细胞形态单一、生长稳定、增殖较快，为进一步应用MSCs促进创伤修复提供了实验基础。     

大鼠骨髓间质干细胞的分离培养及生物学特性研究

目的建立大鼠骨髓间质干细胞(MSCs)最佳的体外分离培养方法,并研究其基本的生物学特性。方法采用密度梯度离心和贴壁培养两种方法分离大鼠的MSCs,进行形态学观察及增殖生长方面的测定;免疫细胞化学染色鉴定表面标志。结果大鼠MSCs体外培养呈梭形;细胞生长曲线为“S”型;密度梯度离心法原代培养15d达到80%～90%的融合,而改良的直接贴壁法只需要5d;细胞周期显示G0/G1期88.29%;免疫细胞化学染色显示CD44表达阳性,CD34、CD45为阴性。结论两种方法均可获得骨髓间质干细胞,但贴壁培养法操作更简便、快速,具有对细胞活性影响较小,更利于其贴壁和增殖的优点。MSCs体外培养条件下生长性状稳定,适于做组织工程的种子细胞。     

SD大鼠骨髓间充质干细胞体外培养、表型鉴定及标记

目的：探讨SD大鼠骨髓间充质干细胞(MSCs)体外分离培养、表型鉴定和标记的方法,为进一步研究MSCs干预器官移植的免疫排斥奠定基础。方法：采用直接贴壁法分离培养MSCs;通过流式细胞术检测细胞表面标志抗原CD90、CD45的表达率对培养的 MSCs进行表型鉴定;DAPI标记第3代MSCs,观察标记效率。结果:体外培养的原代MSCs 48 h内可见有少量贴壁细胞,7～10 d达到90%汇合;流式细胞术检测,第3代MSCs表面标记物CD90、CD45的阳性率分别为99.8%、6.8%, 提示MSCs表达CD90而不表达CD45;用DAPI进行细胞标记后,荧光显微镜下见所有MSCs均已被标记蓝色荧光,提示DAPI标记法敏感性好,标记效率高。结论：贴壁培养法是一种简便易行的培养MSCs方法,操作简单,成功率高,可以作为培养SD大鼠MSCs的常规方法;DAPI标记可作为标记MSCs的一种有效手段。     

兔骨髓间充质干细胞的分离、体外培养、鉴定和体外标记

目的：探讨分离、体外培养、鉴定及体外标记兔骨髓间充质干细胞（bone marrow-derived mesenchymal stemcells,MSCs）的方法,为进一步的实验研究打下基础。方法：选择健康幼龄新西兰大耳白兔,于双侧髂后上嵴及胫骨上端内侧部行骨髓穿刺提取骨髓。采用全骨髓培养法（直接贴壁法）分离培养MSCs,对第2、3、4、5、6代MSCs应用MTT法测定生长曲线,分析MSCs的生长规律。对MSCs进行形态学观察,通过流式细胞术对CD34、CD44、CD45、CD105这4种MSCs表面抗原进行鉴定,证明所培养的细胞为MSCs。采用5-溴脱氧尿嘧啶核苷（5-Bromo-2-deoxyuridine,BrdU）体外标记兔MSCs,检测其标记阳性率。结果：全骨髓培养法培养的原代MSCs接种4天后可以被观察到,形态均匀成梭形,生长增殖迅速,符合MSCs生长的特性,7～8d MSCs融合接近80%,传代培养生长良好。MSCs生长曲线呈S型,由生长曲线分析可知,MSCs在培养第4～8d为高速生长期,MSCs在第3～5代生长最为旺盛。经流式细胞术鉴定,CD34（-）、CD45（-）、CD44（＋）、CD105（＋）,证明所培养的细胞为较纯的MSCs。BrdU体外标记兔MSCs的阳性率达到85%～90%。结论：应用本实验方法,可以分离、纯化培养兔骨髓间充质干细胞且操作简便,效率高,经济实用。所培养的MSCs体外生长稳定、增殖速度快、贴壁率高、可连续传代,可用于MSCs功能及应用的进一步研究。应用BrdU体外标记兔MSCs是安全可靠的。     

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.     

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.     

The clinicopathological and immuohistochemical analysis of solid-pseudopapillary tumor of the pancreas： report of 9 cases

Objective：To investigate the clinical features, pathological characteristics and immunophenotype of solid-pseudopapillary tumor of the pancreas（SPTP）. Methods：Nine surgically treated cases of SPTP were retrospectively reviewed. Hematoxylin and Eosin（HE） staining and immunohistochemical staining were used to analyze all cases, and the general clinical data was collected. Results：Six patients were asymptomatic except for a palpable mass. Two patients complained of vague-epigastric pain. One patient appeared jaundice. The tumor was encapsulated and solid tissues alternately with cystic tissues. Histologically, the histological structure of solid portion was pseudopapillary with a fibrovascular core. Tumor cells were uniform and medium-sized which were arranged in sheets ets or nests or pseudopapillary patterns. Immunohistochemical studies demonstrated that SPTP proved positive in vimentin（9/9 cases）, AAT（9/9 cases）, NSE（9/9 cases）, ACT（7/9 cases）, CK20（2/9 cases）, CgA（1/9 cases）, S-100（3/gcases）, PR（4/gcases）, Syn（3/9 cases） and CD56（5/9cases）, negative in CEA and ER. Conclusion：SPTP is a tumor predominantly occurring in young women frequently without special symptoms. This tumor has various characteristical histological patterns with different immunophenotype.     

Electro-Acupuncture Combined with the Trigger Point Needle-Embedding for Treatment of Primary Trigeminal Neuralgia in 31 Cases

In recent years, the author of this essay has applied electro-acupuncture combined with the trigger point needle-embedding for treatment of primary trigeminal neuralgia in 31 cases, yielding satis- factory results as reported in the following.     

Investigation of Matrix Metalloproteinase -1, 2 and tissue inhibitor of matrix metalloproteinases-1 in Endometriosis

Objective： To explore the role of matrix metalloproteinase-1,2 （MMP-1, MMP-2） and tissue inhibitor of matrix metalloproteinases-1 （TIMP-1） in endometriosis. Methods： The eutopic and ectopic endometria from 40 subjects suffering from endometriosis and regular.endometria from 40 subjects （excluding endometriosis） were collected and examined by in situ hybridization technology and western blot assay. Results： Both expressions of MMP-1 and -2 were stronger in ectopic endometrium and eutopic endometrium than in normal endometrium. On the contrary, the expression of TIMP-1 in ectopic endometrium and eutopic endometrium was lower. The differences were significant （P 〈 0.01 ）. Moreover, there was no relationship among the expressions of MMP-1, 2 and TIMP-1 in ectopic endometrium. Conclusion： The expressions of MMP-1, 2 and TIMP-1 lose balance and lack of periodic changes in ectopic endometrium , which explains the biological invasive behavior of endometriosis. It was suggested-that regulating the balance between the MMPs and TIMP-1 should be an ideal therapeutic target to endometriosis.     

Shi Da-zhuo (史大卓) —An outstanding cardiologist of integrative medicine

Prof. SHI Da-zhuo, Ph.D., male, was born on March 20, 1960. Prof. SHI entered the Ph.D. program in 1990 at the China Academy of Chinese Medical Sciences under the supervision of Prof. CHEN Ke-ji, majoring in the treatment of cardiovascular diseases. After receiving his Ph.D. degree in 1993, Prof. SHI started working at the Cardiovascular Center in Xiyuan Hospital affiliated to China Academy of Chinese Medical sciences.     

Persist in and Carry Forward the Integrative Medical Research——Speech at the 6th National General Congress of Chinese Association of Integrative Medicine by Academician CHEN Zhu,Minister of Ministry of Health

The 6th National General Congress of Chinese Association of Integrative Medicine （CALM） was convened at 19-20, April 2008 in Beijing. Academician CHEN Zhu, the minister of Ministry of Health indicated at the congress that the integration of Chinese and Western medicine is very well in keeping with the situation of our country and the general rule of development in medical science; and as a good integration of Chinese medicine and Western medicine, it is mutually beneficial and advantageous to both of them. Seeing the creativity shown in integrative medical investigation in theoretic and methodological sides, we should and must persist in and develop it.